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 .