Next Article in Journal
Modulation of de Novo Lipogenesis Improves Response to Enzalutamide Treatment in Prostate Cancer
Previous Article in Journal
Experimental Assessment of Color Deconvolution and Color Normalization for Automated Classification of Histology Images Stained with Hematoxylin and Eosin
Previous Article in Special Issue
Blockage of Store-Operated Ca2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca2+ Selective Orai1 Pore
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Hydrogen Sulfide-Evoked Intracellular Ca2+ Signals in Primary Cultures of Metastatic Colorectal Cancer Cells

1
Laboratory of General Physiology, Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, 27100 Pavia, Italy
2
Department of Biology, Cihan University-Erbil, 44001 Erbil, Iraq
3
Laboratory of Immunology Transplantation, Foundation IRCCS Policlinico San Matteo, 27100 Pavia, Italy
4
Laboratory of Biochemistry, Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, 27100 Pavia, Italy
5
Department of Molecular Medicine, University of Pavia, 27100 Pavia, Italy
6
Veneto Institute of Molecular Medicine, Foundation for Advanced Biomedical Research, 35131 Padua, Italy
7
Medical Surgery, Foundation IRCCS Policlinico San Matteo, 27100 Pavia, Italy
8
Faculty of Science, Department of Medical Analysis, Tishk International University-Erbil, 44001 Erbil, Iraq
9
Medical Oncology, Foundation IRCCS Policlinico San Matteo, 27100 Pavia, Italy
10
Diagnostic and Pediatric, Department of Sciences Clinic-Surgical, University of Pavia, 27100 Pavia, Italy
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Cancers 2020, 12(11), 3338; https://doi.org/10.3390/cancers12113338
Submission received: 10 September 2020 / Revised: 5 November 2020 / Accepted: 6 November 2020 / Published: 11 November 2020
(This article belongs to the Special Issue Targeting Calcium Signaling in Cancer Cells)

Abstract

:

Simple Summary

Colorectal cancer (CRC) is the most common type of gastrointestinal cancer and the third most predominant cancer in the world. CRC is potentially curable with surgical resection of the primary tumor. The clinical problem of colorectal cancer, however, is the spread and outgrowth of metastases, which are difficult to eradicate and lead to a patient’s death. The failure of conventional treatment to significantly improved outcomes in mCRC has prompted the search for alternative molecular targets with the goal of ameliorating the prognosis of these patients. The present investigation revealed that exogenous delivery of hydrogen sulfide (H2S) suppresses proliferation in metastatic colorectal cancer cells by inducing an increase in intracellular Ca2+ concentration. H2S was effective on metastatic, but not normal, cells. Therefore, we propose that exogenous administration of H2S to patients affected by metastatic colorectal carcinoma could represent a promising therapeutic alternative.

Abstract

Exogenous administration of hydrogen sulfide (H2S) is emerging as an alternative anticancer treatment. H2S-releasing compounds have been shown to exert a strong anticancer effect by suppressing proliferation and/or inducing apoptosis in several cancer cell types, including colorectal carcinoma (CRC). The mechanism whereby exogenous H2S affects CRC cell proliferation is yet to be clearly elucidated, but it could involve an increase in intracellular Ca2+ concentration ([Ca2+]i). Herein, we sought to assess for the first time whether (and how) sodium hydrosulfide (NaHS), one of the most widely employed H2S donors, induced intracellular Ca2+ signals in primary cultures of human metastatic CRC (mCRC) cells. We provided the evidence that NaHS induced extracellular Ca2+ entry in mCRC cells by activating the Ca2+-permeable channel Transient Receptor Potential Vanilloid 1 (TRPV1) followed by the Na+-dependent recruitment of the reverse-mode of the Na+/Ca2+ (NCX) exchanger. In agreement with these observations, TRPV1 protein was expressed and capsaicin, a selective TRPV1 agonist, induced Ca2+ influx by engaging both TRPV1 and NCX in mCRC cells. Finally, NaHS reduced mCRC cell proliferation, but did not promote apoptosis or aberrant mitochondrial depolarization. These data support the notion that exogenous administration of H2S may prevent mCRC cell proliferation through an increase in [Ca2+]i, which is triggered by TRPV1.

1. Introduction

Colorectal cancer (CRC) is the most common type of gastrointestinal cancer and the third most predominant cancer in the world. In 2018, around 1.8 million cases were reported by the World Health Organization (WHO) and 862,000 deaths were registered (WHO, 2018). These numbers are expected to increase by 80% in year 2035, reaching approximately 2.4 million cases and contributing to 1.3 million deaths worldwide [1]. CRC is potentially curable with surgical resection of the primary tumor [2]. The clinical problem of colorectal cancer, however, is the spread and outgrowth of metastases. Over the last decade, the development of new combinations of chemotherapeutic agents along with the introduction of targeted therapies improved survival of a cohort of metastatic CRC (mCRC) patients. Moreover, treatment of advanced disease is still associated with a poor prognosis and significant morbidity. The failure of conventional treatment to significantly improved outcomes in mCRC has prompted the search for alternative molecular targets with the goal of ameliorating the prognosis of these patients [2]. This makes of pivotal importance the search for alternative molecular targets with the goal of ameliorating the prognosis of these patients.
Exogenous administration of hydrogen sulfide (H2S) is emerging as an alternative anticancer treatment. H2S is the latest addition to the family of gasotransmitters, also including nitric oxide and carbon monoxide. H2S is endogenously generated from the precursor L-cysteine by pyridoxal-5’ phosphate-dependent (PLP) enzymes, including cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) [3,4]. H2S is produced in response to appropriate cell stimulation and regulates a myriad of physiological processes, including vascular tone and blood flow regulation [5,6], angiogenesis [7], synaptic transmission [8], cellular stress, inflammation, apoptosis, and energy metabolism [4,9]. Not surprisingly, disruption of physiological H2S synthesis has been implicated in multiple disorders, including hypertension, Alzheimer’s disease, diabetes mellitus, ulcerative colitis, and end-stage renal disease [9,10], while high cellular or tissue levels of H2S are highly toxic and result in severe cytotoxic effects [11]. The double-edged role played by H2S has recently been highlighted in cancer. Endogenous production of H2S enhances tumor growth and metastasis by stimulating mitochondrial bioenergetics, by eliciting proliferation, migration and invasion, and by promoting angiogenesis [12,13,14]. Nevertheless, exogenous H2S administration through H2S-releasing compounds exerts a strong anticancer effect by suppressing cancer cell proliferation and/or inducing cancer cell apoptosis [12,14,15]. A recent series of studies revealed that exogenously delivered H2S suppressed proliferation in a panel of CRC cell lines, including HT-29, Caco-2, SW1116, HCT116, and DLD1 [16,17,18,19]. The mechanism whereby exogenous H2S affects CRC cell proliferation is yet to be clearly elucidated, although it could involve the expression of the cyclin-dependent kinase inhibitor p21Cip [17] and an increase in intracellular Ca2+ concentration ([Ca2+]i) [20]. It has long been known that H2S is able to elevate [Ca2+]i by inducing either Ca2+ release from the endoplasmic reticulum (ER) [7] or by promoting extracellular Ca2+ entry through multiple Ca2+ entry pathways [21,22], including Transient Receptor Potential Vanilloid 1 (TRPV1) [21,23,24,25], TRPV3, and TRPV6 [26], TRP Ankyrin 1 (TRPA1) [27,28], L- and T-type voltage-gated Ca2+ channels (VGCCs) [21,29,30]. Intracellular Ca2+ signaling plays a crucial role in CRC cell proliferation and migration [31,32,33,34,35]; however, it has been demonstrated that Ca2+-entry pathways which exert a mitogenic effect in commercial CRC cell lines fail to do so in primary cultures derived from CRC patients [36]. It would be, therefore, therapeutically relevant to assess the effect of exogenous H2S on patients-derived CRC cells.
TRPV1 is a polymodal non-selective cation channel, which is gated by multiple stimuli, including the dietary agonist capsaicin [37], noxious heat (>41 °C) [37], extracellular acidification [38], reactive oxygen species [39], and several vanillotoxins [40]. Furthermore, TRPV1 is regarded among the main mediators of H2S-induced extracellular Ca2+ entry in healthy cells [21,23,24,25]. Notably, exogenously delivered H2S may suppress cell proliferation by promoting TRPV1-mediated extracellular Ca2+ entry in multiple cancer cell lines [41], including leukemia, breast cancer, cervical carcinoma, whereas capsaicin-induced TRPV1 activation exerts an anticancer effect in CRC [42], breast cancer [43] and bladder cancer [44] cell lines. Therefore, the present investigation aimed at assessing for the first time whether and how exogenously added H2S exerts an anticancer effect in primary cultures of metastatic CRC (mCRC) cells. We focused on mCRC cells, such as mCRC, it is often not treatable and tends to develop resistance mechanisms towards conventional pharmacological therapies [2,45]. It is, therefore, mandatory to identify alternative strategies, which could be effectively be translated in the clinical practice, to eradicate mCRC cells. To do so, we exploited a multidisciplinary approach, including intracellular Ca2+ imaging, western blotting, immunocytochemistry, flow cytometer and pharmacological manipulation. Our findings demonstrated that H2S was able to trigger extracellular Ca2+ entry in mCRC cells by activating TRPV1 and the reverse (i.e., Ca2+ entry) mode of the Na+/Ca2+ exchanger (NCX). H2S-induced Ca2+ entry was in turn able to suppress mCRC proliferation by arresting the cell cycle in the S-phase, thereby confirming that exogenous delivery of H2S may represent a reliable strategy to treat metastatic CRC patients.

2. Results

2.1. H2S Evokes a Dose-Dependent Increase [Ca2+]i in Primary Cultures of mCRC Cells

H2S was delivered to primary cultures of CRC cells loaded with the Ca2+-sensitive dye Fura-2 in the form of sodium hydrosulfide (NaHS), a water-soluble H2S donor that is widely employed to investigate H2S-induced intracellular Ca2+ signals in normal [46,47,48] as well as cancer [49] cells. Our preliminary experiments revealed that 100 µM NaHS evoked a robust increase in [Ca2+]i in mCRC cells (Figure 1A), whereas it induced a significantly (p < 0.05) smaller Ca2+ response in primary CRC (pCRC) cells (Figure 1A,B) and in cells isolated from the adjacent non-neoplastic tissue, which was used as control (Ctrl) (Figure 1A,B). Similarly, NaHS-evoked intracellular Ca2+ signals were significantly (p < 0.05) larger in pCRC as compared to non-neoplastic cells (Figure 1A,B). As eradicating metastatic cells represents the therapeutic challenge to treat CRC [2,45] and the Ca2+ signals to exogenous H2S was remarkably lower in non-neoplastic cells and pCRC cells, we focused our attention on mCRC cells.
NaHS was found to evoke dose-dependent Ca2+ signals in mCRC cells. NaHS did not induce any discernible increase in [Ca2+]i at concentrations lower than 5 µM, such as 2.5 µM (Figure 2A–C). The Ca2+ response to NaHS indeed appeared at 5 µM (Figure 2A,B), when the majority of mCRC cells produced a single Ca2+ transient in response to agonist stimulation (Figure 2A). A careful examination of the Ca2+ responses to increasing doses of NaHS revealed a U-shaped dose-response relationship, as previously reported in rat aortic endothelial cells [49]. Both the percentage of responding cells and the magnitude of the Ca2+ peak decreased as NaHS concentration raised from to 5 µM up to 50 µM and then increased again for a further elevation in NaHS dose (Figure 2B,C). Our analysis indicated that the highest Ca2+ response was induced by 100 µM NaHS, while there was no significant (p < 0.05) difference in the percentage of responding cells in the concentration range spanning from 75 µM to 300 µM (Figure 2B,C). In aggregate, these data suggest that 100 µM NaHS represent the most suitable dose to explore the mechanisms of H2S-induced intracellular Ca2+ signaling in mCRC.
The kinetics of the Ca2+ response to 100 μM NaHS showed two main patterns even in cells from the same microscopic field. The most frequent pattern observed consisted in a rapid increase in [Ca2+]i which rapidly decayed to the baseline on agonist removal (blue trace in Figure 3A). This transient increase in [Ca2+]i was detected in ≈75% of the cells (Figure 3B). In the remaining 25% (Figure 3B), the initial Ca2+ peak elevation rapidly decayed to a sustained plateau phase that was maintained as long as NaHS was present in the bath (green trace in Figure 3A). We, finally, showed that the transient Ca2+ response to 100 µM NaHS was reversible after a short washout (Figure 3C).

2.2. The Ca2+ Response to H2S in Primary Cultures of mCRC Cells Depends on Extracellular Ca2+ Entry

H2S elicits both endogenous Ca2+ release and extracellular Ca2+ entry in mammalian cells [7,21,22]. In order to assess the Ca2+ source recruited by NaHS to induce intracellular Ca2+ signals in mCRC cells, external Ca2+ was removed from the bath (0Ca2+). Under such conditions, the H2S donor failed to augment [Ca2+]i in 138 out of 138 cells (100%) (Figure 4A–C), whilst it triggered a Ca2+ response in most cells subsequent to Ca2+ re-addition to the extracellular solution (Figure 4A–C). This finding strongly suggests that NaHS-induced Ca2+ signaling required Ca2+ entry from the extracellular milieu in mCRC cells. In agreement with this observation, removal of extracellular Ca2+ in plateauing cells caused the [Ca2+]i to rapidly return to the baseline. Restoration of extracellular Ca2+ resumed the NaHS-evoked increase in [Ca2+]i (Figure 4D). The molecular nature of the membrane pathway mediating this Ca2+ inflow was then investigated by a pharmacological approach.

