Search Results (29)

Background: Lactate dehydrogenase (LDH) activity, producing high levels of lactate from pyruvate in cancer cells, is often associated with poor patient prognosis. We previously showed enhanced LDH/lactate levels in estrogen receptor (ER) compared to ER + breast cancer cells; lactate or pyruvate supplementation to ER + cells significantly enhanced their motile ability, while LDHB gene knockout (KO) or treatment with LDH inhibitors reduced the motility of the highly aggressive ER breast cancer cells. Aims: To investigate the molecular mechanisms by which lactate, LDHB KO, or treatment with LDH inhibitors can modulate the motile capabilities of breast cancer cell lines. Methods: KO experiments were performed using siRNA, and global expression was determined by proteomic profiling with Proteome Profiler Human XL Oncology arrays, Western blot, and immunofluorescence. Results: Lactate supplementation to ER + breast cancer cells enhanced expression of vimentin, N-cadherin, and snail, while reducing the expression of JAM-A, E-cadherin, and nectin-4. This expression profile was reversed with LDHB KO in ER cells. LDHB KO, or treatment with LDH inhibitors in ER cells, also reduced the expression of IL-6, IL-8, and MMP-2. The expressions of other markers such as PECAM-1, CCL20, and ENPP-2 were differentially modulated with LDH B KO in de novo ER cells (MDA-MB-231) vs. those that had ER knockout (pII). Conclusions: Our data show a novel role for lactate in modulating the EMT status in breast cancer cells and highlight the important role of lactate in breast cancer motility in part through modulating EMT status and the expression profile of cytokines, adhesion molecules, MMP-2, and nectin-4. Full article

Ovarian cancer (OC) is the second most common female reproductive cancer and the most lethal gynecological malignancy worldwide. Most human OCs are characterized by high rates of drug resistance and metastasis, leading to poor prognosis. Improving the outcomes of patients with relapsed and treatment-resistant OC remains a challenge. This study aimed to investigate the role of epidermal growth factor-like domain 8 (EGFL8) in human OC by examining the effects of siRNA-mediated EGFL8 knockdown on cancer progression. EGFL8 knockdown in human OC cells promoted aggressive traits associated with cancer progression, including enhanced proliferation, colony formation, migration, invasion, chemoresistance, and reduced apoptosis. Additionally, knockdown upregulated the expression of epithelial–mesenchymal transition (EMT) markers (Snail, Twist1, Zeb1, Zeb2, and vimentin) and cancer stem cell biomarkers (Oct4, Sox2, Nanog, KLF4, and ALDH1A1), and increased the expression of matrix metallopeptidases (MMP-2 and MMP-9), drug resistance genes (MDR1 and MRP1), and Notch1. Low EGFL8 expression also correlated with poor prognosis in human OC. Overall, this study provides crucial evidence that EGFL8 inhibits the proliferation and cancer aggressiveness of human OC cells by suppressing ERK/MAPK signaling. Therefore, EGFL8 may serve as a valuable prognostic biomarker and a potential target for developing novel human OC therapies. Full article

In prostate cancer (PCa), androgens upregulate tumorigenesis, whereas in benign tissue, the revival of androgen receptor (AR) signaling suppresses aggressive behaviors, suggesting therapeutic potential. Dogs, natural PCa models, often lack AR in PCa. We restored AR in dog PCa to investigate resultant characteristics. Three AR-null canine PCa lines (1508, Leo, 1258) were transfected with canine wild-type AR and treated with dihydrotestosterone (DHT). In 1508, AR restoration decreased clonogenicity (p = 0.03), viability (p = 0.004), migration (p = 0.03), invasion (p = 0.01), and increased expression of the tumor suppressor NKX3.1, an AR transcriptional target (p = 0.001). In Leo, AR decreased clonogenicity (p = 0.04) and the expression of another AR transcriptional target FOLH1 (p < 0.001) and increased the expression of NKX3.1 (p = 0.01). In 1258, AR increased migration (p = 0.006) and invasion (p = 0.03). Epithelial–mesenchymal transition (EMT) marker (Vimentin, N-cadherin, SNAIL1) expression increased with AR restoration in Leo and 1258 but not 1508; siRNA vimentin knockdown abrogated AR-induced 1258 migration only. Overall, 1508 showed AR-mediated tumor suppression; AR affected proliferation in Leo but not migration or invasion; and EMT and AR regulated migration and invasion in 1258 but not proliferation. This study highlights the heterogeneous nature of PCa in dogs and cell line-specific effects of AR abrogation on aggressive behaviors. Full article

