Cisplatin is a standard anticancer drug which has shown significant chemotherapeutic potential in treating various solid tumors [
40]. However, cisplatin has shown side effects like renal failure, nausea, ear damage etc. [
41,
42]. In most cancer chemotherapy, tumor cells develop drug resistance culminating into severe side effect leading to worst outcome [
42,
43]. New advances in anticancer drug development by making alternative to standard known drugs for cancer therapeutics using coordination chemistry has been made in the last years [
44,
45]. We reported earlier the development of platinum complex based on salen ligand (PT) having anticancer potential [
20]. Herein, we explored the molecular mechanism of PT by which it inhibited the cellular proliferation in colorectal cancer cells. We demonstrated for the first time that this platinum complex (PT) inhibited the colony forming ability of human colorectal cancer cells in a dose dependent manner. A similar polyamine complex of Pt(II) inhibited colony formation in breast cancer cells [
46] and Pt (II) complex of 1, 10-phenanthroline exhibited antitumor effect in lung cancer [
47]. Inhibition of cell proliferation is mainly mediated by apoptosis that plays an important role in fighting cancer. Therefore, for cancer therapeutic strategies apoptosis is a popular target. Induction of apoptosis by anticancer therapy is considered to be the most popular strategy to kill cancer cells [
48]. In this study, we found that PT could trigger a significant increase in apoptosis in HT-29 and SW620 cells whereas induction of cell death in SW480 cell was found to be mediated by necrosis. ROS production plays an essential role in anticancer drug discovery. Previous evidence has suggested that some organometallic complex induced oxidative stress [
49,
50]. In corroboration with these finding we found that PT induced ROS production and glutathione depletion in HT-29, SW480 and SW620 cells. Interestingly, the ROS production was much higher in HT-29 and SW620 cells than SW480 cells. Cancer cells are known to produce higher amount of ROS than normal cells, treating cancer cells with these ROS generating agents would insult these cells and leads to cell death [
51]. The redox balance has been shown to play important role in cancer progression [
52]. Higher ROS generation leads to alteration in mitochondrial membrane potential. In this study we found that PT treatment inhibited mitochondrial membrane potential in human colorectal cancer cells. Bcl2 family proteins which are grouped into anti-apoptotic protein (Bcl2, BclxL, Mcl1) and pro-apoptotic protein (Bax, Bak, Bid) play essential role in the regulation of early events of apoptosis [
53]. The balance between anti-apoptotic and pro-apoptotic proteins determines the fate of cancer cells. The overexpression of Bcl2 and BclxL are known to block apoptosis and promote cancer cell proliferation, thus making them as attractive targets for cancer drug discovery. In this study we found that PT inhibited the expression of Bcl2, BclxL and XIAP in colorectal cancer cells. Therefore, inhibition of Bcl2 and BclxL makes cancer cells more vulnerable to apoptosis. Cyclins are known to play pivotal role in the regulation of cell cycle [
31]. This platinum complex inhibited cyclin D1 expression and partially upregulated cyclin E1 expression at lower concentration of 5 μM while downregulated 10 μM. The significance of concentration dependent regulation of cyclin E1 by PT needs further exploration. Under stress pro-apoptotic Bax gets activated by oligomerization which leads to the release of cytochrome c from mitochondria into cytosol. The release of cytochrome c leads to the activation of caspase cascade and apoptosis [
33,
53]. PT treated colorectal cancer cells were found to have increased amount of cytochrome c as compared to control cells. Cytochrome c release into cytosol leads to caspase-9 activation which in turn activates caspase-3. Activation of caspase family of proteins play essential role in the initiation and execution of apoptotic process [
53]. Caspase-3 is the major rate limiting caspase resulting in the cleavage of PARP that leads to apoptotic cell death. PARP cleavage was detected in PT-treated colorectal cancer cells. We found that PT complex induced caspase-9, caspase-3 and caspase-7 activation in dose dependent manner. However, no caspase-8 activation was detected suggesting that PT induced intrinsic apoptotic pathway in colorectal cancer cells. Similar findings were obtained with ONS-donor ligand based Pt(II) complex which induced anticancer activity by PARP cleavage, Caspase-3/− 7 activation and autophagy [
54]. Kowalski et al. reported that Oxidovanadium(IV) coordination complex induces cell cycle arrest and autophagy by ROS generation and triggering P53/21 pathway [
55]. Our findings indicate that PT induces cell death by inhibition of anti-apoptotic proteins, increasing the cytochrome c release and PARP cleavage. We provided direct evidence that the production of ROS represented the first step of PT-induced apoptosis. N-acetylecysteine (NAC) is a ROS scavenger known to block ROS-mediated apoptosis. In fact, pre-treatment with NAC attenuated PT-induced cell death in human colorectal cancer cells.
Mitogen-activated protein kinases (MAPKs) belong to a highly conserved family of serine/threonine kinases. MAPK signaling plays a significant role in cancer cell proliferation, differentiation, motility, survival and apoptosis [
17‐
19]. The family of MAPK is made up of JNK, p38 MAPK and ERK1/2 and known to regulate many of the cellular processes associated with growth, survival and cell death. We found that pre-incubation with p38 MAPK inhibitor (SB202190) significantly inhibited the PT-induced cell death in HT-29, SW480 and SW620 cells. AKT inhibitor inhibited PT-induced cell death significantly in HT-29 and SW480; whereas it has no significant effect in SW620 cells. Other inhibitors of MAPK family, like JNK and ERK had no effect on PT-induced apoptosis in colorectal cancer cells. This study thus indicates that PT-induced apoptosis is mediated by the activation of p38 MAPK signaling. AKT pathway plays a role in PT-induced cell death in HT-29 and SW480 but not in SW620 cells suggesting its involvement in a cell context dependent manner. In addition, we confirmed that PT induced the phosphorylation of p38 MAPK and inhibited the AKT phosphorylation in colorectal cancer cells. The phosphorylation of other MAPK like JNK and ERK was unperturbed in PT-treated cells. Silva et al. [
56] showed that Ru(II)-thymine complex causes DNA damage and p38 activation in colorectal cancer similar to our finding. Consistent with these findings we found that PT-induced activation of p38 MAPK signaling leads to the phosphorylation of downstream substrate (HSP27-Ser82) in colorectal cancer cells. In addition, p38 MAPK inhibitor SB202190 blocked PT-induced cytochrome c confirming the role of p38 MAPK in PT-induced apoptosis. Furthermore, silencing of p38 by siRNA also inhibited PT-induced apoptosis in colorectal cancer cells supporting our findings from p38 MAPK inhibitor SB202190. PT may inhibit inflammatory pathway like NFkB and STAT3. NFkB and STAT3 pathway play essential role in inflammatory response and link chronic inflammation to colorectal cancer [
57‐
59]. These pathways are known to regulate cell proliferation, apoptosis, invasion, metastasis and drug resistance. NFkB and STAT3 transcription factors regulate anti-apoptotic gene expression of Bcl2 and BclxL. Our finding indicates that PT inhibited Bcl2 and BclxL protein expression in various colorectal cancer cells indicating that PT may alter NFkB and STAT3 pathway. Zhu et al. have shown that cyclometalated Pt complex inhibited the cell viability of HepG2 cell without affecting normal cells by hijacking the NFkB pathway [
60]. Others have reported the cytotoxic effectiveness of platinum complexes by inhibiting NFkB proteins-platinated DNA interaction [
61].
With increasing incidence and high mortality associated with colorectal cancer specifically with advance disease stages, novel cancer therapeutics are needed urgently. Traditional chemotherapy against colorectal cancer includes oxaliplatin and other analogues of cisplatin. For decades, platinum complexes have been used as gold standard for anti-cancer therapy. However, treatment with platinum complexes resulting in higher side effect causing damage to healthy cells cannot be neglected. Many cancer cells exhibited inherent or acquired resistance to platinum-based drugs and prevent further clinical utilization. PT based on salicylaldimine ligand exhibited anticancer potential not only on adenocarcinoma colorectal cancer but also on metastatic colorectal cancer cells by generating ROS production and regulating MAPK pathway. Therefore, this platinum complex may be explored with improved cytotoxicity and minimal side effects in colorectal cancer patients.