2.3. TRPV1 Protein is Expressed and Mediates Extracellular Ca2+ Entry in Primary Cultures of mCRC Cells

H2S may induce extracellular Ca2+ entry by activating TRPV1 in a growing number of cell types [21,23,24,25]. Herein, we first sought to assess TRPV1 expression by exposing mCRC cells to capsaicin, a selective TRPV1 agonist [37,39,43,50,51], in presence of extracellular Ca2+. Accordingly, capsaicin induced a significant elevation in [Ca2+]i that exhibited different temporal patterns (Figure 5), as previously described for NaHS (Figure 3A,B). The most frequent pattern observed consisted in a rapid increase in [Ca2+]i which rapidly decayed to the baseline in the continuous presence of the agonist (blue trace in Figure 5A). This transient increase in [Ca2+]i was detected in ≈75% of the cells (Figure 5B). In the remaining 25% (Figure 5B), the initial Ca2+ peak rapidly decayed to a sustained plateau phase that was maintained as long as capsaicin was present in the bath (green trace in Figure 5A).
As expected, capsaicin (10 μM) did not evoke endogenous Ca2+ release in the absence of extracellular Ca2+ (0Ca2+), while the Ca2+ response resumed upon Ca2+ restitution to the perfusate (Figure 6A–D). Furthermore, capsaicin-induced extracellular Ca2+ entry was suppressed by preincubating the cells with specific TRPV1 antagonists, capsazepine (10 μM, 20 min), and SB-366791 (10 μM, 20 min) [37,39,50,52] (Figure 7A–C). Similarly, the Ca2+ response to capsaicin was inhibited by the less specific TRPV1 antagonist, ruthenium red (RuR) (10 μM, 20 min) [39,53,54,55] (Figure 7A–C).
Western blot and immunofluorescence analysis confirmed the expression of TRPV1 in mCRC cell (Figure 8). Immunoblots revealed a major band of approximately 75 kDa (Figure 8A and Figure S6), as also detected in other cell types [56,57,58]. Immunocytochemical analysis, performed using a primary anti-TRPV1 antibody and a Tetramethylrhodamine-isothiocyanate (TRITC)-conjugated secondary antibody, confirmed the expression of TRPV1 in mCRC cells (Figure S1). In control samples, stained with secondary antibody in the absence of primary antibody, no significant signal was detected using the same instrument settings (Figure S1). Confocal microscopy analysis revealed that red fluorescently-labeled TRPV1 is partially localized at the same focal plane of the plasma membrane, here labeled with green fluorescent dye (Figure 8B). Altogether, these results demonstrate that TRPV1 is expressed and able to mediate extracellular Ca2+ entry in mCRC cells.

2.4. TRPV1 Mediates H2S-Induced Extracellular Ca2+ Entry in Primary Cultures of mCRC Cells

In order to assess whether TRPV1 mediates H2S-evoked extracellular Ca2+ entry, mCRC cells were pre-treated with capsazepine (10 µM, 20 min) or SB-366791 (10 μM, 20 min) and then challenged with NaHS (100 µM). As shown in Figure 9A, this treatment dramatically impaired NaHS-evoked extracellular Ca2+ entry, thereby confirming that TRPV1 mediates the Ca2+ response to H2S. Capsazepine and SB-366791 dramatically reduced the percentage of responding cells (Figure 9B) and the amplitude of the residual Ca2+ signal in the responding ones (Figure 9C). In agreement with this observation, NaHS-evoked extracellular Ca2+ entry was also erased by the less specific TRPV1 inhibitor, ruthenium red (10 µM, 30 min) (Figure 9).
Subsequently, we silenced TRPV1 expression by using a selective small interfering RNA (siTRPV1) (Figures S2 and S8). The efficacy of TRPV1 deletion in mCRC cells was confirmed by evaluating TRPV1 protein expression as compared to mCRC cells transfected with a scrambled construct (Figure 10). Preliminary experiments revealed that the Ca2+ response to capsaicin (10 µM) was significantly (p < 0.05) reduced in silenced cells (Figure 10A,B). Subsequently, we found that genetic deletion of TRPV1 remarkably reduced also NaHS-evoked extracellular Ca2+ entry (Figure 10C,D). These results indicate that TRPV1 channels play a crucial role in mediating H2S-induced extracellular Ca2+ influx in mCRC cells.
H2S has also been shown to activate VGCCs [21,29,30]. However, the Ca2+ response to NaHS (100 µM) was not affected either by nifedipine (10 µM, 30 min) or by Zn2+ (100 µM, 15 min) (Figure S3), which, respectively, block L- and T-type VGCCs [59,60]. A recent investigation showed that intracellular Ca2+ release from the endoplasmic reticulum (ER) in mCRC cells may fall below the resolution limit of epifluorescence Ca2+ imaging and lead to store-operated Ca2+ entry (SOCE) [36]. However, the pharmacological blockade of SOCE with the selective inhibitor, BTP-2 (20 µM, 30 min) [35,36], did not impair the increase in [Ca2+]i evoked by NaHS (100 µM) (Figure S4). These findings are consistent with the notion that TRPV1 plays a major role in the onset of the Ca2+ response to H2S.
In agreement with these findings, capsaicin-evoked Ca2+ signals were significantly (p < 0.05) larger in mCRC cells as compared to non-neoplastic an pCRC cells (Figures S5A,B and S9). Nevertheless, TRPV1 expression did not significantly differ between mCRC and non-neoplastic cells (Figure S5C,D).

2.5. The Reverse-Mode of NCX Contributes to H2S-Induced Extracellular Ca2+ Entry in Primary Cultures of mCRC Cells

Non-selective cation channels, such as TRPV1, which mediate also Na+ inrush into the cells, may elevate [Ca2+]i by inverting the mode of operation of NCX [61,62]. Indeed, Na+ accumulation beneath the plasma membrane may locally switch the exchanger in the reverse direction (3Na+ out: 1 Ca2+ in). We, therefore, sought to assess whether NCX sustains TRPV1-mediated Ca2+ entry in mCRC cells [63] In order to inhibit NCX activity, extracellular Na+ was replaced by an equimolar amount of N-Methyl-D-glucamine (0Na+), that cannot substitute Na+ to sustain Ca2+ transport across the plasma membrane, as described elsewhere [62] This maneuver caused a transient increase in [Ca2+]i due to the reversal of Na+ gradient and reversal of NCX activity into the reverse (i.e., Ca2+ entry) mode (Figure 11A). At the end of the Ca2+ transient, NCX could no longer mediate extracellular Ca2+ entry as Na+ was no longer present in the extracellular solution. Under this condition, the Ca2+ response to NaHS was abolished in 179 out of 245 cells (73%) (Figure 11A,B), whilst most cells were activated by the H2S donor upon restitution of extracellular Na+ (Figure 11A). The amplitude of the Ca2+ signal in the responding cells (66 out of 245; 27%) was significantly (p < 0.05) smaller as compared to the control (Figure 11C). In order to confirm NCX role in NaHS-dependent Ca2+ signaling, we probed the effect of KB-R 7943 (20 μM, 20 min), a selective inhibitor of the reverse-mode NCX at this concentration [62,64]. In agreement with the results presented above, NaHS failed to elicit an [Ca2+]i elevation in 146 out of 182 (80.2%) cells pre-treated with the blocker (Figure 11D,E). In addition, the amplitude of the Ca2+ response was significantly lower in the fraction of cells activated by the H2S donor in presence of KB-R 7943 (Figure 11F).
Furthermore, removal of extracellular Na+ (0Na+) and KB-R 7943 (20 μM, 20 min) interfered with the Ca2+ response to direct TRPV1 activation with capsaicin (10 μM). Under 0Na+ conditions, capsaicin induced a discernible increase in [Ca2+]i in 81 out of 144 cells (Figure 12A–C), whereas the magnitude of the Ca2+ signal in responding cells was significantly (p < 0.05) reduced (Figure 12D). Likewise, capsaicin-induced intracellular Ca2+ signals were absent in 53 out of 134 cells pretreated with KB-R 7943 (Figure 12E,F), whereas the magnitude of the Ca2+ signal in responding cells was also significantly (p < 0.05) reduced (Figure 12G). Taken together, these findings hint at NCX as a key mediator of NaHS-elicited Ca2+ inflow in mCRC cells upon TRPV1 activation.

2.6. Exogenous H2S Suppresses Proliferation, but Does Not Induce Apoptosis, in mCRC Cells through TRPV1 Activation

Emerging evidence suggested an inhibitory effect of exogenous application of H2S in many cancer cell lines and, in particular, in CRC cell lines [16,17,18,19]. To study the effect of NaHS on mCRC proliferation, we sought to exploit the Trypan blue exclusion assay. Figure 13 showed the effect on mCRC viability of increasing NaHS concentrations (50, 100, and 200 µM). We found a significant (p < 0.05) reduction in mCRC number after 24 h preincubation in the presence of 100 µM and 200 µM NaHS, while 50 µM NaHS did not affect mCRC cell viability (Figure 13A). The proliferation rate underwent a further slight reduction after 72 h preincubation in the presence of 200 µM NaHS (Figure 13A). In order to assess the role of extracellular Ca2+ entry, we assessed the anti-proliferative effect of NaHS in the presence of capsazepine (10 µM), to inhibit TRPV1, and KB-R 7943 (10 µM), to block the reverse mode NCX. As depicted in Figure 13B, neither capsazepine (10 µM, 20 min) nor KB-R 7943 (10 µM, 30 min) alone affected proliferation in mCRC cells as compared to control cells. However, both drugs prevented the inhibitory effect of exogenous NaHS on mCRC cell viability at 100 µM. These results strongly indicate that exogenous administration of H2S could reduce proliferation in mCRC cells by triggering Ca2+ influx through activation of TRPV1 followed by recruitment of the reverse mode NCX.
A biparametric analysis with Annexin V and 7-amino-actinomycin D (7-AAD) was then carried to evaluate whether 100 µM NaHS inhibited mCRC cell proliferation by inducing apoptosis (Figure 13C), as shown in other tumor cell types [12,14,15,41]. Nevertheless, statistical analysis revealed that 72 h preincubation with 100 µM NaHS did not induce mCRC cell apoptosis (Figure 13D). Notably, a significant percentage of mCRC cells was found to be in late apoptosis under control conditions and exposure to 100 µM NaHS did not exacerbate the extent of mCRC cell death (Figure 13C,D). Depolarization of mitochondrial potential (ΔΨm) is regarded an widespread early marker of apoptosis [65,66]. Consistent with previous findings, exposure to 100 µM NaHS for 72 h did not significantly change in ΔΨm mCRC cells loaded with tetramethylrhodamine, methyl ester (TMRM) (Figure 13E). Therefore, exogenous H2S did not cause mCRC cell death.

2.7. Exogenous H2S Does Not Stimulate Phosphorylation Cascades in mCRC Cells

Finally, we evaluated whether the administration of exogenous H2S stimulate a number of intracellular signaling pathways which are known to control mCRC cell proliferation, migration and survival [34,68]. However, NaHS (100 µM) did not promote the phosphorylation of Akt, extracellular signal-regulated kinases 1 and 2 (Erk 1 and Erk 2, respectively) and of the mammalian target of rapamycin (mTOR) (Figure 14 and Figure S7). These data are consistent with the observation that H2S suppresses, rather than enhancing, mCRC cell proliferation.

3. Discussion

Exogenous delivery of H2S is emerging as an alternative strategy to treat multiple types of malignancies [12,14,15], including CRC [69]. The mechanism whereby exogenous H2S inhibits CRC proliferation is still unclear, although it could involve an increase in [Ca2+]i [20]. Furthermore, the anticancer effect of H2S remains to be confirmed in patients-derived CRC cells, as recent studies revealed that intracellular Ca2+ signals may drive cell fate in multiple commercially available cancer cell lines, but not in primary cultures established from tumors as different as glioblastoma [70], renal cellular carcinoma [71], and CRC [36]. In addition, only a few studies described H2S-induced intracellular Ca2+ signals in cancer cells [20,49], but they failed to identify the main mediator(s) of the Ca2+ response. The present investigation provided the first evidence that H2S inhibits proliferation in primary cultures of mCRC cells by inducing extracellular Ca2+ entry through TRPV1. TRPV1-mediated Ca2+ entry is, in turn, sustained by the reverse mode NCX. This finding endorses the view that exogenous delivery of H2S represents a promising strategy to treat CRC.

3.1. H2S-Evoked Intracellular Ca2+ Signals in Primary Cultures of mCRC Cells

Exogenous administration of H2S in the form of the widely employed H2S-donor, NaHS, reliably increased the [Ca2+]i in primary cultures of mCRC cells. Conversely, the Ca2+ responses to NaHS were remarkably smaller in non-neoplastic and pCRC cells. This preliminary investigation revealed that mCRC cells displayed a higher sensitivity of exogenous delivery of H2S. An increase in [Ca2+]i was also induced by H2S release from another donor, GYY4137, in the CRC cell line, DLD1 [20]. While this investigation did not investigate the dose-response relationship and the kinetics of the Ca2+ response to H2S release [20], herein we first found that, while the Ca2+ signal arose at low NaHS concentration ([NaHS]) (i.e., 5 µM), the percentage of responding cells and the magnitude of the initial Ca2+ peak decreased by further reducing the [NaHS] to 25–50 µM. Nevertheless, the Ca2+ response was fully restored by increasing the [NaHS] to 75-300 µM. A similar U-shaped dose-response relationship has been described for NaHS-evoked intracellular Ca2+ signals in the native endothelium of rat aorta [62] and EA.hy926 cells, while a bell-shaped pattern has been reported in breast tumor-derived endothelial cells (B-TECs) [7]. The peculiar U-shaped pattern of the dose-response relationship that H2S exhibits in several cellular models has been attributed to the wide array of signaling pathways recruited by this gasotransmitter [72,73]. It should also be noticed that H2S may differently affect the Ca2+ handling machinery even within the same cell type [21,22]. For instance, NaHS stimulated inositol-1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) in Ea.hy926 cells [7] and saphenous vein-derived endothelial cells [74], while it inhibited InsP3-dependent Ca2+ release in rat aortic endothelium [7] and was ineffective in human endothelial colony forming cells [7]. Furthermore, NaHS was found to both inhibit [75] and activate [76] voltage-gated Ca2+ entry in rat cardiac myocytes. Likewise, NaHS was able to facilitate [77] or block [78] CaV3.2 ectopically expressed in HEK-293 cells. Such a variable effect exerted by H2S could be due to the different cysteine residues that can be sulfhydrated within the same protein channel, thereby exerting distinct effects on its activity [22]. We, therefore, focused on 100 µM NaHS, as mCRC cells displayed the highest Ca2+ sensitivity to this [NaHS] and preliminary experiments showed that NaHS did not affect mCRC cell viability at lower doses.
One hundred µM NaHS evoked two main intracellular Ca2+ signatures in mCRC cells: a transient increase in [Ca2+]i, which occurred in ≈75% of the cells, and a biphasic Ca2+ signal, which arose in the remaining lower fraction of cells. Different patterns of intracellular Ca2+ signals were also elicited by NaHS in rat aortic endothelium [62] and endothelial cells harvested from human saphenous vein (SVECs) [74]. Unlike non-cancer cells [62,74], the Ca2+ response to NaHS was reversible and did not desensitize after the first stimulation, which suggests that the molecular trigger of the Ca2+ signal is different and/or that the recovery from H2S-induced modifications (most likely, sulfhydration) is faster in mCRC cells. As discussed in more detail in the next paragraph, our evidence hints at TRPV1 as the main responsible for the onset of the Ca2+ response to NaHS in mCRC cells.

