The molecular mechanisms of apoptosis induction by RY10-4 in HepG2 cells.

<p>(A) HepG2 cells were treated with indicated concentrations of RY10-4, and the expression of p-STAT3 and p21 was analyzed by Western blot. (B) HepG2 cells were treated with RY10-4 with or without IL-6 stimulation; expression of p-STAT3 and cyclin E was analyzed by Western blot. (C) HepG2 cells were treated with RY10-4 in the absence or presence of NAC pretreatment. The expression of p-STAT3 and p53 was analyzed by Western blot.</p

The anti-proliferative effects of RY10-4 on other hepatocellular carcinoma cells.

<p>(A) Hep3B or HuH-7 cells were treated with series concentrations of RY10-4, and cell growth inhibition ratio was determined by SRB assay. (B) Hep3B cells were treated with RY10-4, and the expressions of apoptosis-related proteins p53, Bcl-2 and Bax were analyzed by Western blot.</p

RY10-4 Inhibits the Proliferation of Human Hepatocellular Cancer HepG2 Cells by Inducing Apoptosis <i>In Vitro</i> and <i>In Vivo</i>

<div><p>This study aimed to investigate the anti-tumor activity of RY10-4, a small molecular that was designed and synthesized based on the structure of protoapigenone. A previous screening study showed that RY10-4 possessed anti-proliferative effects against HepG2 human hepatocellular carcinoma cells. However, the full range of RY10-4 anti-cancer effects on liver tumors and the underlying mechanisms have not been identified. Herein, employing flow cytometry, and Western blot analysis, we demonstrate that RY10-4 can induce cell cycle arrest, intracellular reactive oxygen species (ROS) production and apoptosis in HepG2 cells. In HepG2 cell xenograft tumor model, RY10-4 significantly inhibited the growth of tumors and induced apoptosis in tumor cells, with little side effects. Moreover, RY10-4 caused the suppression of STAT3 activation, which may be involved the apoptosis induction. In addition, RY10-4 inhibited the proliferation of Hep3B and HuH-7 human hepatocellular carcinoma cells in a concentration-dependent manner. Taken together, our results suggest that RY10-4 has a great potential to develop as chemotherapeutic agent for liver cancer.</p></div

The effect of RY10-4 on cell cycle distribution of HepG2 cells.

<p>(A) Cells were treated with 0, 0.9, 1.8, and 2.7μM RY10-4 for 24 h, and the DNA content was analyzed using propidium iodide staining and flow cytometry. (B) Cell cycle distributions, presented as mean ± SD of three independent experiments. (C) After the treatment with RY10-4, expression of cell cycle-related proteins cyclin E and CDK2 was analyzed by Western blot.</p

RY10-4 induced apoptosis in HepG2 cells.

<p>(A) Cells were treated with indicated concentrations of RY10-4 for 24 h, and the nuclei were stained by Hoechst 33342. Arrows indicate condensed and fragmented nuclei. (B) Flow cytometry assay to detect apoptosis in HepG2 cells using Annexin V/PI staining. Representative flow cytometry profiles are shown. (C) The percentage of apoptotic cells, presented as mean ± SD of three independent experiments. (D) Expression of apoptosis-related proteins was analyzed by Western blot in HepG2 cells untreated or treated with RY10-4.</p

The effect of RY10-4 on intracellular ROS production.

<p>(A) HepG2 cells were treated with indicated concentrations of RY10-4, and the intracellular ROS production was detected as described in Methods. (B) The intracellular ROS levels (% of control), presented as mean ± SD of three independent experiments. (C) HepG2 cells were treated with RY10-4 in the absence or presence of NAC pretreatment; the cell viability was determined by SRB assay.</p