Hepatocellular carcinoma is one of the most common malignant tumors in the world and shows strong metastatic potential. Current medicine for hepatocellular carcinoma therapy is invalid, while Scutellaria baicalensis Georgi exhibits the pharmaceutical potential to treat liver diseases and liver cancer. Herein, we verified the inhibitory properties and the pivotal molecules regimented by Scutellaria baicalensis on advanced hepatocellular carcinoma. At first, the viability of SK-Hep-1 cells was significantly reduced under treatment of Scutellaria baicalensis extract in a dose-dependent manner without affecting the growth of normal hepatocyte. Scutellaria baicalensis extract application could remarkably cause apoptosis of SK-Hep-1 cells through p53/cytochrome C/poly-ADP ribose polymerase cascades and arrest the cell cycle at the G1/S phase by downregulating cyclin-dependent kinases. Meanwhile, administration of Scutellaria baicalensis extract remarkably attenuated the migration capability as well as suppressed matrix metalloproteinase activity of advanced hepatocellular carcinoma cells. The proteome profiles and network analysis particularly implied that exposure to Scutellaria baicalensis extract downregulated the expression of HSP90β, and the clinical stage of hepatocellular carcinoma is also positively correlated with the HSP90β level. Combined treatment of Scutellaria baicalensis extract and HSP90β siRNAs could markedly enhance the ubiquitination activity and the degradation of vimentin to subsequently inhibit the metastatic property of SK-Hep-1 cells. Moreover, application of Scutellaria baicalensis extract and HSP90β siRNAs depleted phosphorylation of AKT, which stimulated the expression of p53 and consecutively triggered cell apoptosis. These findings suggest that HSP90β may be a prospective target for the effective therapy of advanced hepatocellular carcinoma via accelerating apoptosis of hepatocellular carcinoma cells and eliciting mesenchymal–epithelial transition with the administration of Scutellaria baicalensis extract. Full article

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer type characterized by a marked desmoplastic tumor stroma that is formed under the influence of transforming growth factor (TGF)-β. Data from mouse models of pancreatic cancer have revealed that transcriptionally active p73 (TAp73) impacts the TGF-β pathway through activation of Smad4 and secretion of biglycan (Bgn). However, whether this pathway also functions in human PDAC cells has not yet been studied. Here, we show that RNA interference-mediated silencing of TAp73 in PANC-1 cells strongly reduced the stimulatory effect of TGF-β1 on BGN. TAp73-mediated regulation of BGN, and inhibition of TGF-β signaling through a (Smad-independent) ERK pathway, are reminiscent of what we previously observed for the small GTPase, RAC1b, prompting us to hypothesize that in human PDAC cells TAp73 and RAC1b are part of the same tumor-suppressive pathway. Like TAp73, RAC1b induced SMAD4 protein and mRNA expression. Moreover, siRNA-mediated knockdown of RAC1b reduced TAp73 mRNA levels, while ectopic expression of RAC1b increased them. Inhibition of BGN synthesis or depletion of secreted BGN from the culture medium reproduced the promigratory effect of RAC1b or TAp73 silencing and was associated with increased basal and TGF-β1-dependent ERK activation. BGN also phenocopied the effects of RAC1b or TAp73 on the expression of downstream effectors, like the EMT markers E-cadherin, Vimentin and SNAIL, as well as on negative regulation of the ALK2-SMAD1/5 arm of TGF-β signaling. Collectively, we showed that tumor-suppressive TAp73-Smad4-Bgn signaling also operates in human cells and that RAC1b likely acts as an upstream activator of this pathway. Full article

Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in these cells, blocking ACE2 by antibody treatment did not prevent infection by SARS-CoV-2 spike pseudovirions, nor did antibody blockade of extracellular vimentin and other cholesterol-rich lipid raft proteins. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, is it not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 μM GW3965. We probed the role of endocytic pathways but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 µM chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 µM at the 48 h time point. Finally, we found that anti-LDLR antibodies block pseudovirion infection to a similar degree as anti-caveolin-1 and anti-dynamin I/II antibodies, while transfection with LDLR-specific siRNA led to a decrease in spike pseudotyped lentiviral infection, compared to scrambled control siRNAs. Thus, we conclude that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR. Full article

2-(4-Benzyloxy-3-methoxyphenyl)-5-(carbethoxyethylene)-7-methoxy-benzofuran (BMBF), a benzofuran derivative, is an intermediate found in the process of total synthesis of ailanthoidol. Benzofuran derivatives are a class of compounds that possess various biological and pharmacological activities. The present study explored the anti-metastasis effects of BMBF in hepatocellular carcinoma (HCC). Our preliminary findings indicate that BMBF suppresses the proliferation and changes the morphology of Huh7—an HCC cell line with a mutated p53 gene (Y220C). According to a scratching motility assay, non-cytotoxic concentrations of BMBF significantly inhibited the motility and migration in Huh7 cells. BMBF upregulated the expression of E-cadherin and downregulated the expression of vimentin, Slug, and MMP9, which are associated with epithelial–mesenchymal transition (EMT) and metastasis in Huh7 cells. BMBF decreased the expression of integrin α7, deactivated its downstream signal FAK/AKT, and inhibited p53 protein levels. Cell transfection with p53 siRNA resulted in the prevention of cell invasion because of the reduction in integrin α7, Slug, and MMP-9 in Huh7 cells. BMBF had anti-metastatic effects in PLC/PRF/5—an HCC cell line with R249S, a mutated p53 gene. Our findings indicate that BMBF has anti-metastatic effects in downregulating p53 and mediating the suppression of integrin α7, EMT, and MMP-9 in HCC cells with a mutated p53 gene. Full article

Epithelial-Mesenchymal Transition (EMT), triggered by external and internal cues in several physiological and pathological conditions, elicits the transformation of epithelial cells into a mesenchymal-like phenotype. During EMT, epithelial cells lose cell-to-cell contact and acquire unusual motility/invasive capabilities. The associated architectural and functional changes destabilize the epithelial layer consistency, allowing cells to migrate and invade the surrounding tissues. EMT is a critical step in the progression of inflammation and cancer, often sustained by a main driving factor as the transforming growth factor-β1 (TGF-β1). Antagonizing EMT has recently gained momentum as an attractive issue in cancer treatment and metastasis prevention. Herein, we demonstrate the capability of myo-inositol (myo-Ins) to revert the EMT process induced by TGF-β1 on MCF-10A breast cells. Upon TGF-β1 addition, cells underwent a dramatic phenotypic transformation, as witnessed by structural (disappearance of the E-cadherin–β-catenin complexes and the emergence of a mesenchymal shape) and molecular modifications (increase in N-cadherin, Snai1, and vimentin), including the release of increased collagen and fibronectin. However, following myo-Ins, those changes were almost completely reverted. Inositol promotes the reconstitution of E-cadherin–β-catenin complexes, decreasing the expression of genes involved in EMT, while promoting the re-expression of epithelial genes (keratin-18 and E-cadherin). Noticeably, myo-Ins efficiently inhibits the invasiveness and migrating capability of TGF-β1 treated cells, also reducing the release of metalloproteinase (MMP-9) altogether with collagen synthesis, allowing for the re-establishment of appropriate cell-to-cell junctions, ultimately leading the cell layer back towards a more compact state. Inositol effects were nullified by previous treatment with an siRNA construct to inhibit CDH1 transcripts and, hence, E-cadherin synthesis. This finding suggests that the reconstitution of E-cadherin complexes is an irreplaceable step in the inositol-induced reversion of EMT. Overall, such a result advocates for the useful role of myo-Ins in cancer treatment. Full article