3.2. Evidence that TRPV1 and Reverse Mode NCX Mediate H2S-Evoked Intracellular Ca2+ Signals in Primary Cultures of mCRC Cells

It has been demonstrated that H2S may increase the [Ca2+]i by both mobilizing ER stored Ca2+ through InsP3Rs and ryanodine receptors (RyRs) and activating extracellular Ca2+ entry [7,21,22,74]. However, NaHS evoked failed to induce any discernible Ca2+ signal upon removal of extracellular Ca2+, while the Ca2+ response immediately resumed upon Ca2+ restitution to the bathing solution. While this finding does not rule out the possibility that a local Ca2+ signal is induced by NaHS, and is missed by our epifluorescence detection system, it does demonstrate that the bulk increase in [Ca2+]i is triggered by extracellular Ca2+ entry. The same observation has been reported in another cancer-derived cell line, i.e., B-TECs [49], although the underlying signaling pathway has not been uncovered.

3.2.1. TRPV1 Triggers the Ca2+ Response to NaHS in Primary Cultures of mCRC Cells

The following pieces of evidence indicate that NaHS-evoked extracellular Ca2+ entry in primary cultures of mCRC cells is triggered by TRPV1. First, the Ca2+ response to NaHS was erased by blocking TRPV1 with the selective antagonists, capsazepine and SB-366791 [37,39,50,52], and the less selective blocker ruthenium red [39,40]. Moreover, NaHS-evoked extracellular Ca2+ entry was significantly reduced by the genetic deletion of TRPV1 by a selective siTRPV1. Second, TRPV1 protein was abundantly expressed in mCRC cells, as demonstrated by immunoblotting and confocal microscopy analysis of TRPV1 expression. Likewise, TRPV1 transcript has been reported in the CRC cell line [79], HT29, and TRPV1 protein has been detected in CRC tissue [42]. The theoretical molecular weight (MW) of TRPV1 protein is 90 kDa [37]. Our TRPV1 antibody revealed a protein species of 75 kDa, as previously reported in rodent urothelium [58], retinal ganglion cells [57] and astrocytes [56], normal human bronchial epithelial cells [80], mouse and chicken chondrogenic cells [81]. As discussed in [81], multiple TRPV1 splice variants were detected [82,83,84,85,86]. Furthermore, TRPV1 proteins presents several sites for post-translational modifications, which induce alterations in the MW of the channel protein [86,87,88,89]. Third, the dietary agonist capsaicin, which is widely exploited to monitor TRPV1 activation [37,39,43,50,51], induced an increase in [Ca2+]i that was also sensitive to capsazepine, SB-366791 and ruthenium red, and to genetic deletion of TRPV1. Notably, capsaicin evoked two distinct patterns of intracellular Ca2+ signals that strongly resembled those induced by NaHS: a transient Ca2+ elevation in ≈75% cells and a biphasic Ca2+ response in the remaining ≈25% cells. One to ten µM capsaicin was shown to elicit both transient [52,90] and long-lasting [51,91,92,93,94] intracellular Ca2+ signals upon TRPV1 activation. The variable kinetics of the Ca2+ response to capsaicin (and NaHS) is likely to depend on the different extent of TRPV1 inactivation. As widely discussed in [40], extracellular Ca2+ entry could inactivate the channel by inducing TRPV1 dephosphorylation [95], depleting phosphatidylinositol 4,5-bisphosphate levels [96], or calmodulin binding [97]. It is, therefore, conceivable that the Ca2+-dependent mechanisms of TRPV1 inactivation are uncoupled from extracellular Ca2+ entry in the majority of mCRC cells. In addition, it has been suggested that increasing the strength of capsaicin stimulation results in TRPV1 pore dilation, thereby increasing single channel conductance and prolonging the duration of the ensuing increase in [Ca2+]i [93,98]. However, this mechanism is unlikely to explain the heterogeneity in the Ca2+ response to capsaicin reported in the present investigation as this was observed by presenting mCRC cells with the same agonist concentration. Ten µM capsaicin was found to stimulate TRPV1 and induce long-lasting elevations in in MCF-7 breast cancer cells [43], while the effect of TRPV1 stimulation on prostate cancer is more controversial. Indeed, 50 µM capsaicin induced intracellular Ca2+ oscillations in some prostate cancer cell lines (DU 145 and PC-3) in one study [50], while it failed to increase the [Ca2+]I in another one [51]. Capsaicin increased resting also in CRC HCT116 cells [42], but this study did not evaluate the kinetics and underlying mechanisms of the Ca2+ signal.

3.2.2. The Reverse Mode NCX Sustains the Ca2+ Response to NaHS in Primary Cultures of mCRC Cells

The following pieces of evidence indicate that the reverse (i.e., Ca2+ entry) mode NCX sustains NaHS- and capsaicin-induced extracellular Ca2+ entry. First, replacement of extracellular Na+ with an equimolar amount of choline induced a transient increase in [Ca2+]i, which reflects the directional switch of NCX from the forward (i.e., Ca2+ exit) to the reverse (i.e., Ca2+ entry) mode upon reversal of Na+ gradient across the plasma membrane [62,99]. Second, only a modest fraction (≈25%) of mCRC cells displayed a Ca2+ signal in response to NaHS and capsaicin under 0Na+ conditions. The amplitude of the Ca2+ elevation was, however, significantly lower whereas its duration was dramatically curtailed. Third, the selective blockade of the reverse mode NCX with KB-R mimicked the effect of Na+ withdrawal on NaHS- and capsaicin-induced intracellular Ca2+ signals [62,64], thereby confirming that the reverse mode NCX is recruited by TRPV1-mediated extracellular Na+ entry to sustain the ensuing increase in [Ca2+]i. These data are consistent with those reported in native endothelium of rat aorta [62] and confirm a recent report on in the CRC cell line, DLD1 [20]. In these cells, exogenous delivery of H2S through GYY4137 induced an increase in resting [Ca2+]i that was associated to the switch of NCX activity into the reverse mode. Furthermore, earlier work demonstrated that H2S release was able to increase the expression levels of NCX1 [100], the main NCX isoform expressed in CRC cells [20]. It is, therefore, possible to conclude that TRPV1 and NCX may by physically coupled in primary mCRC cells, as also described for TRP Melastatin 4 [101] and TRP Canonical 6 [102]. This feature might contribute to explaining why capsaicin- and NaHS-evoked extracellular Ca2+ entry is larger in mCRC cells although TRPV1 protein expression was similar in mCRC and non-neoplastic cells. We speculate that TRPV1 channels may be uncoupled from NCX (possibly due to lower NCX expression) in non-neoplastic and pCRC cells, thereby attenuating the amplitude of the Ca2+ response to TRPV1 activation. Experiments in our laboratories are under way to assess this important issue.

3.3. H2S Inhibits Proliferation in Primary Cultures of mCRC Cells

Exogenous delivery of H2S has been shown to suppress proliferation in several types of cancer cell lines, including hepatocellular carcinoma (Hep G2), human cervical carcinoma (HeLa), breast adenocarcinoma (MCF-7), osteosarcoma (U2OS), human thyroid carcinoma (Nthy-ori3-1), and erythroleukemia (K562) [19,41,103]. In addition, exogenously added H2S impaired cell viability in a wide number of CRC cell lines, including HT-29, Caco-2, HCT-116, SW1116, HCT116, and DLD1 [16,17,18,19]. Herein, we confirmed that exogenous H2S significantly reduced proliferation in primary cultures of mCRC cells exposed to 100–200 µM NaHS for 24 h. Of note, there was not further decrease in mCRC cell survival when the exposure to NaHS was prolonged to 72 h, which suggests the activation of protective signaling pathways. For instance, 24–48 h treatment with 400–1000 µM NaHS induced protective autophagy by inhibiting the mammalian target of rapamycin, thereby hampering the concurrent anti-mitogenic effect on CRC cell lines [17]. The anti-proliferative effect of NaHS was rescued by preventing TRPV1-induced extracellular Ca2+ entry wither with capsazepine or KB-R 7943, as recently reported in leukemia, breast cancer, cervical carcinoma cell lines [41]. TRPV1 has been shown to inhibit cancer cell proliferation by promoting apoptosis [42]. However, 72 h treatment with NaHS did not increase the basal rate of mCRC cell apoptosis, neither promoted mitochondrial depolarization, which represents an established marker of apoptosis. Conversely, TRPV1-mediated Ca2+ entry recruited calcineurin to activate p53 and induce apoptosis in the CRC HCT116 cell line [42]. As discussed elsewhere [35,104], the discrepancy between this report and our findings is consistent with the emerging notion that the same components of the Ca2+ toolkit could induce distinct outcomes in primary cultures of mCRC cells [36] and commercially available CRC cell lines [31,105]. Furthermore, the Ca2+ response evoked by exogenous administration of H2S displayed transient kinetics in the majority of mCRC cells. Conversely, pro-apoptotic Ca2+ signals usually consist in prolonged elevations in [Ca2+]i, which persist as long as the stimulus is presented to the cells [66,106,107,108,109], as we have recently shown in cisplatin-treated glioblastoma cells [65].
Nevertheless, data obtained from both primary cultures and immortalized cell lines support the anticancer effect of exogenous H2S delivery in CRC. Interestingly, a recent report showed that, in bladder cancer cell lines, TRPV1-mediated extracellular Ca2+ entry prevented the nuclear translocation of proliferating cell nuclear antigen (PCNA), a 29 kDa protein which serves as auxiliary protein for accessory protein for DNA polymerase δ (Polδ) and DNA polymerase ε (Polε) [110,111]. Future work will have to assess whether this signaling pathway is recruited by exogenous H2S to inhibit proliferation also in mCRC cells. Currently, we provided the evidence that the administration of exogenous H2S did not activate the Akt, Erk 1/2, and mTOR signaling pathways.

4. Materials and Methods

4.1. Expansion of Tumor Cells

Patients (>18 years) suffering from mCRC who had undergone surgery intervention to dissect primary tumor and/or liver metastases were enrolled. All the patients signed an informed consent. Tumor specimens were processed as previously described [36,112]. The present investigation has been approved by the Ethical Committee of the IRCCS Foundation Policlinico San Matteo (protocol number: 20190069408). Briefly, tumor samples were treated with Tumor dissociation Kit (Miltenyi BIOTEC, Bergisch Gladbach, Germany) and then disaggregated with the gentleMACS Dissociator (Miltenyi BIOTEC, Bergisch Gladbach, Germany) according to the manufacturers’ instructions. Tumor cells recovered from filtration for removing were resuspended at a concentration of 0.5–1 × 106 cells/mL of CellGro SCGM (CellGenix, Freiburg, Germany), supplemented with 20% FBS, 2 mM L-glutamine (complete medium) (Life Technologies, Inc. Monza, Italy) and cultured in 25 cm2 tissue flasks (Corning, Stone, England) at 37 °C and 5% CO2. The culture medium was changed twice a week and cellular homogeneity evaluated microscopically every 24–48 h, when cultures reaching about 75–100% confluence were subjected to trypsinization with 0.25% trypsin and 0.02% EDTA (Life Technologies, Inc.) in a calcium/magnesium-free balanced solution. The culture medium was changed twice a week and cellular homogeneity was confirmed by using a light microscope every 24–48 h. In particular, we cryopreserved several vials at passage T0 (after disaggregation) and at passage T1. These vials were then further thawed for further experiments. Using this approach, the experiments were carried out with tumor cells that underwent no more than 3–4 in vitro passages, to avoid problems related to long-term culture of cancer cells. Cells were cryopreserved in 90% FBS and 10% dimethyl sulfoxide and stored in liquid nitrogen for further experiments. To confirm the neoplastic origin of cultured cells passaged 3–5 times, we carried out morphological and immunocytochemical analysis, as described in [112,113]. Non-neoplastic cells were derived from tissue a sample of healthy colon tissue near the tumor. Cells were expanded and their non-neoplastic nature was assessed by morphological and immunocytochemical analysis.

4.2. Solutions for Intracellular Ca2+ Recordings

The composition of the physiological salt solution (PSS) was the following (in mM): 150 NaCl, 6 KCl, 1.5 CaCl2, 1 MgCl2, 10 Glucose, 10 HEPES. In Ca2+-free solution (0Ca2+), Ca2+ was replaced with 2 mM NaCl, and 0.5 mM EGTA was added. Solutions were titrated to pH 7.4 with NaOH. The osmolality of PSS was measured with an osmometer (WESCOR 5500, Logan, UT, USA) and was equal to 338 mmol/kg.

4.3. [Ca2+]i Measurements

Ca2+ imaging in mCRC cells was carried out as described elsewhere [34,36]. Briefly, mCRC cells were loaded with 4 µM fura-2 acetoxymethyl ester (Fura-2/AM; 1 mM stock in dimethyl sulfoxide) in PSS for 30 min at 37 °C and 5% CO2. The cells were maintained in the presence of Fura-2 for 30 min at 37 °C and 5% CO2 saturated humidity. After de-esterification in PSS for 15 min, the coverslip (8 mm) was mounted on the bottom of a Petri dish and the cells observed by an upright epifluorescence Axiolab microscope (Carl Zeiss, Oberkochen, Germany) equipped with a Zeiss ×40 Achroplan objective (water-immersion, 2.0 mm working distance, 0.9 numerical aperture). The cells were alternately excited at 340 nM and 380 nm by using a filter wheel (Lambda 10, Sutter Instrument, Novato, CA, USA). The emitted fluorescence was detected at 510 nm by using an Extended-ISIS CCD camera (Photonic Science, Millham, UK). Custom software, working in the LINUX environment, was used to drive the camera and the filter wheel, and to measure and plot on-line the fluorescence from 10 up to 40 rectangular “regions of interest” (ROI), each corresponding to a well-defined single cell. The [Ca2+]i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (Ratio (F340/F380)). An increase in [Ca2+]i causes an increase in the ratio [36]. Ratio measurements were performed and plotted on-line every 3 s. The experiments were performed at room temperature (22 °C).

