Ziyu II induces apoptosis in CRC cells both in vitro and in vivo
Apoptosis is a major form of cell death induced by chemotherapy drugs and has been widely documented. Previous studies suggested that Ziyu II exerted antitumor activity by inducing apoptosis in human breast cancer and gastric cancer cells [20,22]. To get more insights into the mechanism of Ziyu II-induced cell death, we detected whether Ziyu II induced apoptosis in CRC cells. Firstly, Ziyu II treatment resulted in increased levels of cleaved-caspase3, a prototypical caspase that becomes activated during apoptosis [23].and cleaved-PARP and decreased expression of anti-apoptotic protein Bcl2 (Fig. S2A). Consistently, the flow cytometry analysis exerted remarkable apoptotic effect in Ziyu II treated CRC cells (Fig. 2A). To further validate Ziyu II induced apoptosis, CRC cells were treated with Ziyu II combination with the apoptosis inhibitor-zVAD. As shown in Fig. S2B, combinational treatment resulted in reduced levels of cleaved-PARP. Consistently, the pro-apoptotic effect of Ziyu II was partially rescued under the combinational treatment, as evidenced by MTT assay and colony formation (Fig. 2B-D). As the accumulation of reactive oxygen species (ROS) plays a key role in apoptosis induction, we detected whether Ziyu II treatment promoted ROS accumulation. The flow cytometry analysis showed that Ziyu II markedly increased the level of ROS in a dose-dependent manner in CRC cells (Fig. 2E). What’s more, MTT assay showed that N-acetylcysteine (NAC, a ROS scavenger) significantly reversed Ziyu II-induced growth inhibition (Fig. 2F). In brief, these results demonstrate the pro-apoptotic effect of Ziyu II in CRC cells in vitro .
To evaluate the pro-apoptotic effect of Ziyu II on CRC cells in vivo , xenografts were stained for cleaved-caspase3. As shown in Fig. S2C and D, Ziyu II-treated xenografts displayed stronger cleaved-caspase3 staining than that of placebo-treated xenografts. In summary, these data indicate that apoptosis is involved in Ziyu II against CRC cells both in vitro and in vivo .