Enterovirus A71, a non-enveloped single-stranded (+) RNA virus, enters host cells through three stages: attachment, endocytosis and uncoating. In recent years, receptors/co-receptors anchored on the host cell membrane and involved in this process have been continuously identified. Among these, hSCARB-2 was the first receptor revealed to specifically bind to a definite site of the EV-A71 viral capsid and plays an indispensable role during viral entry. It actually acts as the main receptor due to its ability to recognize all EV-A71 strains. In addition, PSGL-1 is the second EV-A71 receptor discovered. Unlike hSCARB-2, PSGL-1 binding is strain-specific; only 20% of EV-A71 strains isolated to date are able to recognize and bind it. Some other receptors, such as sialylated glycan, Anx 2, HS, HSP90, vimentin, nucleolin and fibronectin, were discovered successively and considered as “co-receptors” because, without hSCARB-2 or PSGL-1, they are not able to mediate entry. For cypA, prohibitin and hWARS, whether they belong to the category of receptors or of co-receptors still needs further investigation. In fact, they have shown to exhibit an hSCARB-2-independent entry. All this information has gradually enriched our knowledge of EV-A71’s early stages of infection. In addition to the availability of receptors/co-receptors for EV-A71 on host cells, the complex interaction between the virus and host proteins and various intracellular signaling pathways that are intricately connected to each other is critical for a successful EV-A71 invasion and for escaping the attack of the immune system. However, a lot remains unknown about the EV-A71 entry process. Nevertheless, researchers have been continuously interested in developing EV-A71 entry inhibitors, as this study area offers a large number of targets. To date, important progress has been made toward the development of several inhibitors targeting: receptors/co-receptors, including their soluble forms and chemically designed compounds; virus capsids, such as capsid inhibitors designed on the VP1 capsid; compounds potentially interfering with related signaling pathways, such as MAPK-, IFN- and ATR-inhibitors; and other strategies, such as siRNA and monoclonal antibodies targeting entry. The present review summarizes these latest studies, which are undoubtedly of great significance in developing a novel therapeutic approach against EV-A71. Full article

Oral squamous cell carcinoma (OSCC) frequently carries high epidermal growth factor receptor (EGFR) expression. Erlotinib, a small molecule tyrosine kinase inhibitor (TKI), is an effective inhibitor of EGFR activity; however, resistance to this drug can occur, limiting therapeutic outcomes. Therefore, in the current study, we aimed to unveil key intracellular molecules and adjuvant reagents to overcome erlotinib resistance. First, two HSC-3-derived erlotinib-resistant cell lines, ERL-R5 and ERL-R10, were established; both exhibited relatively higher growth rates, glucose utilization, epithelial-mesenchymal transition (EMT), and invasiveness compared with parental cells. Cancer aggressiveness-related proteins, such as N-cadherin, Vimentin, Twist, MMP-2, MMP-9, and MMP-13, and the glycolytic enzymes PKM2 and GLUT1 were upregulated in ERL-R cells. Notably, ERL-R cells were sensitive to quercetin, a naturally-existing flavonol phytochemical with anti-cancer properties against various cancer cells. At a concentration of 5 μM, quercetin effectively arrested cell growth, reduced glucose utilization, and inhibited cellular invasiveness. An ERL-R5-derived xenograft mouse model confirmed the growth-inhibitory efficacy of quercetin. Additionally, knock-down of PKM2 by siRNA mimicked the effect of quercetin and re-sensitized ERL-R cells to erlotinib. Furthermore, adding quercetin blocked the development of erlotinib-mediated resistance by enhancing apoptosis. In conclusion, our data support the application of quercetin in anti-erlotinib-resistant OSCC and indicate that PKM2 is a determinant factor in erlotinib resistance and quercetin sensitivity. Full article