4.4. SDS-PAGE and Immunoblotting

Cells were lysed in ice-cold RIPA buffer (50 mM TRIS/HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P40, 1 mM EDTA, 0.25% sodium deoxycholate, 0.1% SDS) added of protease and phosphatase inhibitors. Upon protein quantification, the samples were dissociated by addition of half volume of SDS-sample buffer 3× (37.5 mM TRIS, pH 8.3, 288 mM glycine, 6% SDS, 1.5% DTT, 30% glycerol, and 0.03% bromophenol blue), separated by SDS-PAGE on a 10% or 6% polyacrylamide gel, and blotted on a PVDF membrane. Membrane probing was performed using the different antibodies diluted 1:1000 in TBS (20 mM Tris, 500 mM NaCl, pH 7.5) containing 5% BSA and 0.1% Tween-20 in combination with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000 in Phosphate Buffered Saline (PBS) plus 0.1% Tween-20). The following antibodies were used: anti-TRPV1 (ab3487) from ABCAM (Cambridge, UK), HRP-conjugated anti-GAPDH (sc365062), anti-Akt 1/2/3 (sc8312), anti-Erk 2 (sc-154) and anti-mTOR (sc1549) from Santa Cruz Biotechnology (Dallas, TX, USA); the specific phospho-antibodies: phospho-Akt (Ser473) (#9271), phospho-Erk 1/2 (Thr 202/Tyr 204) (#9101) and phosphor-mTOR (Ser 2481) (#2974) from Cell Signaling Technology (Danvers, MA, USA). The chemiluminescence reaction was performed using Immobilon Western (Millipore, Burlington, MA, USA) and images were acquired by ChemiDoc XRS (Bio-Rad, Hercules, CA, USA). Membrane was then re-probed with an HRP-conjugated anti-GAPDH antibody as equal loading control.

4.5. Confocal Microscopy and Immunofluorescence

Cells were grown for 24 h on 15 mm glass coverslips in a 12-well plate. Samples were then fixed with 3% PFA in PBS, permeabilized with 0.25% ice-cold TRITON X100 in PBS and blocked with 1% BSA in PBS. The coverslips were stained with a rabbit anti-human TRPV1 antibody (1:250, ab3487-ABCAM; Cambridge, UK) for 1 h at room temperature and then incubated with TRITC-anti rabbit secondary antibody (1:500, ab6718-ABCAM; Cambride, UK) in the dark for 1 h at room temperature. Negative controls were prepared by omitting the primary antibody. Nuclei were stained with 1 µg/mL Hoechst 33342 (#4082-Cell Signaling Technologies; Danvers, MA, USA) and cell membrane with 2 µM PKH67 (MINI67–Sigma-Aldrich; Milan, Italy). Finally, the coverslips were mounted on glass slides using ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA, USA) as mounting medium. Immunofluorescence images were acquired using an Olympus BX51 microscope, whereas the confocal microscopy images were captured using a Leica TCS SP8 Microscope and analyzed with LAS X software (Leica Microsystems GmbH, Wetzlar, Germany).

4.6. Gene Silencing

The siRNA targeting TRPV1 was purchased from Sigma-Aldrich Inc. MISSION esiRNA (human TRPV1) (EHU073721). Scrambled siRNA was used as negative control. In brief, once the seeded mCRC cells had reached 50% confluency, the medium was replaced with Opti-MEM, the serum in the medium reduced without antibiotics (Life Technologies, Milan, Italy). siRNAs (100 nM final concentration) were diluted with Opti-MEM I reduced serum medium and mixed with Lipofectamine™ RNAiMAX transfection reagent (Life Technologies, Milan, Italy) pre-diluted in Opti-MEM), according to the manufacturer’s instructions. After 20 min incubation at room temperature, the mixtures were added to the cells and incubated at 37 °C for 5 h. Transfection mixes were then completely removed and fresh culture media was added again and silenced cells were used 48 h after transfection. The effectiveness of silencing was determined by immunoblotting (see Figure S2).

4.7. Identification of Apoptotic Cells

Metastatic CRC cells at confluence were detached by trypsinization and incubated with FITC-conjugated Annexin V (5 µL/5 × 105 cells) (Annexin V apoptosis Detection Kit, Invitrogen, Carlsbad, California, USA) and with 7-ADD (5 µL/5 × 105 cells) (BD Pharmingen, San Diego, CA, USA). Metastatic CRC cells maintained in the absence and presence of NaHS (100 µM, 72 h) were incubated for 15 min at resting temperature and then analyzed by Beckman Coulter Navios according to manufactures’ instructions.

4.8. Measurement of Mitochondrial Membrane Potential (ΔΨm)

ΔΨm was evaluated with TMRM at a loading concentration sufficient to cause dye aggregation within the mitochondrial matrix and by using the same fluorescence detection system used to measure changes in Fura-2 fluorescence. The mCRC cells were loaded with 25 nM TMRM and 200 nM of cyclosporine H, in PSS for 30 min at 37 °C and 5% CO2. After washing in PSS, the coverslip was fixed to the bottom of a Petri dish and the cells were excited 480 nm, while the emitted light was detected at 510 nm. TRITC filter for live imaging has been utilized to examine the TMRM red-orange fluorescence, and round diaphragm was employed to augment the contrast. Measurements were carried out and plotted on-line every 10 s. All the recordings were carried out at 22 °C.

4.9. Statistics

All the data have been obtained from mCRC cells plated on at least three coverslips and deriving from three distinct donors. Each trace shown is representative of multiple cells displaying a similar Ca2+ activity and deriving from at least three distinct donors. The peak amplitude of NaHs- and capsaicin-evoked Ca2+ signals was measured by evaluating the difference between the F340/F380 ratio at the peak of the Ca2+ response, and the mean F340/F380 ratio of 1 min baseline recording before agonist addition. Pooled data are presented as mean ± SE, and statistical significance (p < 0.05) was evaluated by the Student’s t-test for unpaired observations or one-way ANOVA analysis followed by the post-hoc Dunnett’s or Bonferroni tests, as required. The number of cells measured for each experimental condition is indicated in, or above, the corresponding bar histogram.

5. Conclusions

This study demonstrates for the first time that the H2S-releasing compounds NaHS elicit intracellular Ca2+ signals in primary cultures of mCRC, but not of non-neoplastic and pCRC cells, by stimulating TRPV1 to mediate extracellular Ca2+ entry and induce NCX to switch into the reverse (Ca2+ entry) mode. NaHS-induced TRPV1 activation, in turn, inhibited mCRC cell proliferation. The anti-proliferative effect of NaHS was not due to mCRC cell apoptosis. These data lend further support to the exogenous delivery of H2S as a novel therapeutic strategy to treat mCRC.

Supplementary Materials

The following are available online at https://www.mdpi.com/2072-6694/12/11/3338/s1, Figure S1: Representative immunofluorescence analysis of TRPV1 expression in mCRC cells, Figure S2: Genetic silencing of TRPV1 in silenced mCRC cells, Figure S3: The pharmacological blockade of voltage-gated Ca2+ channels does not affect NaHS-evokes intracellular Ca2+ signals in CRC and non-neoplastic cells, Figure S4: The pharmacological blockade of SOCE does not affect NaHS-evokes intracellular Ca2+ signals in CRC and non-neoplastic cells, Figure S5: TRPV1-mediated extracellular Ca2+ entry is larger in mCRC cells, Figure S6: Uncropped blots corresponding to Western blot in the Figure 8A in the main text, Figure S7: Uncropped blots corresponding to Western blot in the Figure 14 in the main text, Figure S8: Uncropped blots corresponding to Western blot in the Figure S2 shown above, Figure S9: Uncropped blots corresponding to Western blot in the Figure S5 shown above.

Author Contributions

Conceptualization, F.M.; methodology, P.F., F.F., M.V., M.T., S.N., A.R.; formal analysis, P.F., F.F., M.V., M.T. and S.N.; resources, K.L., M.M., M.S., P.P., G.F.G., D.M., and F.M.; writing—original draft preparation, F.M.; writing—review and editing, G.F.G., D.M., F.M.; writing—final approval, G.F.G., D.M., and F.M.; funding acquisition, P.P., D.M., and F.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by: Italian Ministry of Education, University and Research (MIUR): Dipartimenti di Eccellenza Program (2018–2022)-Dept. of Biology and Biotechnology "L. Spallanzani", University of Pavia (F.M.), Fondo Ricerca Giovani from the University of Pavia (F.M.), Italian Ministry of Health Grants RF-2010-2316319 (D.M.), and by program “Ricerca Corrente” of the IRCCS Policlinico San Matteo: RC/08059815B (D.M.) and RF-2011-02352315 (P.P.).

