*2.6. Anti-Proliferative and Pro-Apoptotic Effects of the Ru(II) Naphthalimide-NHC Complex in HCT116 Cells Proceed via p38 MAPK Signaling, Involving ROS*

To further elucidate the role of p38 in the anti-tumor properties of MC6, which triggered the strongest p38 activation across the three complexes (Figure 6A,B), we knocked-down the expression of *p38α* (*MAPK14*) in HCT116 cells while using siRNA (Figure 7D). As determined by SRB cytotoxicity assay, upon 24 h treatment with 12 μM of MC6, cellular viability was significantly increased in cells that were transfected with anti-*p38α* siRNA as compared to that of the negative control (siRNA NC) and non-transfected HCT116 cells (Figure 7A). We then aimed at investigating the influence of p38 knock-down on the MC6-mediated apoptosis using annexinV/propidium iodide (AV/PI) staining. As depicted in Figure 7B,C, 24 h treatment with 12 μM of MC6 resulted in the transition of cells through the early apoptotic (AV+/PI−) quadrant in HCT116 and HCT116 siRNA NC, as determined by ~1.8- and ~1.4-fold increase, respectively. This effect was found to be attenuated in response to the p38 knock-down, as the fold change of early apoptotic- as well as late apoptotic/necrotic (AV+/PI+) cells were significantly less than that of HCT116 and cells transfected with the non-targeting siRNA (Figure 7B). Similar results were obtained in the presence of two well-known chemical inhibitors of p38α, VX-702, and Ralimetinib. As shown in Figure 7E,F, the number of apoptotic cells upon 24 h of MC6 treatment was significantly decreased when combined with p38α inhibitors, as detected by AV/PI staining. Additionally, the pro-apoptotic response of HCT116 cells to MC6 was evaluated in the absence/presence of p38α activity using flow cytometric analysis of caspase 3 activation where significantly less cleaved caspase 3 levels were observed upon both knock-down of p38α (Figure 7G), as well as its pharmacological inhibition (Figure 7H,I). Moreover, we monitored the MC6-induced apoptosis using TUNEL assay and found significantly more apoptotic cells with 24 h of MC6 treatment, an effect that was clearly abrogated upon VX-702-mediated p38α inhibition (Figure 7J,K).

**Figure 7.** p38 signaling appears to be crucial for the MC6-mediated cytotoxic- and pro-apoptotic effects. (**A**) siRNA-mediated repression of *p38α* was found to hamper the growth inhibitory effects of MC6 in HCT116 cells treated with 12 μM of the compound for 24 h, measured by SRB assay. Percentage cell viability was calculated by normalizing the values of MC6-treated cells to those of the corresponding mock (0.1% DMSO) treatments. Error bars ± SD; *n* = 3; (**B**) Knock-down of *p38α* attenuates the pro-apoptotic response to MC6 (12 μM, 24 h), assessed by flow cytometric analysis of AV/PI staining. Percentage cell population in each quadrant was normalized to the respective mock (0.1% DMSO) treatment. Error bars ± SD. Multiple comparisons were performed using two-way ANOVA followed by a post-hoc Tukey test; (**C**) Density plots representative of three biological replicates illustrate increased population of AV+/PI<sup>−</sup> and AV+/PI+ with treatment, however, to a lesser extent in case of cells transfected with anti-*p38α* siRNA; (**D**) Confirmation of knock-down efficiency, as determined by immunoblotting; (**E**) chemical inhibition of p38α abrogates the MC6-mediated apoptosis. HCT116 cells were treated with 12 μM of MC6 in the absence/presence of p38α inhibitors, VX-702 and Ralimetinib at a concentration of 0.5 μM for 24 h. Error bars ± SD, *n* = 3. Asterisks show significance in the amount of early- and late apoptotic population between cells treated with MC6 and each of the two inhibitors, and MC6 as a single agent, determined by two-tailed student's *t*-test; (**F**) Representative density plots of one out of three biological replicates demonstrate reduced number of AV+/PI<sup>−</sup> and AV+/PI+ cells when p38α activity is inhibited; (**G**) Flow cytometric analysis of caspase 3 activation shows significantly less cleaved caspase 3 expression in cells transfected with anti-*p38α* siRNA as compared to that of the negative control. Error bars ± SD, *n* = 6. Statistical significance between the two groups was made using two-tailed student's *t*-test; (**H**) Chemical inhibition of p38α was found to decrease the levels of active caspase 3 in a similar manner to that of *p38α* knock-down. Error bars ± SD, *n* = 6. Statistical comparison was performed between combination treatments and MC6 using two-tailed student's *t*-test; (**I**) Representative density plots of one out of six biological replicates; (**J**) Detection of apoptotic cells using TUNEL assay. HCT116 cells were treated with the indicated concentrations of MC6 for 24 h in the absence/presence of VX-702 (0.5 μM). Statistical significance was calculated between mock and the respective treatment as well as MC6 as single agent and in combination with VX-702 using two-tailed student's *t*-test; (**K**) Representative fluorescence images of TUNEL reaction. Scale bar: 100 μm. *p*-values less than or equal to 0.05, 0.01, 0.001, and 0.0001 are denoted as \*, \*\*, \*\*\*, and \*\*\*\*, respectively.