Chronic rhinosinusitis (CRS) pathogenesis is closely related to tissue remodeling, including epithelial–mesenchymal transition (EMT). Epigenetic mechanisms play key roles in EMT. DNA methylation, mediated by DNA methyltransferases (DNMTs), is an epigenetic marker that is critical to EMT. The goal of this study was to determine whether DNMTs were involved in TGF-β1-induced EMT and elucidate the underlying mechanisms in nasal epithelial cells and air–liquid interface cultures. Global DNA methylation and DNMT activity were quantified. DNMT expression was measured using real-time PCR (qRT–PCR) in human CRS tissues. mRNA and protein levels of DNMTs, E-cadherin, vimentin, α-SMA, and fibronectin were determined using RT–PCR and Western blotting, respectively. DNMT1, DNMT3A, and DNMT3B gene expression were knocked down using siRNA transfection. MAPK phosphorylation and EMT-related transcription factor levels were determined using Western blotting. Signaling pathways were analyzed using specific inhibitors of MAPK. We demonstrated these data in primary nasal epithelial cells and air–liquid interface cultures. Global DNA methylation, DNMT activity, and DNMT expression increased in CRS tissues. DNMT expression was positively correlated with Lund–McKay CT scores. TGF-β1 dose-dependently induced DNMT expression. Further, 5-Aza inhibited TGF-β1-induced DNMT, Snail, and Slug expression related to EMT, as well as p38 and JNK phosphorylation in A549 cells and TGF-β1-induced DNMT expression and EMT in primary nasal epithelial cells and air–liquid interface cultures. TGF-β1-induced DNMT expression leads to DNA methylation and EMT via p38, JNK, Snail, and Slug signaling pathways. Inhibition of DNMT suppressed the EMT process and therefore is potentially a CRS therapeutic strategy. Full article

Background: Collagen type XI α1 (COL11A1) is associated with tumorigenesis and development in many human malignancies. Previous reports indicate that COL11A1 may be a significant diagnostic marker for pancreatic ductal adenocarcinoma (PDAC); however, its biological role in PDAC progression remains unclear. In this study, we investigated the influence of COL11A1 on the invasion and migration abilities of pancreatic cancer cells and explored its potential molecular mechanisms. Methods: Cell migration and invasion were assessed using Transwell assays in pancreatic cancer cells transfected with siCOL11A1 and pCNV3-COL11A1 plasmids. The protein and mRNA expression levels of N-cadherin, E-cadherin, Vimentin, cluster of differentiation (CD)-24, CD44, serine–threonine kinase (AKT), glycogen synthase kinase (GSK)-3β, phospho (p)-AKT^Ser473, p-GSK-3β^Ser9, and Snail were analyzed using Western blotting and real-time polymerase chain reaction (PCR). The effect of COL11A1 on cell stemness was tested using flow cytometry and clone formation assays. Results: These results demonstrated that COL11A1 significantly promoted the invasion and migration abilities of PDAC cells. Furthermore, COL11A1 facilitated the occurrence of epithelial–mesenchymal transition (EMT) and cell stemness by upregulating the expression levels of p-AKT^Ser473, p-GSK-3β^Ser9, and Snail. Conclusions: This study suggests that the activation of the AKT/GSK-3β/Snail signaling pathway induced by COL11A1 plays a major role in the progression of PDAC. Therefore, COL11A1 could serve as a potential target for PDAC treatment. Full article