Acknowledgments

The authors thank Maria Grazia Bottone and Valentina Astesana, Department of Biology and Biotechnology “L. Spallanzani”, Laboratory of Cell Biology, for the use of the fluorescence microscope for Lysotracker Red experiments.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Douaiher, J.; Ravipati, A.; Grams, B.; Chowdhury, S.; Alatise, O.; Are, C. Colorectal cancer-global burden, trends, and geographical variations. J. Surg. Oncol. 2017, 115, 619–630. [Google Scholar] [CrossRef] [PubMed]
  2. Carrato, A. Adjuvant treatment of colorectal cancer. Gastrointest. Cancer Res. 2008, 2, S42–S46. [Google Scholar] [PubMed]
  3. Paul, B.D.; Snyder, S.H. H2S signalling through protein sulfhydration and beyond. Nat. Rev. Mol. Cell Biol. 2012, 13, 499–507. [Google Scholar] [CrossRef] [PubMed]
  4. Li, L.; Rose, P.; Moore, P.K. Hydrogen sulfide and cell signaling. Annu. Rev. Pharmacol. Toxicol. 2011, 51, 169–187. [Google Scholar] [CrossRef] [Green Version]
  5. Yang, G.; Wu, L.; Jiang, B.; Yang, W.; Qi, J.; Cao, K.; Meng, Q.; Mustafa, A.K.; Mu, W.; Zhang, S.; et al. H2S as a physiologic vasorelaxant: Hypertension in mice with deletion of cystathionine gamma-lyase. Science 2008, 322, 587–590. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Altaany, Z.; Moccia, F.; Munaron, L.; Mancardi, D.; Wang, R. Hydrogen sulfide and endothelial dysfunction: Relationship with nitric oxide. Curr. Med. Chem. 2014, 21, 3646–3661. [Google Scholar] [CrossRef] [Green Version]
  7. Potenza, D.M.; Guerra, G.; Avanzato, D.; Poletto, V.; Pareek, S.; Guido, D.; Gallanti, A.; Rosti, V.; Munaron, L.; Tanzi, F.; et al. Hydrogen sulphide triggers VEGF-induced intracellular Ca2+ signals in human endothelial cells but not in their immature progenitors. Cell Calcium 2014, 56, 225–234. [Google Scholar] [CrossRef] [Green Version]
  8. Austgen, J.R.; Hermann, G.E.; Dantzler, H.A.; Rogers, R.C.; Kline, D.D. Hydrogen sulfide augments synaptic neurotransmission in the nucleus of the solitary tract. J. Neurophysiol. 2011, 106, 1822–1832. [Google Scholar] [CrossRef] [Green Version]
  9. Wang, R. Physiological implications of hydrogen sulfide: A whiff exploration that blossomed. Physiol. Rev. 2012, 92, 791–896. [Google Scholar] [CrossRef] [Green Version]
  10. Whiteman, M.; Le Trionnaire, S.; Chopra, M.; Fox, B.; Whatmore, J. Emerging role of hydrogen sulfide in health and disease: Critical appraisal of biomarkers and pharmacological tools. Clin. Sci. 2011, 121, 459–488. [Google Scholar] [CrossRef]
  11. Jiang, J.; Chan, A.; Ali, S.; Saha, A.; Haushalter, K.J.; Lam, W.L.; Glasheen, M.; Parker, J.; Brenner, M.; Mahon, S.B.; et al. Hydrogen Sulfide—Mechanisms of Toxicity and Development of an Antidote. Sci. Rep. 2016, 6, 20831. [Google Scholar] [CrossRef] [PubMed]
  12. Hellmich, M.R.; Szabo, C. Hydrogen Sulfide and Cancer. Handb. Exp. Pharmacol. 2015, 230, 233–241. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Katsouda, A.; Bibli, S.I.; Pyriochou, A.; Szabo, C.; Papapetropoulos, A. Regulation and role of endogenously produced hydrogen sulfide in angiogenesis. Pharmacol. Res. 2016, 113, 175–185. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Szabo, C. Gasotransmitters in cancer: From pathophysiology to experimental therapy. Nat. Rev. Drug Discov. 2016, 15, 185–203. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Cao, X.; Ding, L.; Xie, Z.Z.; Yang, Y.; Whiteman, M.; Moore, P.K.; Bian, J.S. A Review of Hydrogen Sulfide Synthesis, Metabolism, and Measurement: Is Modulation of Hydrogen Sulfide a Novel Therapeutic for Cancer? Antioxid. Redox Signal. 2019, 31, 1–38. [Google Scholar] [CrossRef]
  16. Olah, G.; Modis, K.; Toro, G.; Hellmich, M.R.; Szczesny, B.; Szabo, C. Role of endogenous and exogenous nitric oxide, carbon monoxide and hydrogen sulfide in HCT116 colon cancer cell proliferation. Biochem. Pharmacol. 2018, 149, 186–204. [Google Scholar] [CrossRef] [PubMed]
  17. Wu, Y.C.; Wang, X.J.; Yu, L.; Chan, F.K.; Cheng, A.S.; Yu, J.; Sung, J.J.; Wu, W.K.; Cho, C.H. Hydrogen sulfide lowers proliferation and induces protective autophagy in colon epithelial cells. PLoS ONE 2012, 7, e37572. [Google Scholar] [CrossRef] [Green Version]
  18. Sakuma, S.; Minamino, S.; Takase, M.; Ishiyama, Y.; Hosokura, H.; Kohda, T.; Ikeda, Y.; Fujimoto, Y. Hydrogen sulfide donor GYY4137 suppresses proliferation of human colorectal cancer Caco-2 cells by inducing both cell cycle arrest and cell death. Heliyon 2019, 5, e02244. [Google Scholar] [CrossRef] [Green Version]
  19. Lee, Z.W.; Zhou, J.; Chen, C.S.; Zhao, Y.; Tan, C.H.; Li, L.; Moore, P.K.; Deng, L.W. The slow-releasing hydrogen sulfide donor, GYY4137, exhibits novel anti-cancer effects in vitro and in vivo. PLoS ONE 2011, 6, e21077. [Google Scholar] [CrossRef] [Green Version]
  20. Szadvari, I.; Hudecova, S.; Chovancova, B.; Matuskova, M.; Cholujova, D.; Lencesova, L.; Valerian, D.; Ondrias, K.; Babula, P.; Krizanova, O. Sodium/calcium exchanger is involved in apoptosis induced by H2S in tumor cells through decreased levels of intracellular pH. Nitric Oxide 2019, 87, 1–9. [Google Scholar] [CrossRef]
  21. Munaron, L.; Avanzato, D.; Moccia, F.; Mancardi, D. Hydrogen sulfide as a regulator of calcium channels. Cell Calcium 2013, 53, 77–84. [Google Scholar] [CrossRef] [PubMed]
  22. Zhang, W.; Xu, C.; Yang, G.; Wu, L.; Wang, R. Interaction of H2S with Calcium Permeable Channels and Transporters. Oxid. Med. Cell. Longev. 2015, 2015, 323269. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Yang, R.; Liu, Y.; Yu, T.; Liu, D.; Shi, S.; Zhou, Y.; Zhou, Y. Hydrogen sulfide maintains dental pulp stem cell function via TRPV1-mediated calcium influx. Cell Death Discov. 2018, 4, 1. [Google Scholar] [CrossRef] [PubMed]
  24. Patacchini, R.; Santicioli, P.; Giuliani, S.; Maggi, C.A. Hydrogen sulfide (H2S) stimulates capsaicin-sensitive primary afferent neurons in the rat urinary bladder. Br. J. Pharmacol. 2004, 142, 31–34. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Bhatia, M.; Zhi, L.; Zhang, H.; Ng, S.W.; Moore, P.K. Role of substance P in hydrogen sulfide-induced pulmonary inflammation in mice. Am. J. Physiol. Lung Cell Mol. Physiol. 2006, 291, L896–L904. [Google Scholar] [CrossRef]
  26. Liu, Y.; Yang, R.; Liu, X.; Zhou, Y.; Qu, C.; Kikuiri, T.; Wang, S.; Zandi, E.; Du, J.; Ambudkar, I.S.; et al. Hydrogen sulfide maintains mesenchymal stem cell function and bone homeostasis via regulation of Ca2+ channel sulfhydration. Cell Stem Cell 2014, 15, 66–78. [Google Scholar] [CrossRef] [Green Version]
  27. Hajna, Z.; Saghy, E.; Payrits, M.; Aubdool, A.A.; Szoke, E.; Pozsgai, G.; Batai, I.Z.; Nagy, L.; Filotas, D.; Helyes, Z.; et al. Capsaicin-Sensitive Sensory Nerves Mediate the Cellular and Microvascular Effects of H2S via TRPA1 Receptor Activation and Neuropeptide Release. J. Mol. Neurosci. 2016, 60, 157–170. [Google Scholar] [CrossRef]
  28. Pozsgai, G.; Hajna, Z.; Bagoly, T.; Boros, M.; Kemeny, A.; Materazzi, S.; Nassini, R.; Helyes, Z.; Szolcsanyi, J.; Pinter, E. The role of transient receptor potential ankyrin 1 (TRPA1) receptor activation in hydrogen-sulphide-induced CGRP-release and vasodilation. Eur. J. Pharmacol. 2012, 689, 56–64. [Google Scholar] [CrossRef]
  29. Tang, G.; Wu, L.; Wang, R. Interaction of hydrogen sulfide with ion channels. Clin. Exp. Pharmacol. Physiol. 2010, 37, 753–763. [Google Scholar] [CrossRef]
  30. Elies, J.; Scragg, J.L.; Boyle, J.P.; Gamper, N.; Peers, C. Regulation of the T-type Ca2+ channel Cav3.2 by hydrogen sulfide: Emerging controversies concerning the role of H2 S in nociception. J. Physiol. 2016, 594, 4119–4129. [Google Scholar] [CrossRef] [Green Version]
  31. Nunez, L.; Valero, R.A.; Senovilla, L.; Sanz-Blasco, S.; Garcia-Sancho, J.; Villalobos, C. Cell proliferation depends on mitochondrial Ca2+ uptake: Inhibition by salicylate. J. Physiol. 2006, 571, 57–73. [Google Scholar] [CrossRef] [PubMed]
  32. Weiss, H.; Amberger, A.; Widschwendter, M.; Margreiter, R.; Ofner, D.; Dietl, P. Inhibition of store-operated calcium entry contributes to the anti-proliferative effect of non-steroidal anti-inflammatory drugs in human colon cancer cells. Int. J. Cancer 2001, 92, 877–882. [Google Scholar] [CrossRef] [PubMed]
  33. Valero, R.A.; Senovilla, L.; Nunez, L.; Villalobos, C. The role of mitochondrial potential in control of calcium signals involved in cell proliferation. Cell Calcium 2008, 44, 259–269. [Google Scholar] [CrossRef] [PubMed]
  34. Faris, P.; Pellavio, G.; Ferulli, F.; Di Nezza, F.; Shekha, M.; Lim, D.; Maestri, M.; Guerra, G.; Ambrosone, L.; Pedrazzoli, P.; et al. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Induces Intracellular Ca2+ Release through the Two-Pore Channel TPC1 in Metastatic Colorectal Cancer Cells. Cancers 2019, 11, 542. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Moccia, F.; Zuccolo, E.; Poletto, V.; Turin, I.; Guerra, G.; Pedrazzoli, P.; Rosti, V.; Porta, C.; Montagna, D. Targeting Stim and Orai Proteins as an Alternative Approach in Anticancer Therapy. Curr. Med. Chem. 2016, 23, 3450–3480. [Google Scholar] [CrossRef]
  36. Zuccolo, E.; Laforenza, U.; Ferulli, F.; Pellavio, G.; Scarpellino, G.; Tanzi, M.; Turin, I.; Faris, P.; Lucariello, A.; Maestri, M.; et al. Stim and Orai mediate constitutive Ca2+ entry and control endoplasmic reticulum Ca2+ refilling in primary cultures of colorectal carcinoma cells. Oncotarget 2018, 9, 31098–31119. [Google Scholar] [CrossRef] [Green Version]
  37. Caterina, M.J.; Schumacher, M.A.; Tominaga, M.; Rosen, T.A.; Levine, J.D.; Julius, D. The capsaicin receptor: A heat-activated ion channel in the pain pathway. Nature 1997, 389, 816–824. [Google Scholar] [CrossRef]
  38. Caterina, M.J.; Leffler, A.; Malmberg, A.B.; Martin, W.J.; Trafton, J.; Petersen-Zeitz, K.R.; Koltzenburg, M.; Basbaum, A.I.; Julius, D. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000, 288, 306–313. [Google Scholar] [CrossRef]
  39. Lodola, F.; Rosti, V.; Tullii, G.; Desii, A.; Tapella, L.; Catarsi, P.; Lim, D.; Moccia, F.; Antognazza, M.R. Conjugated polymers optically regulate the fate of endothelial colony-forming cells. Sci. Adv. 2019, 5, eaav4620. [Google Scholar] [CrossRef] [Green Version]
  40. Bevan, S.; Quallo, T.; Andersson, D.A. Trpv1. Handb. Exp. Pharmacol. 2014, 222, 207–245. [Google Scholar] [CrossRef]
  41. Gao, M.; Li, J.; Nie, C.; Song, B.; Yan, L.; Qian, H. Design, synthesis and biological evaluation of novel hydrogen sulfide releasing capsaicin derivatives. Bioorg. Med. Chem. 2018, 26, 2632–2639. [Google Scholar] [CrossRef] [PubMed]
  42. Hou, N.; He, X.; Yang, Y.; Fu, J.; Zhang, W.; Guo, Z.; Hu, Y.; Liang, L.; Xie, W.; Xiong, H.; et al. TRPV1 Induced Apoptosis of Colorectal Cancer Cells by Activating Calcineurin-NFAT2-p53 Signaling Pathway. BioMed Res. Int. 2019, 2019, 6712536. [Google Scholar] [CrossRef] [PubMed]
  43. Wu, T.T.; Peters, A.A.; Tan, P.T.; Roberts-Thomson, S.J.; Monteith, G.R. Consequences of activating the calcium-permeable ion channel TRPV1 in breast cancer cells with regulated TRPV1 expression. Cell Calcium 2014, 56, 59–67. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Zheng, L.; Chen, J.; Ma, Z.; Liu, W.; Yang, F.; Yang, Z.; Wang, K.; Wang, X.; He, D.; Li, L.; et al. Capsaicin enhances anti-proliferation efficacy of pirarubicin via activating TRPV1 and inhibiting PCNA nuclear translocation in 5637 cells. Mol. Med. Rep. 2016, 13, 881–887. [Google Scholar] [CrossRef] [Green Version]
  45. Gonzalez-Villarreal, C.A.; Quiroz-Reyes, A.G.; Islas, J.F.; Garza-Trevino, E.N. Colorectal Cancer Stem Cells in the Progression to Liver Metastasis. Front. Oncol. 2020, 10, 1511. [Google Scholar] [CrossRef]
  46. Hennig, B.; Diener, M. Actions of hydrogen sulphide on ion transport across rat distal colon. Br. J. Pharmacol. 2009, 158, 1263–1275. [Google Scholar] [CrossRef] [Green Version]
  47. Zhang, Y.; Wang, Y.; Read, E.; Fu, M.; Pei, Y.; Wu, L.; Wang, R.; Yang, G. Golgi stress response, H2S metabolism, and intracellular calcium homeostasis. Antioxid. Redox Signal. 2019, 32, 583–601. [Google Scholar] [CrossRef]
  48. De Pascual, R.; Baraibar, A.M.; Mendez-Lopez, I.; Perez-Ciria, M.; Polo-Vaquero, I.; Gandia, L.; Ohia, S.E.; Garcia, A.G.; de Diego, A.M.G. Hydrogen sulphide facilitates exocytosis by regulating the handling of intracellular calcium by chromaffin cells. Pflugers Arch. 2018, 470, 1255–1270. [Google Scholar] [CrossRef]
  49. Pupo, E.; Pla, A.F.; Avanzato, D.; Moccia, F.; Cruz, J.E.; Tanzi, F.; Merlino, A.; Mancardi, D.; Munaron, L. Hydrogen sulfide promotes calcium signals and migration in tumor-derived endothelial cells. Free Radic. Biol. Med. 2011, 51, 1765–1773. [Google Scholar] [CrossRef]
  50. Pecze, L.; Blum, W.; Henzi, T.; Schwaller, B. Endogenous TRPV1 stimulation leads to the activation of the inositol phospholipid pathway necessary for sustained Ca2+ oscillations. Biochim. Biophys. Acta 2016, 1863, 2905–2915. [Google Scholar] [CrossRef]