**Figure 8.** MC6-induced cytotoxicity and pro-apoptotic effects are mediated via the ROS-p38 signaling axis. (**A**) Treatment with GSH and NAC at the indicated concentrations 1 h prior to the addition of MC6 (12 μM) for 24 h blocked the activation of p38 as well as cleavages of caspase 3 and PARP, detected by immunoblotting; (**B**) Densitometric analyses show a significant reduction in the MC6-induced pp38 (T180/Y182) accumulation in the presence of anti-oxidants. Error bars indicate the SEM of three independent experiments, one of those is presented in (**A**); (**C**) Increased cellular survival of HCT116 cells pre-treated with either 5 or 10 mM of GSH 1 h before the addition of MC6 (12 μM) for 24 h, as determined by SRB assay. Data represent mean ± SD of three biological replicates, normalized to mock (0.1% DMSO) and the respective GSH treatment; (**D**–**F**) HCT116 cells pre-incubated with either 5 or 10 μM of Mito TEMPO (MT) for 2 h were treated with MC6 (12 μM) for 24 h. The mitochondria-targeted ROS scavenger was found to attenuate the MC6-mediated p38 activation as well as PARP cleavage at the highest used concentration (10 μM), as detected by immunoblotting (**D**) and the associated densitometric quantification (**E**), obtained from three independent experiments. Error bars ± SEM. Additionally, it rescued the MC6-mediated cytotoxic effects, as determined by SRB assay (**F**). Percentage cell viability of MC6-treated cells was normalized to mock (0.1% DMSO) and the respective MT treatment. Error bars ± SD; *n* = 3; (**G**) Flow cytometric analysis of caspase 3 activation illustrates significantly lower levels of the cleaved form of the protein in cells pre-treated for 1 h with either GSH (10 mM) or NAC (10 mM) as compared to that of MC6-treated cells (12 μM, 24 h). Error bars ± SD, *n* = 3; (**H**) Representative histogram of one out of three biological replicates presented in (**G**) demonstrates a left-ward shift in caspase 3 activity in the presence of ROS scavengers. Statistical significance between the MC6-treated cells in the absence/presence of anti-oxidants was calculated using two-tailed student's *t*-test. \*, \*\*, \*\*\*, and \*\*\*\* on the figures represent *p*-values that are less than or equal to 0.05, 0.01, 0.001, and 0.0001, respectively.

Activation of the pro-apoptotic p38 MAPK signaling can happen as result of ROS generation [19], and it has been previously implicated in the anti-cancer mechanism of metal NHC complexes [13]. We hence sought to investigate whether pp38 MAPK induction and subsequent growth inhibitory/pro-apoptotic effects of MC6 are due the elevated ROS levels. As shown in Figure 8A,B, pre-incubation with the ROS scavengers, reduced GSH as well as NAC, blocked the MC6-induced p38 activation as well as apoptosis, determined by the absence of PARP and caspase 3 cleavages, markers of apoptotic cell death. With respect to MC6-mediated cytotoxicity, we observed increased cellular survival percentages with GSH treatment, however, the difference failed to reach statistical significance (Figure 8C). Similarly, pre-treatment with a mitochondria-targeted scavenger, Mito TEMPO, hampered the effects of MC6 on p38 activation and PARP cleavage (Figure 8D,E), as well as its cytotoxicity (Figure 8F) in HCT116 cells. We further confirmed the protective effect of anti-oxidant treatment on MC6-mediated apoptosis by quantification of cleaved caspase 3 levels using flow cytometry. As shown in Figure 8G,H, pre-treatment of HCT116 cells with GSH and NAC rescued the MC6-mediated caspase 3 cleavage. In view of these findings, we propose that the in vitro anti-tumor activity of MC6 in HCT116 CRC cells may be regulated through mtROS-induced activation of the p38 MAPK pathway.

#### **3. Discussion**

Regulation of the redox state plays an important role in tumor cell survival. Although elevated ROS levels allow cancer cells to promote pro-tumorigenic signaling, excessive ROS production is usually associated with anti-tumorigenic pathways, which can trigger oxidative stress-induced cancer cell death [19]. The latter effects of ROS are mainly mediated through the ASK1/JNK and ASK1/p38 axes, leading to cell cycle arrest, growth inhibition, and apoptosis [19]. Here, we demonstrate that a novel Ru(II) naphthalimide-NHC complex is able of causing a remarkable increase in mtROS generation, which in turn activates the pro-apoptotic p38 signaling in HCT116 CRC cells.