Glioblastoma (GBM) is the most malignant glioma with an extremely poor prognosis. It is characterized by high vascularization and its growth depends on the formation of new blood vessels. We have previously demonstrated that TRPML2 mucolipin channel expression increases with the glioma pathological grade. Herein by ddPCR and Western blot we found that the silencing of TRPML2 inhibits expression of the VEGFA/Notch2 angiogenic pathway. Moreover, the VEGFA/Notch2 expression increased in T98 and U251 cells stimulated with the TRPML2 agonist, ML2-SA1, or by enforced-TRPML2 levels. In addition, changes in TRPML2 expression or ML2-SA1-induced stimulation, affected Notch2 activation and VEGFA release. An increased invasion capability, associated with a reduced VEGF/VEGFR2 expression and increased vimentin and CD44 epithelial-mesenchymal transition markers in siTRPML2, but not in enforced-TRPML2 or ML2-SA1-stimulated glioma cells, was demonstrated. Furthermore, an increased sensitivity to Doxorubicin cytotoxicity was demonstrated in siTRPML2, whereas ML2-SA1-treated GBM cells were more resistant. The role of proteasome in Cathepsin B-dependent and -independent pRB degradation in siTRPML2 compared with siGLO cells was studied. Finally, through Kaplan-Meier analysis, we found that high TRPML2 mRNA expression strongly correlates with short survival in GBM patients, supporting TRPML2 as a negative prognostic factor in GBM patients. Full article

Air pollution presents a major environmental problem, inducing harmful effects on human health. Particulate matter of 10 μm or less in diameter (PM₁₀) is considered an important risk factor in lung carcinogenesis. Epithelial–mesenchymal transition (EMT) is a regulatory program capable of inducing invasion and metastasis in cancer. In this study, we demonstrated that PM₁₀ treatment induced phosphorylation of SMAD2/3 and upregulation of SMAD4. We also reported that PM₁₀ increased the expression and protein levels of TGFB1 (TGF-β), as well as EMT markers SNAI1 (Snail), SNAI2 (Slug), ZEB1 (ZEB1), CDH2 (N-cadherin), ACTA2 (α-SMA), and VIM (vimentin) in the lung A549 cell line. Cell exposed to PM₁₀ also showed a decrease in the expression of CDH1 (E-cadherin). We also demonstrated that expression levels of these EMT markers were reduced when cells are transfected with small interfering RNAs (siRNAs) against TGFB1. Interestingly, phosphorylation of SMAD2/3 and upregulation of SMAD induced by PM₁₀ were not affected by transfection of TGFB1 siRNAs. Finally, cells treated with PM₁₀ exhibited an increase in the capacity of invasiveness because of EMT induction. Our results provide new evidence regarding the effect of PM₁₀ in EMT and the acquisition of an invasive phenotype, a hallmark necessary for lung cancer progression. Full article

Many anti-cancer drugs, including paclitaxel and etoposide, have originated and been developed from natural products, and traditional herbal medicines have fewer adverse effects and lesser toxicity than anti-tumor reagents. Therefore, we developed a novel complex herbal medicine, JI017, which mediates endoplasmic reticulum (ER) stress and apoptosis through the Nox4–PERK–CHOP signaling pathway in ovarian cancer cells. JI017 treatment increases the expression of GRP78, ATF4, and CHOP and the phosphorylation of PERK and eIF2α via the upregulation of Nox4. Furthermore, it increases the release of intracellular reactive oxygen species (ROS), the production of intracellular Ca²⁺, and the activation of exosomal GRP78 and cell lysate GRP78. Combination treatment using the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (TG) and JI017 reportedly induces increased ER stress and cell death in comparison to the control; however, knockdown experiments of PERK and CHOP indicated suppressed apoptosis and ER stress in JI017-treated ovarian cancer cells. Furthermore, targeting Nox4 using specific siRNA and pharmacological ROS inhibitors, including N-acetylcystein and diphenylene iodonium, blocked apoptosis and ER stress in JI017-treated ovarian cancer cells. In the radioresistant ovarian cancer model, when compared to JI017 alone, JI017 co-treatment with radiation induced greater cell death and resulted in overcoming radioresistance by inhibiting epithelial–mesenchymal-transition-related phenomena such as the reduction of E-cadherin and the increase of N-cadherin, vimentin, Slug, and Snail. These findings suggest that JI017 is a powerful anti-cancer drug for ovarian cancer treatment and that its combination treatment with radiation may be a novel therapeutic strategy for radioresistant ovarian cancer. Full article