  51. Pecze, L.; Josvay, K.; Blum, W.; Petrovics, G.; Vizler, C.; Olah, Z.; Schwaller, B. Activation of endogenous TRPV1 fails to induce overstimulation-based cytotoxicity in breast and prostate cancer cells but not in pain-sensing neurons. Biochim. Biophys. Acta 2016, 1863, 2054–2064. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  52. Ramirez-Barrantes, R.; Cordova, C.; Gatica, S.; Rodriguez, B.; Lozano, C.; Marchant, I.; Echeverria, C.; Simon, F.; Olivero, P. Transient Receptor Potential Vanilloid 1 Expression Mediates Capsaicin-Induced Cell Death. Front Physiol 2018, 9, 682. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Dragoni, S.; Guerra, G.; Fiorio Pla, A.; Bertoni, G.; Rappa, A.; Poletto, V.; Bottino, C.; Aronica, A.; Lodola, F.; Cinelli, M.P.; et al. A functional Transient Receptor Potential Vanilloid 4 (TRPV4) channel is expressed in human endothelial progenitor cells. J. Cell. Physiol. 2015, 230, 95–104. [Google Scholar] [CrossRef] [PubMed]
  54. Parpaite, T.; Cardouat, G.; Mauroux, M.; Gillibert-Duplantier, J.; Robillard, P.; Quignard, J.F.; Marthan, R.; Savineau, J.P.; Ducret, T. Effect of hypoxia on TRPV1 and TRPV4 channels in rat pulmonary arterial smooth muscle cells. Pflugers Arch. 2016, 468, 111–130. [Google Scholar] [CrossRef] [PubMed]
  55. Pan, L.; Song, K.; Hu, F.; Sun, W.; Lee, I. Nitric oxide induces apoptosis associated with TRPV1 channel-mediated Ca2+ entry via S-nitrosylation in osteoblasts. Eur. J. Pharmacol. 2013, 715, 280–285. [Google Scholar] [CrossRef]
  56. Ho, K.W.; Lambert, W.S.; Calkins, D.J. Activation of the TRPV1 cation channel contributes to stress-induced astrocyte migration. Glia 2014, 62, 1435–1451. [Google Scholar] [CrossRef] [Green Version]
  57. Sappington, R.M.; Sidorova, T.; Ward, N.J.; Chakravarthy, R.; Ho, K.W.; Calkins, D.J. Activation of transient receptor potential vanilloid-1 (TRPV1) influences how retinal ganglion cell neurons respond to pressure-related stress. Channels 2015, 9, 102–113. [Google Scholar] [CrossRef]
  58. Yu, W.; Hill, W.G.; Apodaca, G.; Zeidel, M.L. Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium. Am. J. Physiol. Renal Physiol. 2011, 300, F49–F59. [Google Scholar] [CrossRef] [Green Version]
  59. Rebuzzini, P.; Zuccolo, E.; Civello, C.; Fassina, L.; Arechaga, J.; Izquierdo, A.; Faris, P.; Zuccotti, M.; Moccia, F.; Garagna, S. Polychlorinated biphenyls reduce the kinematics contractile properties of embryonic stem cells-derived cardiomyocytes by disrupting their intracellular Ca2+ dynamics. Sci. Rep. 2018, 8, 17909. [Google Scholar] [CrossRef]
  60. Hazzaz Abouamal, T.; Choukairi, Z.; Taoufiq, F. Functional Exploration Of T-Type Calcium Channels (Cav3.2 and Cav3.3) And Their Sensitivity to Zinc. Open Microbiol. J. 2018, 12, 280–287. [Google Scholar] [CrossRef]
  61. Batista-Silva, H.; Dambros, B.F.; Rodrigues, K.; Cesconetto, P.A.; Zamoner, A.; Sousa de Moura, K.R.; Gomes Castro, A.J.; Van Der Kraak, G.; Mena Barreto Silva, F.R. Acute exposure to bis(2-ethylhexyl)phthalate disrupts calcium homeostasis, energy metabolism and induces oxidative stress in the testis of Danio rerio. Biochimie 2020, 175, 23–33. [Google Scholar] [CrossRef] [PubMed]
  62. Moccia, F.; Bertoni, G.; Pla, A.F.; Dragoni, S.; Pupo, E.; Merlino, A.; Mancardi, D.; Munaron, L.; Tanzi, F. Hydrogen sulfide regulates intracellular Ca2+ concentration in endothelial cells from excised rat aorta. Curr. Pharm. Biotechnol. 2011, 12, 1416–1426. [Google Scholar] [CrossRef] [PubMed]
  63. Tsumura, M.; Sobhan, U.; Muramatsu, T.; Sato, M.; Ichikawa, H.; Sahara, Y.; Tazaki, M.; Shibukawa, Y. TRPV1-mediated calcium signal couples with cannabinoid receptors and sodium-calcium exchangers in rat odontoblasts. Cell Calcium 2012, 52, 124–136. [Google Scholar] [CrossRef] [PubMed]
  64. Di Giuro, C.M.L.; Shrestha, N.; Malli, R.; Groschner, K.; van Breemen, C.; Fameli, N. Na+/Ca2+ exchangers and Orai channels jointly refill endoplasmic reticulum (ER) Ca2+ via ER nanojunctions in vascular endothelial cells. Pflugers Arch. 2017, 469, 1287–1299. [Google Scholar] [CrossRef] [Green Version]
  65. Astesana, V.; Faris, P.; Ferrari, B.; Siciliani, S.; Lim, D.; Biggiogera, M.; De Pascali, S.A.; Fanizzi, F.P.; Roda, E.; Moccia, F.; et al. [Pt(O,O’-acac)(gamma-acac)(DMS)]: Alternative Strategies to Overcome Cisplatin-Induced Side Effects and Resistance in T98G Glioma Cells. Cell. Mol. Neurobiol. 2020. [Google Scholar] [CrossRef]
  66. Giorgi, C.; Romagnoli, A.; Pinton, P.; Rizzuto, R. Ca2+ signaling, mitochondria and cell death. Curr. Mol. Med. 2008, 8, 119–130. [Google Scholar]
  67. De, P.; Carlson, J.H.; Leyland-Jones, B.; Williams, C.; Dey, N. Triple Fluorescence staining to Evaluate Mechanism-based Apoptosis following Chemotherapeutic and Targeted Anti-cancer Drugs in Live Tumor Cells. Sci. Rep. 2018, 8, 13192. [Google Scholar] [CrossRef] [Green Version]
  68. Li, C.; Zhan, Y.; Ma, X.; Fang, H.; Gai, X. B7-H4 facilitates proliferation and metastasis of colorectal carcinoma cell through PI3K/Akt/mTOR signaling pathway. Clin. Exp. Med. 2020, 20, 79–86. [Google Scholar] [CrossRef]
  69. Guo, F.F.; Yu, T.C.; Hong, J.; Fang, J.Y. Emerging Roles of Hydrogen Sulfide in Inflammatory and Neoplastic Colonic Diseases. Front. Physiol. 2016, 7, 156. [Google Scholar] [CrossRef] [Green Version]
  70. Motiani, R.K.; Hyzinski-Garcia, M.C.; Zhang, X.; Henkel, M.M.; Abdullaev, I.F.; Kuo, Y.H.; Matrougui, K.; Mongin, A.A.; Trebak, M. STIM1 and Orai1 mediate CRAC channel activity and are essential for human glioblastoma invasion. Pflugers Arch. 2013, 465, 1249–1260. [Google Scholar] [CrossRef] [Green Version]
  71. Dragoni, S.; Turin, I.; Laforenza, U.; Potenza, D.M.; Bottino, C.; Glasnov, T.N.; Prestia, M.; Ferulli, F.; Saitta, A.; Mosca, A.; et al. Store-operated Ca2+ entry does not control proliferation in primary cultures of human metastatic renal cellular carcinoma. BioMed Res. Int. 2014, 2014, 739494. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  72. Coavoy-Sanchez, S.A.; Costa, S.K.P.; Muscara, M.N. Hydrogen sulfide and dermatological diseases. Br. J. Pharmacol. 2019. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Hellmich, M.R.; Coletta, C.; Chao, C.; Szabo, C. The therapeutic potential of cystathionine beta-synthetase/hydrogen sulfide inhibition in cancer. Antioxid. Redox Signal. 2015, 22, 424–448. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  74. Bauer, C.C.; Boyle, J.P.; Porter, K.E.; Peers, C. Modulation of Ca2+ signalling in human vascular endothelial cells by hydrogen sulfide. Atherosclerosis 2010, 209, 374–380. [Google Scholar] [CrossRef] [PubMed]
  75. Zhang, R.; Sun, Y.; Tsai, H.; Tang, C.; Jin, H.; Du, J. Hydrogen sulfide inhibits L-type calcium currents depending upon the protein sulfhydryl state in rat cardiomyocytes. PLoS ONE 2012, 7, e37073. [Google Scholar] [CrossRef]
  76. Avanzato, D.; Merlino, A.; Porrera, S.; Wang, R.; Munaron, L.; Mancardi, D. Role of calcium channels in the protective effect of hydrogen sulfide in rat cardiomyoblasts. Cell. Physiol. Biochem. 2014, 33, 1205–1214. [Google Scholar] [CrossRef]
  77. Sekiguchi, F.; Miyamoto, Y.; Kanaoka, D.; Ide, H.; Yoshida, S.; Ohkubo, T.; Kawabata, A. Endogenous and exogenous hydrogen sulfide facilitates T-type calcium channel currents in Cav3.2-expressing HEK293 cells. Biochem. Biophys. Res. Commun. 2014, 445, 225–229. [Google Scholar] [CrossRef]
  78. Elies, J.; Scragg, J.L.; Huang, S.; Dallas, M.L.; Huang, D.; MacDougall, D.; Boyle, J.P.; Gamper, N.; Peers, C. Hydrogen sulfide inhibits Cav3.2 T-type Ca2+ channels. FASEB J. 2014, 28, 5376–5387. [Google Scholar] [CrossRef] [Green Version]
  79. Perez-Riesgo, E.; Gutierrez, L.G.; Ubierna, D.; Acedo, A.; Moyer, M.P.; Nunez, L.; Villalobos, C. Transcriptomic Analysis of Calcium Remodeling in Colorectal Cancer. Int. J. Mol. Sci. 2017, 18, 922. [Google Scholar] [CrossRef]
  80. Deering-Rice, C.E.; Stockmann, C.; Romero, E.G.; Lu, Z.; Shapiro, D.; Stone, B.L.; Fassl, B.; Nkoy, F.; Uchida, D.A.; Ward, R.M.; et al. Characterization of Transient Receptor Potential Vanilloid-1 (TRPV1) Variant Activation by Coal Fly Ash Particles and Associations with Altered Transient Receptor Potential Ankyrin-1 (TRPA1) Expression and Asthma. J. Biol. Chem. 2016, 291, 24866–24879. [Google Scholar] [CrossRef] [Green Version]
  81. Somogyi, C.S.; Matta, C.; Foldvari, Z.; Juhasz, T.; Katona, E.; Takacs, A.R.; Hajdu, T.; Dobrosi, N.; Gergely, P.; Zakany, R. Polymodal Transient Receptor Potential Vanilloid (TRPV) Ion Channels in Chondrogenic Cells. Int. J. Mol. Sci. 2015, 16, 18412–18438. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  82. Wang, C.; Hu, H.Z.; Colton, C.K.; Wood, J.D.; Zhu, M.X. An alternative splicing product of the murine trpv1 gene dominant negatively modulates the activity of TRPV1 channels. J. Biol. Chem. 2004, 279, 37423–37430. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  83. Tian, W.; Fu, Y.; Wang, D.H.; Cohen, D.M. Regulation of TRPV1 by a novel renally expressed rat TRPV1 splice variant. Am. J. Physiol. Renal Physiol. 2006, 290, F117–F126. [Google Scholar] [CrossRef] [PubMed]
  84. Eilers, H.; Lee, S.Y.; Hau, C.W.; Logvinova, A.; Schumacher, M.A. The rat vanilloid receptor splice variant VR.5’sv blocks TRPV1 activation. Neuroreport 2007, 18, 969–973. [Google Scholar] [CrossRef]
  85. Schumacher, M.A.; Eilers, H. TRPV1 splice variants: Structure and function. Front. Biosci. 2010, 15, 872–882. [Google Scholar] [CrossRef] [Green Version]
  86. Mistry, S.; Paule, C.C.; Varga, A.; Photiou, A.; Jenes, A.; Avelino, A.; Buluwela, L.; Nagy, I. Prolonged exposure to bradykinin and prostaglandin E2 increases TRPV1 mRNA but does not alter TRPV1 and TRPV1b protein expression in cultured rat primary sensory neurons. Neurosci. Lett. 2014, 564, 89–93. [Google Scholar] [CrossRef] [Green Version]
  87. Kedei, N.; Szabo, T.; Lile, J.D.; Treanor, J.J.; Olah, Z.; Iadarola, M.J.; Blumberg, P.M. Analysis of the native quaternary structure of vanilloid receptor 1. J. Biol. Chem. 2001, 276, 28613–28619. [Google Scholar] [CrossRef] [Green Version]
  88. Veldhuis, N.A.; Lew, M.J.; Abogadie, F.C.; Poole, D.P.; Jennings, E.A.; Ivanusic, J.J.; Eilers, H.; Bunnett, N.W.; McIntyre, P. N-glycosylation determines ionic permeability and desensitization of the TRPV1 capsaicin receptor. J. Biol. Chem. 2012, 287, 21765–21772. [Google Scholar] [CrossRef] [Green Version]
  89. Winter, Z.; Buhala, A.; Otvos, F.; Josvay, K.; Vizler, C.; Dombi, G.; Szakonyi, G.; Olah, Z. Functionally important amino acid residues in the transient receptor potential vanilloid 1 (TRPV1) ion channel—An overview of the current mutational data. Mol. Pain 2013, 9, 30. [Google Scholar] [CrossRef] [Green Version]
  90. Song, S.; Ayon, R.J.; Yamamura, A.; Yamamura, H.; Dash, S.; Babicheva, A.; Tang, H.; Sun, X.; Cordery, A.G.; Khalpey, Z.; et al. Capsaicin-induced Ca2+ signaling is enhanced via upregulated TRPV1 channels in pulmonary artery smooth muscle cells from patients with idiopathic PAH. Am. J. Physiol. Lung Cell Mol. Physiol. 2017, 312, L309–L325. [Google Scholar] [CrossRef]
  91. Medvedeva, Y.V.; Kim, M.S.; Usachev, Y.M. Mechanisms of prolonged presynaptic Ca2+ signaling and glutamate release induced by TRPV1 activation in rat sensory neurons. J. Neurosci. 2008, 28, 5295–5311. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  92. Fenwick, A.J.; Fowler, D.K.; Wu, S.W.; Shaffer, F.J.; Lindberg, J.E.M.; Kinch, D.C.; Peters, J.H. Direct Anandamide Activation of TRPV1 Produces Divergent Calcium and Current Responses. Front. Mol. Neurosci. 2017, 10, 200. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  93. Tian, Q.; Hu, J.; Xie, C.; Mei, K.; Pham, C.; Mo, X.; Hepp, R.; Soares, S.; Nothias, F.; Wang, Y.; et al. Recovery from tachyphylaxis of TRPV1 coincides with recycling to the surface membrane. Proc. Natl. Acad. Sci. USA 2019, 116, 5170–5175. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  94. Stueber, T.; Eberhardt, M.J.; Caspi, Y.; Lev, S.; Binshtok, A.; Leffler, A. Differential cytotoxicity and intracellular calcium-signalling following activation of the calcium-permeable ion channels TRPV1 and TRPA1. Cell Calcium 2017, 68, 34–44. [Google Scholar] [CrossRef] [PubMed]
  95. Mohapatra, D.P.; Nau, C. Regulation of Ca2+-dependent desensitization in the vanilloid receptor TRPV1 by calcineurin and cAMP-dependent protein kinase. J. Biol. Chem. 2005, 280, 13424–13432. [Google Scholar] [CrossRef] [Green Version]
  96. Yao, J.; Qin, F. Interaction with phosphoinositides confers adaptation onto the TRPV1 pain receptor. PLoS Biol. 2009, 7, e46. [Google Scholar] [CrossRef] [Green Version]
  97. Grycova, L.; Lansky, Z.; Friedlova, E.; Obsilova, V.; Janouskova, H.; Obsil, T.; Teisinger, J. Ionic interactions are essential for TRPV1 C-terminus binding to calmodulin. Biochem. Biophys. Res. Commun. 2008, 375, 680–683. [Google Scholar] [CrossRef]
  98. Chung, M.K.; Guler, A.D.; Caterina, M.J. TRPV1 shows dynamic ionic selectivity during agonist stimulation. Nat. Neurosci. 2008, 11, 555–564. [Google Scholar] [CrossRef]
  99. Berra-Romani, R.; Guzman-Silva, A.; Vargaz-Guadarrama, A.; Flores-Alonso, J.C.; Alonso-Romero, J.; Trevino, S.; Sanchez-Gomez, J.; Coyotl-Santiago, N.; Garcia-Carrasco, M.; Moccia, F. Type 2 Diabetes Alters Intracellular Ca2+ Handling in Native Endothelium of Excised Rat Aorta. Int. J. Mol. Sci. 2019, 21, 250. [Google Scholar] [CrossRef] [Green Version]
  100. Markova, J.; Hudecova, S.; Soltysova, A.; Sirova, M.; Csaderova, L.; Lencesova, L.; Ondrias, K.; Krizanova, O. Sodium/calcium exchanger is upregulated by sulfide signaling, forms complex with the beta1 and beta3 but not beta2 adrenergic receptors, and induces apoptosis. Pflugers Arch. 2014, 466, 1329–1342. [Google Scholar] [CrossRef]