The p38 pathway is a major regulator of stress responses, influencing various biological processes, including cellular proliferation and survival [8]. Studies have demonstrated that the role of p38 MAPK signaling in cancer therapy is contextual, depending on the nature of the stimuli, cancer type, as well as the status of other MAPKs (ERKs and JNKs) [30,31]. On the one hand, p38 MAPK activation mediates the sensitivity of tumor cells to a variety of chemotherapeutics, for example, cisplatin [9–11,32], oxaliplatin [33], and auranofin [12]. In particular, cisplatin has been reported to induce apoptosis in HCT116 CRC cells through the ROS-p38α axis downstream of p53 activation [10]. On the other hand, cancer cell lines with a high basal expression of pp38 MAPK tend to lose the tumor-suppressing functions of this molecule, possibly because of the inability to further activate p38 MAPK in response to anti-cancer treatments [26]. In light of this, we detected a clear induction of pp38 MAPK (T180/Y182) levels in response to all three naphthalimide-NHC derivatives in HCT116 and MCF-7 cells, with the Ru(II) analogue (MC6) exhibiting the strongest effect. However, MC6 was hardly able to promote p38 activation in the triple-negative MDA-MB-231 breast cancer cells in which the basal levels of phospho-p38 are elevated (Figure 6F,G). This is in line with a reduced cytotoxic response of MDA-MB-231 as compared with that observed for HCT116 and MCF-7 (Figure 1B), suggesting that the lack of p38 activation renders this cell line resistant to the anti-proliferative effects of naphthalimide-NHC conjugates. In this regard, several reports have shown the beneficial outcome of p38 MAPK inhibition rather than its activation for the treatment of invasive breast cancer models. For example, it has been proposed that p38 MAPK inhibition has synergistic effects with cisplatin for the treatment of breast cancer through the activation of ROS-mediated JNK signaling [27]. Our results suggest an anti-tumorigenic role for p38 signaling in response to MC6 as its genetic as well as chemical inhibition attenuates the cytotoxic- and pro-apoptotic effects of the compound in HCT116 cells (Figure 7). However, this requires further investigation in other cellular contexts, in particular, in cancer cells with enhanced basal p38 MAPK phosphorylation. To further address whether other MAPKs, including JNK and/or ERK signaling, are implicated in these processes, we also analyzed the regulatory state of these kinases in response to treatment with the three naphthalimide-NHC derivatives, as, for

instance, JNK activation and/or ERK inhibition, which has been previously reported in the anti-cancer efficacy of several other organometallic drugs [27,34,35]. We, however, were not able to observe a profound regulation of MAPKs other than p38α in response to the naphthalimide-NHC analogues (Figure S5), further demonstrating the important role of p38α activation in sensitizing tumor cells to MC6-mediated apoptosis.

In this study, we show an essential role for mtROS generation in the cytotoxicity of naphthalimide-NHC analogues. There are two main sources of the signaling-associated ROS in cells: (i) membrane bound-NADPH oxidases (known as NOX enzymes) and (ii) mitochondrial electron transport chain (ETC) [19]. Our results demonstrate that intracellular ROS levels are minimally affected by the naphthalimide-NHC derivatives, whereas mitochondrial superoxide production is markedly increased in response to the molecules, with the Ru(II) complex showing the highest fold change. MtROS have been frequently mentioned in the regulation of pro-inflammatory/apoptotic responses via multiple mechanisms, among others, is the activation of MAPKs [20]. In view of this, we observed that treatment with a mitochondria-targeted ROS scavenger and MC6 blocks the latter's effect on p38 MAPK activity (Figure 8D,E), suggesting that mtROS generation may be acting upstream of MC6-mediated p38 induction. It is known that mitochondrial superoxide species sustain MAPK activity through the oxidation and inactivation of MAPK phosphatases (MKPs) [36]. In line with this, we found reduced MKP6 expression in response to the naphthalimide-NHC conjugates (data not shown), further supporting the involvement of mtROS in the induction of p38 signaling. Importantly, the cytotoxic and pro-apoptotic effects of MC6 were hampered upon general- and mitochondria-specific anti-oxidant treatments (Figure 8), implicating a ROS-mediated pathway underlying the in vitro anti-tumor efficacy of the complex.

One suggested mode of action of metal NHC complexes and naphthalimide derivatives is via mitochondrial accumulation, and perturbations in the MMP (Δψm) [13,37,38]. We here report that Δψm is differentially regulated by the three naphthalimide-NHC conjugates, showing all possible options; a timely decrease in case of MC5, a transient increase in case of MC6 followed by a decrease at 24 h, and no significant change in case of MC7. With regards to the relationship between mitochondria-driven ROS and Δψm, the general concept is that more polarized membrane (high Δψm) is associated with greater ROS production [20]. This is consistent with the observation of MC6 showing the highest MMP along with the highest mtROS generation as compared to the other two complexes. However, it fails to explain the influence of MC5 treatment on mitochondrial parameters, wherein, despite reduced Δψm, mtROS production increases. These disparate observations reconcile by a "redox-optimized ROS balance" model proposed by Aon et al. who demonstrated that oxidative stress can occur at either extreme of MMP (high Δψm or low Δψm), meaning that under oxidizing conditions mtROS can increase because of the compromised anti-oxidant defense mechanisms [21]. Taking all the mentioned observations into consideration, we suggest that the mode of anti-cancer activity of naphthalimide-NHC compounds is most likely through mitochondrial localization, leading to ROS-mediated activation of the pro-apoptotic p38 MAPK pathway.

#### **4. Materials and Methods**
