We recently discovered a novel quinazolin-4-sulphomide derivative (3c) as a potential anti-tumor agent [
19]. However the mechanism by which this compound acts remains unknown. In this study we shed new light on the mechanism by which 3c exerts its anti-cancer activity. Our results show that 3c induces apoptosis in human colorectal cancer cells by increased ROS production. ROS, which includes hydrogen peroxide, hydroxyl radical, and superoxides, are chemically active prooxidant molecules generated by incomplete reduction of oxygen [
26]. Reactive oxygen species are involved in a variety of cellular physiology and pathology [
27]. Anti-cancer agents-induced ROS production that mediates the induction of apoptosis has been exploited in designing effective strategies for cancer therapeutics [
26]. Many studies investigated the effects of both natural and synthetic anti-cancer agents which modulate ROS in colorectal cancer [
28,
29]. 3c was found to increase ROS in colorectal cancer cells and was blocked by NAC. Our results support a model in which 3c-induced ROS production stimulates the intrinsic mitochondrial apoptotic pathway by decreasing outer mitochondrial membrane potential and thereby releasing cytochrome c that further activates caspase dependent signaling [
30]. ROS are well known mediators of the intracellular signaling cascade known to play an important role in cancer drug discovery [
31,
32]. They play critical role in the regulation of diverse functions, such as proliferation, apoptosis and transformation. When ROS levels reach a threshold point beyond the capacity of cellular antioxidant machinery it leads to oxidative stress, which in turn modulate mitochondrial membrane potential [
33]. Several studies suggest that cancer cells are under increased oxidative stress associated with oncogenic transformation and increased ROS generation possibly due to its higher metabolic activity [
34,
35]. A further increase in the ROS levels can make these malignant cells more vulnerable to cell death relative to untransformed cells. Thus, in cancer therapeutics, ROS signaling can be exploited to develop novel drugs to inhibit or kill cancer cells through its specific ROS signaling mechanism. Our newly developed compound 3c utilizes this mechanism in killing tumor cells. 3c treatment resulted in high level of ROS in HT-29 cells whereas NAC combined with 3c reduced ROS production and subsequently inhibited apoptosis. The molecular mechanism by which 3c induces elevated ROS production is presently not understood. Previous findings have reported that sulphonamide derivatives inhibit carbonic anhydrases activity [
36‐
38]. 3c is known to inhibit the carbonic anhydrase IX and XII expression [
19]. Some carbonic anhydrases have been reported to possess antioxidant property. Specifically, carbonic anhydrase III (CA III) and VII (CA VII) are known to have antioxidant characteristics [
39‐
42]. This compound 3c may inhibit CA III and/ or CAVII expression and thereby increases ROS generation. Other mechanism may be involved as well like acting on electron transport chain or by abrogating key antioxidant systems in cells like depleting glutathione pool and/ or inhibiting superoxide dismutase (SOD) [
43]. There are some anticancer agents for example elesclomol which is in clinical trial exerts its effect by inducing ROS but mechanism for ROS generation is not known [
44]. Oxidative stress leads to decreased mitochondrial membrane potential. In this study 3c was also found to decrease mitochondrial membrane potential. We further showed that 3c induced PARP cleavage is blocked in the presence of NAC. Cytochrome c release induced by 3c was also inhibited by NAC, proving that indeed 3c induced apoptotic markers are ROS dependent. NADPH is required for the conversion of oxidized glutathione to reduced glutathione. 3c treatment resulted in decreased levels of NADPH and GSH reducing the cells antioxidation capacity and thus increasing oxidative stress.
We further uncovered the molecular pathway underlying 3c-induced apoptosis through upregulation of pro-apoptotic protein, Bax leading to the cytochrome c release from mitochondria. Cytochrome c release from mitochondria leads to the activation of caspase cascade which is essential in initiating apoptosis by anti-cancer agents [
45]. 3c treatment resulted in an increase in caspase-9 and effector caspase-3 and 6 activities. Caspase-9 is an initiator caspase in mitochondria mediated apoptosis pathway [
46]. These findings indicate that 3c induces apoptosis through the intrinsic pathway. Certain caspases also target PARP for its cleavage into 24 kDa and 89 kDa fragments, rendering them incapable of DNA repair and leading to cell death. In this study, 3c was found to increase cleaved PARP levels. Cancer cells express series of anti-apoptotic proteins such as Bcl2 and BclxL. Overexpression of these anti-apoptotic proteins inhibits apoptosis and promotes cancer cell survival [
47]. The anti-apoptotic Bcl2 protein has been demonstrated to be overexpressed in colorectal cancers [
48]. According to Bonnotte et al., Bcl2 mediated apoptosis inhibition restores the tumorigenicity of colon tumors [
49]. The reduced expression of Bcl2 and BclxL in our investigation suggests that 3c-induced apoptosis is mediated by an inhibition of these proteins. Zhu and colleagues have shown that induction of Bax expression is essential for death-receptor mediated apoptosis in colon cancer cells [
50]. Tumor suppressor p53 is a known regulator of Bcl2 and Bax gene expression [
24]. p53 may be involved in the 3c-induced Bax expression and downregulation of Bcl2 expression. 3c was found to increase p53 and Bax expression in a time dependent manner similarly decreasing Bcl2 expression. This finding may provide a mechanism for 3c-induced alterations of Bcl family protein by involving p53 transcription factor. Our observation of elevated Bax expression and an increase in caspase-8 activity in 3c treated colorectal cancer cells may suggest involvement of extrinsic apoptotic pathway as well. However, it is possible that caspase-8 through cleavage of BH3-only protein, BID also activates intrinsic apoptosis pathway [
51]. Additionally we observed an important property of 3c to inhibit cancer cell migration that would have a significant clinical benefit in controlling invasion and metastasis. Epithelial to mesenchymal transition (EMT) is essential process for cell invasion and migration [
25]. 3c was found to alter the expression of EMT markers like E-Cadherin and Vimentin. TGFβ-smad pathway is known to induce EMT in cancer cells. This compound of interest, 3c inhibits TGFβ-induced smad pathway in colorectal cancer cells. These findings thus indicate that 3c-induced inhibition of cell migration is mediated by TGFβ-smad pathway.