  101. Cantero-Recasens, G.; Butnaru, C.M.; Brouwers, N.; Mitrovic, S.; Valverde, M.A.; Malhotra, V. Sodium channel TRPM4 and sodium/calcium exchangers (NCX) cooperate in the control of Ca2+-induced mucin secretion from goblet cells. J. Biol. Chem. 2019, 294, 816–826. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  102. Poburko, D.; Fameli, N.; Kuo, K.H.; van Breemen, C. Ca2+ signaling in smooth muscle: TRPC6, NCX and LNats in nanodomains. Channels 2008, 2, 10–12. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  103. Wu, D.; Li, J.; Zhang, Q.; Tian, W.; Zhong, P.; Liu, Z.; Wang, H.; Wang, H.; Ji, A.; Li, Y. Exogenous Hydrogen Sulfide Regulates the Growth of Human Thyroid Carcinoma Cells. Oxid. Med. Cell. Longev. 2019, 2019, 6927298. [Google Scholar] [CrossRef] [Green Version]
  104. Tanwar, J.; Arora, S.; Motiani, R.K. Orai3: Oncochannel with therapeutic potential. Cell Calcium 2020, 90, 102247. [Google Scholar] [CrossRef] [PubMed]
  105. Villalobos, C.; Sobradillo, D.; Hernandez-Morales, M.; Nunez, L. Calcium remodeling in colorectal cancer. Biochim. Biophys. Acta 2017, 1864, 843–849. [Google Scholar] [CrossRef]
  106. Marchi, S.; Rimessi, A.; Giorgi, C.; Baldini, C.; Ferroni, L.; Rizzuto, R.; Pinton, P. Akt kinase reducing endoplasmic reticulum Ca2+ release protects cells from Ca2+-dependent apoptotic stimuli. Biochem. Biophys. Res. Commun. 2008, 375, 501–505. [Google Scholar] [CrossRef] [Green Version]
  107. Giorgi, C.; Ito, K.; Lin, H.K.; Santangelo, C.; Wieckowski, M.R.; Lebiedzinska, M.; Bononi, A.; Bonora, M.; Duszynski, J.; Bernardi, R.; et al. PML regulates apoptosis at endoplasmic reticulum by modulating calcium release. Science 2010, 330, 1247–1251. [Google Scholar] [CrossRef] [Green Version]
  108. Al-Taweel, N.; Varghese, E.; Florea, A.M.; Busselberg, D. Cisplatin (CDDP) triggers cell death of MCF-7 cells following disruption of intracellular calcium ([Ca2+]i) homeostasis. J. Toxicol. Sci. 2014, 39, 765–774. [Google Scholar] [CrossRef] [Green Version]
  109. Busselberg, D.; Florea, A.M. Targeting Intracellular Calcium Signaling ([Ca2+]i) to Overcome Acquired Multidrug Resistance of Cancer Cells: A Mini-Overview. Cancers 2017, 9, 48. [Google Scholar] [CrossRef] [Green Version]
  110. Kelman, Z. PCNA: Structure, functions and interactions. Oncogene 1997, 14, 629–640. [Google Scholar] [CrossRef] [Green Version]
  111. Bravo, R.; Frank, R.; Blundell, P.A.; Macdonald-Bravo, H. Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta. Nature 1987, 326, 515–517. [Google Scholar] [CrossRef] [PubMed]
  112. Turin, I.; Schiavo, R.; Maestri, M.; Luinetti, O.; Dal Bello, B.; Paulli, M.; Dionigi, P.; Roccio, M.; Spinillo, A.; Ferulli, F.; et al. In Vitro Efficient Expansion of Tumor Cells Deriving from Different Types of Human Tumor Samples. Med. Sci. 2014, 2, 70–81. [Google Scholar] [CrossRef]
  113. Turin, I.; Delfanti, S.; Ferulli, F.; Brugnatelli, S.; Tanzi, M.; Maestri, M.; Cobianchi, L.; Lisini, D.; Luinetti, O.; Paulli, M.; et al. In Vitro Killing of Colorectal Carcinoma Cells by Autologous Activated NK Cells is Boosted by Anti-Epidermal Growth Factor Receptor-induced ADCC Regardless of RAS Mutation Status. J. Immunother. 2018, 41, 190–200. [Google Scholar] [CrossRef] [PubMed]
Figure 1. NaHS evokes intracellular Ca2+ signals in colorectal cancer (CRC) and non-neoplastic cells. (A), NaHS (100 µM) evoked intracellular Ca2+ signals in non-neoplastic (Control, Ctrl), primary CRC (pCRC) and metastatic CRC (mCRC) cells. (B), mean ± SE of the amplitude of the peak Ca2+ response induced by NaHS in the different cell types. One-way A analysis followed by the post-hoc Bonferroni test was used for Statistical comparison. In Panels B: *** p ≤ 0.001.
Figure 1. NaHS evokes intracellular Ca2+ signals in colorectal cancer (CRC) and non-neoplastic cells. (A), NaHS (100 µM) evoked intracellular Ca2+ signals in non-neoplastic (Control, Ctrl), primary CRC (pCRC) and metastatic CRC (mCRC) cells. (B), mean ± SE of the amplitude of the peak Ca2+ response induced by NaHS in the different cell types. One-way A analysis followed by the post-hoc Bonferroni test was used for Statistical comparison. In Panels B: *** p ≤ 0.001.
Cancers 12 03338 g001
Figure 2. Dose-dependent effect of NaHS on [Ca2+]i in mCRC cells. (A), intracellular Ca2+ signals evoked by increasing concentrations of NaHS in mCRC cells. Each dose-response relationship was carried out on cells from the same batch in three separate experiments. (B), mean ± SE of the percentage of cells presenting a discernible increase in [Ca2+]i in the presence of different concentrations of NaHS. (C), mean ± SE of the amplitude of the peak Ca2+ response to different concentration of NaHS. One-way ANOVA analysis followed by the post-hoc Bonferroni test was used for Statistical comparison. In Panels B and C: *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns: not significant.
Figure 2. Dose-dependent effect of NaHS on [Ca2+]i in mCRC cells. (A), intracellular Ca2+ signals evoked by increasing concentrations of NaHS in mCRC cells. Each dose-response relationship was carried out on cells from the same batch in three separate experiments. (B), mean ± SE of the percentage of cells presenting a discernible increase in [Ca2+]i in the presence of different concentrations of NaHS. (C), mean ± SE of the amplitude of the peak Ca2+ response to different concentration of NaHS. One-way ANOVA analysis followed by the post-hoc Bonferroni test was used for Statistical comparison. In Panels B and C: *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns: not significant.
Cancers 12 03338 g002
Figure 3. Distinct Ca2+ signals induced by NaHS in mCRC cells. (A), administration of 100 µM NaHS in the presence of extracellular Ca2+ triggered heterogeneous Ca2+ signals in mCRC cells, such as transient and long-lasting increases in [Ca2+]i. (B), mean ± SE of the percentage of cells presenting transient and long-lasting Ca2+ signals in 287 mCRC cells in response to 100 µM NaHS. (C), repetitive applications of NaHS (100 μM) resulted in Ca2+ signals similar amplitude and kinetics. Student’s t-test has been used for statistical comparison. The asterisk indicates p < 0.05.
Figure 3. Distinct Ca2+ signals induced by NaHS in mCRC cells. (A), administration of 100 µM NaHS in the presence of extracellular Ca2+ triggered heterogeneous Ca2+ signals in mCRC cells, such as transient and long-lasting increases in [Ca2+]i. (B), mean ± SE of the percentage of cells presenting transient and long-lasting Ca2+ signals in 287 mCRC cells in response to 100 µM NaHS. (C), repetitive applications of NaHS (100 μM) resulted in Ca2+ signals similar amplitude and kinetics. Student’s t-test has been used for statistical comparison. The asterisk indicates p < 0.05.
Cancers 12 03338 g003
Figure 4. Extracellular Ca2+ entry mediates the Ca2+ response to NaHS. (A), NaHS (100 μM) failed to elicit intracellular Ca2+ levels when the cells were bathed in the absence of extracellular Ca2+ (0Ca2+), the [Ca2+]i raised following re-addition of extracellular Ca2+. (B), mean ± SE of the percentage of mCRC cells presenting a discernible Ca2+ release or Ca2+ entry in response to 100 μM NaHS. (C), mean ± SE of the amplitude of Ca2+ release and Ca2+ entry induced by NaHS in mCRC cells. (D), removal of extracellular Ca2+ (0Ca2+) during the plateau phase caused the [Ca2+]i to undergo a reversible decline to pre-stimulation levels.
Figure 4. Extracellular Ca2+ entry mediates the Ca2+ response to NaHS. (A), NaHS (100 μM) failed to elicit intracellular Ca2+ levels when the cells were bathed in the absence of extracellular Ca2+ (0Ca2+), the [Ca2+]i raised following re-addition of extracellular Ca2+. (B), mean ± SE of the percentage of mCRC cells presenting a discernible Ca2+ release or Ca2+ entry in response to 100 μM NaHS. (C), mean ± SE of the amplitude of Ca2+ release and Ca2+ entry induced by NaHS in mCRC cells. (D), removal of extracellular Ca2+ (0Ca2+) during the plateau phase caused the [Ca2+]i to undergo a reversible decline to pre-stimulation levels.
Cancers 12 03338 g004
Figure 5. Capsaicin induces extracellular Ca2+ influx in mCRC cells. (A), administration of capsaicin (10 µM), a specific TRPV1 agonist, in the presence of extracellular Ca2+ induced both transient and long-lasting intracellular Ca2+ signals. (B), mean ± SE of the percentage of cells presenting transient and long-lasting Ca2+ signals in 170 mCRC cells in response to 10 µM capsaicin. Student’s t-test has been used for statistical comparison. The asterisk indicates p < 0.05. (C), repetitive applications of capsaicin (10 μM) resulted in Ca2+ signals with similar amplitude and kinetics.
Figure 5. Capsaicin induces extracellular Ca2+ influx in mCRC cells. (A), administration of capsaicin (10 µM), a specific TRPV1 agonist, in the presence of extracellular Ca2+ induced both transient and long-lasting intracellular Ca2+ signals. (B), mean ± SE of the percentage of cells presenting transient and long-lasting Ca2+ signals in 170 mCRC cells in response to 10 µM capsaicin. Student’s t-test has been used for statistical comparison. The asterisk indicates p < 0.05. (C), repetitive applications of capsaicin (10 μM) resulted in Ca2+ signals with similar amplitude and kinetics.
Cancers 12 03338 g005
Figure 6. Extracellular Ca2+ entry mediates the Ca2+ response to capsaicin in mCRC cells. (A), application of capsaicin (10 µM) in the absence of extracellular Ca2+ (0Ca2+) did not elicit any detectable increase in [Ca2+]i, while the Ca2+ signal was recorded upon Ca2+ restitution to the bath. (B), mean ± SE of the percentage of mCRC cells presenting a discernible Ca2+ release or Ca2+ entry in response to 10 μM capsaicin. (C), mean ± SE of the amplitude of Ca2+ release and Ca2+ entry induced by capsaicin in mCRC cells. (D), removal of extracellular Ca2+ (0Ca2+) during the plateau phase caused the [Ca2+]i to undergo a reversible decline to pre-stimulation levels.
Figure 6. Extracellular Ca2+ entry mediates the Ca2+ response to capsaicin in mCRC cells. (A), application of capsaicin (10 µM) in the absence of extracellular Ca2+ (0Ca2+) did not elicit any detectable increase in [Ca2+]i, while the Ca2+ signal was recorded upon Ca2+ restitution to the bath. (B), mean ± SE of the percentage of mCRC cells presenting a discernible Ca2+ release or Ca2+ entry in response to 10 μM capsaicin. (C), mean ± SE of the amplitude of Ca2+ release and Ca2+ entry induced by capsaicin in mCRC cells. (D), removal of extracellular Ca2+ (0Ca2+) during the plateau phase caused the [Ca2+]i to undergo a reversible decline to pre-stimulation levels.
Cancers 12 03338 g006
Figure 7. Pharmacological blockade of Transient Receptor Potential Vanilloid 1 (TRPV1) inhibited capsaicin-induced Ca2+ entry in mCRC. (A), transient Ca2+ signals induced by 10 µM of capsaicin were abrogated upon preincubation with the specific TRPV1 antagonists: capsazepine (CPZ; 10 µM, 20 min) and SB 366791 (SB; 10 µM, 20 min) and with the pan-specific TRPV antagonist ruthenium red (RuR; 10 µM, 20 min). (B), mean ± SE of the percentage of cells presenting a discernible Ca2+ response to capsaicin in the absence (Ctrl) and presence of TRPV1 blockers. (C), mean ± SE the amplitude of the Ca2+ response to capsaicin in the absence (Ctrl) and presence of TRPV1 blockers. One-way ANOVA analysis followed by the post-hoc Dunnett’s test was used for Statistical comparison. In Panels B and C: *** p ≤ 0.001; ** p ≤ 0.01.
Figure 7. Pharmacological blockade of Transient Receptor Potential Vanilloid 1 (TRPV1) inhibited capsaicin-induced Ca2+ entry in mCRC. (A), transient Ca2+ signals induced by 10 µM of capsaicin were abrogated upon preincubation with the specific TRPV1 antagonists: capsazepine (CPZ; 10 µM, 20 min) and SB 366791 (SB; 10 µM, 20 min) and with the pan-specific TRPV antagonist ruthenium red (RuR; 10 µM, 20 min). (B), mean ± SE of the percentage of cells presenting a discernible Ca2+ response to capsaicin in the absence (Ctrl) and presence of TRPV1 blockers. (C), mean ± SE the amplitude of the Ca2+ response to capsaicin in the absence (Ctrl) and presence of TRPV1 blockers. One-way ANOVA analysis followed by the post-hoc Dunnett’s test was used for Statistical comparison. In Panels B and C: *** p ≤ 0.001; ** p ≤ 0.01.
Cancers 12 03338 g007
Figure 8. Expression and subcellular distribution of TRPV1 in mCRC cells. (A), immunoblotting analysis of TRPV1 expression in two samples of mCRC cells. Total lysates (15 µg) were separated by SDS-PAGE, blotted on polyvinylidene fluoride (PVDF) membrane and stained with anti TRPV1 antibody. Subsequent reprobing with anti-GAPDH antibody was performed as an equal loading control. (B), Confocal microscopy analysis of the TRPV1 distribution (red signal). Cell nuclei were stained with Hoechst 33342 (blue) and plasma membrane was labeled with PKH67 (green). Representative confocal middle z-section and orthogonal views are reported. Scale bar: 10 μm.
Figure 8. Expression and subcellular distribution of TRPV1 in mCRC cells. (A), immunoblotting analysis of TRPV1 expression in two samples of mCRC cells. Total lysates (15 µg) were separated by SDS-PAGE, blotted on polyvinylidene fluoride (PVDF) membrane and stained with anti TRPV1 antibody. Subsequent reprobing with anti-GAPDH antibody was performed as an equal loading control. (B), Confocal microscopy analysis of the TRPV1 distribution (red signal). Cell nuclei were stained with Hoechst 33342 (blue) and plasma membrane was labeled with PKH67 (green). Representative confocal middle z-section and orthogonal views are reported. Scale bar: 10 μm.
