Combination of AP and CDDP exhibits synergistic anti-cancer effects
We first determined the effect of combined administration of AP and CDDP on viability of colon cancer cells. As shown in Figs. 1A-B, AP treatment alone caused no obvious changes in the viability of HCT116 and HT29 cells, whereas combination of CDDP and AP showed higher antiproliferative activities on the cancer cells than the single agent. We next investigated the underlying mechanisms of synergistic effect of AP and CDDP on HCT116 cells. To this end, we performed flow cytometric assays to comparatively analyze the cell cycle distribution among different groups. As illustrated in Figs. 1C-D, AP in combination with CDDP caused a marked increase in the proportion of cells in G2/M phase compared with AP or CDDP alone, as reflected by the increase of the proportion from 16.5% (CDDP) or 25.3% (AP) to 43.2% (combination of CDDP and AP). Consistent with the flow cytometry, western blot analysis revealed that AP markedly enlarged CDDP-induced decrease in the expression of cell cycle-related proteins MDM2, Cyclin B1 and CDC2 in HCT116 cells (Figs. 1E-F). All these data suggested that AP enhances CDDP-induced cell growth inhibition in HCT116 cells possibly through inducing a cell cycle arrest at G2/M phase.
Ap Potentiates Pro-apoptotic Effect Of Cddp On Hct116 Cells
To determine whether apoptosis mediates growth inhibition of colon cancer cells caused by the combined treatment, we examined pro-apoptotic effects of AP, CDDP and AP plus CDDP on HCT116 cells, respectively. As shown in Figs. 2A-B, combined treatment with AP and CDDP resulted in a significant increase in apoptosis rate as compared to AP or CDDP alone. Moreover, western blot analysis revealed that treatment with AP obviously enhanced cisplatin-induced activation of caspase-3 (cleaved caspase3), while reducing the expression level of Pro-Caspase3 (Figs. 2C-E). Together, these data indicated that combined treatment of AP and CDDP activates caspase-dependent apoptosis pathways, suggesting that AP treatment could promote cisplatin-induced apoptosis in HCT116 cells.
Ap Potentiates Cisplatin-induced Apoptosis Via Ros Accumulation
Previous studies have shown that ROS generation could trigger cell apoptosis[25, 26]. Notably, AP has been found to induce oxidative stress mediated apoptosis via ROS generation in multiple cancer cell lines[23, 27, 28]. We, therefore, analyzed intracellular ROS generation in HCT116 cells by using 2’,7’–dichlorofluorescin diacetate (DCFH-DA) staining. As depicted in Figs. 3A-B, treatment of HCT116 cells with AP or cisplatin alone led to a slight increase in ROS levels, while the ROS level was drastically increased in the cells treated with AP and cisplatin. Consistently, the above observations were reflected in the fluorescence intensity assay (Figs. 3C-D). To determine whether the ROS signal is involved in apoptosis induced by combined treatment of AP and CDDP, we pre-treated the cancer cells with NAC, a ROS inhibitor, and performed further studies. As shown in Figs. 3E-F, pretreatment of cells with 5 mM NAC prevented apoptosis in HCT116 cells caused by combined administration of AP and CDDP. Collectively, these results demonstrated that increased ROS levels contribute to cytotoxicity of the combined treatment against colon cancer cells.
ER stress contributes to apoptosis in colon cancer cells induced by the combination of AP and CDDP
Elevated ROS levels have been reported to induce apoptosis through several downstream pathways including endoplasmic reticulum (ER) stress-related pathway. Indeed, ER stress acts as an important modulator in various pathologic conditions. Besides, activating ER stress progress usually contributing to cancer cell death by using therapeutic agents[30, 31]. Notably, AP could selectively kill cancer cells by activating ER stress. Here, we sought to investigate whether ER stress contributes to cell apoptosis induced by combined treatment of AP and CDDP. For this purpose, we first examined the expression of ER stress-related proteins in HCT116 cells treated with AP and CDDP. As depicted in Figs. 4A-B, treatment of the cells with AP or CDDP for 3 h led to a slight increase in the expression level of p-EIF2α and ATF4, while the expression of p-EIF2α and ATF4 was dramatically increased in the cells treated with AP and CDDP for 3 h. Similarly, we found that combined treatment of AP and CDDP for 10 h resulted in a marked increase in the expression level of CHOP. We next investigated whether ER stress is involved in the apoptosis induced by the combined treatment. In the experiments, we knocked down the expression of ATF4 and then challenged the cells to AP and CDDP (Figs. 4C-D). As shown in Fig. 4E, silencing ATF4 led to an inhibition in the cell death following the combined treatment. Taken together, these data indicated that ER stress plays a key role in apoptosis of the cancer cells treated with AP and CDDP.
The combined treatment inhibits STAT3 phosphorylation in colon cancer cells
Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that is continuously activated in various types of solid tumors, including colon cancer[32–34]. It has been reported that AP improves chemosensitivity of cancer cells to doxorubicin by inhibiting JAK/STAT3 pathway. We further examined whether combined treatment of AP and CDDP modulates STAT3 activation in HCT116 cells. As shown in Figs. 5A-B, compared with the control, cells treated with AP or CDDP displayed a slightly reduced expression of p-STAT3, while a marked decrease in the expression level of p-STAT3 was detected in cells treated with AP and CDDP. Notably, pretreatment of cells with 5 mM NAC relieved the reduced expression of p-STAT3 caused by the combined treatment (Figs. 5C-D). These data indicated that the combined treatment inhibits STAT3 phosphorylation possibly through ROS generation. Altogether, these findings suggested a unique approach for targeting STAT3 signaling pathway by ROS-inducing agents (AP combined with CDDP).
The combined treatment inhibits xenograft tumor growth of HCT116 cells
To determine in vivo anti-cancer effect of the combined treatment, we performed functional assays on a subcutaneous xenograft tumor model of HCT116 cells. As illustrated in Figs. 6A-C, combined administration of AP and CDDP led to an effective inhibition in the growth of tumor xenograft. Clearly, the combined treatment showed a stronger inhibitory effect on tumor weight and volume. Further studies revealed that the combined treatment significantly increased the level of lipid peroxidation product (MDA), a ROS marker, comparing with single treatment group (Fig. 6D). Moreover, immunohistochemical assays identified a marked decrease in the expression of Ki-67 as well as an increase in the expression of cleaved caspase3 in the xenograft tumor model administered with AP and CDDP (Fig. 6E). Taken together, these results demonstrated that AP synergizes with CDDP in inhibiting the tumor growth in vivo possibly by increasing ROS levels.