Cancers 12 03338 g008
Figure 9. NaHS induces Ca2+ influx by activating TRPV1 in mCRC cells. (A), NaHS (100 µM) evoked an increase in [Ca2+]i in the absence (Ctrl), but not in the presence, of the specific TRPV1 antagonists: capsazepine (CPZ; 10 µM, 20 min) and SB 366791 (SB; 10 µM, 20 min) and of the less specific TRPV1 blocker ruthenium red (RuR; 10 µM, 20 min). (B), mean ± SE of the percentage of cells presenting a discernible Ca2+ response to NaHS under the designated treatments. (C), mean ± SE the amplitude of NaHS-evoked Ca2+ influx under the designated treatments. One-way ANOVA analysis followed by the post-hoc Dunnett’s test was used for Statistical comparison. In Panels B and C: *** p ≤ 0.001.
Figure 9. NaHS induces Ca2+ influx by activating TRPV1 in mCRC cells. (A), NaHS (100 µM) evoked an increase in [Ca2+]i in the absence (Ctrl), but not in the presence, of the specific TRPV1 antagonists: capsazepine (CPZ; 10 µM, 20 min) and SB 366791 (SB; 10 µM, 20 min) and of the less specific TRPV1 blocker ruthenium red (RuR; 10 µM, 20 min). (B), mean ± SE of the percentage of cells presenting a discernible Ca2+ response to NaHS under the designated treatments. (C), mean ± SE the amplitude of NaHS-evoked Ca2+ influx under the designated treatments. One-way ANOVA analysis followed by the post-hoc Dunnett’s test was used for Statistical comparison. In Panels B and C: *** p ≤ 0.001.
Cancers 12 03338 g009
Figure 10. Genetic deletion of TRPV1 reduced NaHS-evoked extracellular Ca2+ influx in mCRC cells. (A), the increase in [Ca2+]i evoked by capsaicin (10 µM) in mCRC cells transfected with a scrambled construct (Ctrl) was remarkably reduced in mCRC cells transfected with a selective siTRPV1. (B), mean ± SE of the percentage of the Ca2+ response to capsaicin under the designated treatments. (C), the increase in [Ca2+]i evoked by NaHS (100 µM) in mCRC cells transfected with a scrambled construct (Ctrl) was remarkably reduced in mCRC cells transfected with a selective siTRPV1. (D), mean ± SE of the percentage of the Ca2+ response to NaHS under the designated treatments. Student’s t-test has been used for statistical comparison. In Panel B and D: *** p ≤ 0.001.
Figure 10. Genetic deletion of TRPV1 reduced NaHS-evoked extracellular Ca2+ influx in mCRC cells. (A), the increase in [Ca2+]i evoked by capsaicin (10 µM) in mCRC cells transfected with a scrambled construct (Ctrl) was remarkably reduced in mCRC cells transfected with a selective siTRPV1. (B), mean ± SE of the percentage of the Ca2+ response to capsaicin under the designated treatments. (C), the increase in [Ca2+]i evoked by NaHS (100 µM) in mCRC cells transfected with a scrambled construct (Ctrl) was remarkably reduced in mCRC cells transfected with a selective siTRPV1. (D), mean ± SE of the percentage of the Ca2+ response to NaHS under the designated treatments. Student’s t-test has been used for statistical comparison. In Panel B and D: *** p ≤ 0.001.
Cancers 12 03338 g010
Figure 11. NaHS-evoked Ca2+ entry requires the reverse mode of NCX in mCRC cells. (A), the Ca2+ response to NaHS (100 µM) was prevented by removal of extracellular Na+ (0Na+) and resumed upon adding back Na+ to the perfusate. Please note that removal of extracellular Na+ resulted in a transient increase in [Ca2+]i due to reversal of NCX activity. (B), mean ± SE of the percentage of cells presenting a discernible Ca2+ response to NaHS in the absence (Ctrl) and presence of extracellular Na+. (C), mean ± SE of the amplitude of NaHS-evoked Ca2+ influx in the presence (Ctrl) and absence of extracellular Na+. (D), pre-incubating mCRC cells with KB-R 7943 (KB-R; 20 µM, 20 min), a selective inhibitor of the forward-mode of NCX, abrogated the Ca2+ signal generated by NaHS (100 µM). (E), mean ± SE of the percentage of cells presenting a discernible Ca2+ response to NaHS in the absence (Ctrl) and presence of KB-R 7943 (KB-R). (F), mean ± SE the amplitude of NaHS-evoked Ca2+ influx in the absence (Ctrl) and presence of KB-R 7943 (KB-R). Student’s t-test has been used for statistical comparison. In Panels B, C, E and F, *** p ≤ 0.001.
Figure 11. NaHS-evoked Ca2+ entry requires the reverse mode of NCX in mCRC cells. (A), the Ca2+ response to NaHS (100 µM) was prevented by removal of extracellular Na+ (0Na+) and resumed upon adding back Na+ to the perfusate. Please note that removal of extracellular Na+ resulted in a transient increase in [Ca2+]i due to reversal of NCX activity. (B), mean ± SE of the percentage of cells presenting a discernible Ca2+ response to NaHS in the absence (Ctrl) and presence of extracellular Na+. (C), mean ± SE of the amplitude of NaHS-evoked Ca2+ influx in the presence (Ctrl) and absence of extracellular Na+. (D), pre-incubating mCRC cells with KB-R 7943 (KB-R; 20 µM, 20 min), a selective inhibitor of the forward-mode of NCX, abrogated the Ca2+ signal generated by NaHS (100 µM). (E), mean ± SE of the percentage of cells presenting a discernible Ca2+ response to NaHS in the absence (Ctrl) and presence of KB-R 7943 (KB-R). (F), mean ± SE the amplitude of NaHS-evoked Ca2+ influx in the absence (Ctrl) and presence of KB-R 7943 (KB-R). Student’s t-test has been used for statistical comparison. In Panels B, C, E and F, *** p ≤ 0.001.
Cancers 12 03338 g011
Figure 12. NCX contributes to capsaicin-induced extracellular Ca2+ entry in mCRC cells. (A), control Ca2+ response to capsaicin in the presence of extracellular Na+. (B), the Ca2+ response to capsaicin (10 µM) was dramatically reduced by removal of extracellular Na+ (0Na+) and resumed upon adding back Na+ to the perfusate. As described in Figure 11A, removal of extracellular Na+ resulted in a transient increase in [Ca2+]i due to reversal of NCX activity. (C), mean ± SE of the percentage of cells presenting a discernible Ca2+ response to capsaicin in the presence (Ctrl) and absence of extracellular Na+. (D), mean ± SE of the amplitude of capsaicin-induced Ca2+ influx in the presence (Ctrl) and absence of extracellular Na+. (E), pre-incubating mCRC cells with KB-R 7943 (KB-R; 20 µM, 20 min) reduced the amplitude of the Ca2+ response capsaicin (10 µM). (F), mean ± SE of the percentage of cells presenting a discernible Ca2+ response to capsaicin in the absence (Ctrl) and presence of KB-R 7943 (KB-R). (G), mean ± SE of the amplitude of capsaicin-induced Ca2+ influx in the absence (Ctrl) and presence of KB-R 7943 (KB-R). Student’s t-test has been used for statistical comparison. In Panels C, D, F and G, *** p ≤ 0.001; ** p ≤ 0.01.
Figure 12. NCX contributes to capsaicin-induced extracellular Ca2+ entry in mCRC cells. (A), control Ca2+ response to capsaicin in the presence of extracellular Na+. (B), the Ca2+ response to capsaicin (10 µM) was dramatically reduced by removal of extracellular Na+ (0Na+) and resumed upon adding back Na+ to the perfusate. As described in Figure 11A, removal of extracellular Na+ resulted in a transient increase in [Ca2+]i due to reversal of NCX activity. (C), mean ± SE of the percentage of cells presenting a discernible Ca2+ response to capsaicin in the presence (Ctrl) and absence of extracellular Na+. (D), mean ± SE of the amplitude of capsaicin-induced Ca2+ influx in the presence (Ctrl) and absence of extracellular Na+. (E), pre-incubating mCRC cells with KB-R 7943 (KB-R; 20 µM, 20 min) reduced the amplitude of the Ca2+ response capsaicin (10 µM). (F), mean ± SE of the percentage of cells presenting a discernible Ca2+ response to capsaicin in the absence (Ctrl) and presence of KB-R 7943 (KB-R). (G), mean ± SE of the amplitude of capsaicin-induced Ca2+ influx in the absence (Ctrl) and presence of KB-R 7943 (KB-R). Student’s t-test has been used for statistical comparison. In Panels C, D, F and G, *** p ≤ 0.001; ** p ≤ 0.01.
Cancers 12 03338 g012
Figure 13. NaHS inhibits proliferation, but does not induce apoptosis, in mCRC cells. (A), incubation of mCRC cells for 24 h and for 72 h in presence of increasing concentrations of NaHS (50, 100 and 200 µM) reduced cell proliferation as determined by direct cell counting with the Trypan blue assay. (B), the anti-proliferative effect of NaHS (100 µM, 72 h) was prevented by pretreating mCRC cells with capsazepine (CPZ; 10 µM, 20 min) and KB-R 7943 (KB-R; 10 µM, 30 min). One-way ANOVA analysis followed by the post-hoc Dunnett’s test was used for Statistical comparison. In Panels A and B: ** p ≤ 0.001; * p ≤ 0.05. Each experiment was repeated three times. (C), Flow cytometry analysis of Annexin and 7-ADD staining. Representative cytogram of three independent experiments with the percentage of distribution of different population of mCRC cells in the absence (Ctrl) and in the presence of NaHS (100 µM, 72 h). Living cells (Annexin V−/7−ADD−) are represented in dark grey, early apoptotic cells (Annexin V+/7−ADD−) in black, late apoptotic/dead cells (Annexin V+/7−ADD+) in white, and dead cells (Annexin V+/7−ADD+) in light grey. (D), mean ± SE of the percentage of apoptotic mCRC cells in the absence (Ctrl) and presence of NaHS. (E), mean ± SE of ΔΨm in the absence (Ctrl) and presence of NaHS (100 µM, 72 h). ΔΨm was measured by evaluating tetramethylrhodamine, methyl ester (TMRM) fluorescence [67].
Figure 13. NaHS inhibits proliferation, but does not induce apoptosis, in mCRC cells. (A), incubation of mCRC cells for 24 h and for 72 h in presence of increasing concentrations of NaHS (50, 100 and 200 µM) reduced cell proliferation as determined by direct cell counting with the Trypan blue assay. (B), the anti-proliferative effect of NaHS (100 µM, 72 h) was prevented by pretreating mCRC cells with capsazepine (CPZ; 10 µM, 20 min) and KB-R 7943 (KB-R; 10 µM, 30 min). One-way ANOVA analysis followed by the post-hoc Dunnett’s test was used for Statistical comparison. In Panels A and B: ** p ≤ 0.001; * p ≤ 0.05. Each experiment was repeated three times. (C), Flow cytometry analysis of Annexin and 7-ADD staining. Representative cytogram of three independent experiments with the percentage of distribution of different population of mCRC cells in the absence (Ctrl) and in the presence of NaHS (100 µM, 72 h). Living cells (Annexin V−/7−ADD−) are represented in dark grey, early apoptotic cells (Annexin V+/7−ADD−) in black, late apoptotic/dead cells (Annexin V+/7−ADD+) in white, and dead cells (Annexin V+/7−ADD+) in light grey. (D), mean ± SE of the percentage of apoptotic mCRC cells in the absence (Ctrl) and presence of NaHS. (E), mean ± SE of ΔΨm in the absence (Ctrl) and presence of NaHS (100 µM, 72 h). ΔΨm was measured by evaluating tetramethylrhodamine, methyl ester (TMRM) fluorescence [67].
Cancers 12 03338 g013
Figure 14. NaHS does not activate phosphorylation cascades in mCRC cells. Phosphorylation of the indicated selected signaling proteins in mCRC cells incubated with 100 µM NaHS for 24 h. Representative immunoblots with specific anti-phosphoprotein antibodies directed against the different substrates are reported (i). Akt, Erk and mTOR staining were used for equal loading control for corresponding specific phosphoprotein. GAPDH staining was used as equal loading control. Quantification of the results performed by densitometric scanning is reported in (ii), as fold increase (A.U.) of phosphorylation over basal (NT). Results are the mean ± SD of three different experiments.
Figure 14. NaHS does not activate phosphorylation cascades in mCRC cells. Phosphorylation of the indicated selected signaling proteins in mCRC cells incubated with 100 µM NaHS for 24 h. Representative immunoblots with specific anti-phosphoprotein antibodies directed against the different substrates are reported (i). Akt, Erk and mTOR staining were used for equal loading control for corresponding specific phosphoprotein. GAPDH staining was used as equal loading control. Quantification of the results performed by densitometric scanning is reported in (ii), as fold increase (A.U.) of phosphorylation over basal (NT). Results are the mean ± SD of three different experiments.
Cancers 12 03338 g014
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Faris, P.; Ferulli, F.; Vismara, M.; Tanzi, M.; Negri, S.; Rumolo, A.; Lefkimmiatis, K.; Maestri, M.; Shekha, M.; Pedrazzoli, P.; et al. Hydrogen Sulfide-Evoked Intracellular Ca2+ Signals in Primary Cultures of Metastatic Colorectal Cancer Cells. Cancers 2020, 12, 3338. https://doi.org/10.3390/cancers12113338

AMA Style

Faris P, Ferulli F, Vismara M, Tanzi M, Negri S, Rumolo A, Lefkimmiatis K, Maestri M, Shekha M, Pedrazzoli P, et al. Hydrogen Sulfide-Evoked Intracellular Ca2+ Signals in Primary Cultures of Metastatic Colorectal Cancer Cells. Cancers. 2020; 12(11):3338. https://doi.org/10.3390/cancers12113338

Chicago/Turabian Style

Faris, Pawan, Federica Ferulli, Mauro Vismara, Matteo Tanzi, Sharon Negri, Agnese Rumolo, Kostantinos Lefkimmiatis, Marcello Maestri, Mudhir Shekha, Paolo Pedrazzoli, and et al. 2020. "Hydrogen Sulfide-Evoked Intracellular Ca2+ Signals in Primary Cultures of Metastatic Colorectal Cancer Cells" Cancers 12, no. 11: 3338. https://doi.org/10.3390/cancers12113338

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop