Published online May 7, 2018. doi: 10.3748/wjg.v24.i17.1901
Peer-review started: March 2, 2018
First decision: March 30, 2018
Revised: April 4, 2018
Accepted: April 9, 2018
Article in press: April 9, 2018
Published online: May 7, 2018
Processing time: 66 Days and 3 Hours
Hepatocellular carcinoma (HCC) is a highly prevalent disease worldwide, with poor general prognosis. To develop highly effective natural treatments with limited toxicity for HCC is important. The α-hederin saponin is reported to have antitumor activity. However, the effect of α-hederin on HCC remains to be examined. We evaluated the effect and possible mechanism of α-hederin on HCC cells both in vitro and in vivo.
Developing new, effective and nontoxic chemotherapeutic drugs will contribute to the treatment and prognosis for HCC patients in clinic.
To investigate the antitumor activity of α-hederin in HCC cells and its underlying mechanisms in vitro and in vivo.
Three HCC cells lines (SMMC-7721, HepG-2 and Huh-7 HCC cells) were used to detect the effect of α-hederin on HCC. Cell viability was detected by Cell Counting Kit-8 assay after cells were treated with α-hederin. (BSO) N-acetylcysteine (NAC) and DL-buthionine-S,R-sulfoximine (BSO) were used to interfere with the synthesis of glutathione (GSH) in the SMMC-7721 cells, then, the effects of α-hederin on cell proliferation, cell apoptosis, adenosine triphosphate (ATP) and reactive oxygen species (ROS) and mitochondrial membrane potential were detected. The protein levels of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor (AIF) and cytochrome C (Cyt C) were detected by western blotting. The antitumor efficacy of α-hederin on HCC was also evaluated in nude mice with xenograft tumor. The apoptosis of cancer cells in xenograft tumor were examined by TUNEL staining. In this research, as we used NAC and BSO to interfere with the synthesis of GSH, the mechanism we explored was more persuasive.
The α-hederin treatment inhibited cell growth of the three cell lines in a dose- and time-dependent manner. The IC50 values at 24 h for SMMC-7721, HepG-2 and Huh-7 cells were 13.88, 18.45 and 25.52 μmol/L, respectively, so we used SMMC-7721 cells for the on-going experiments. The results showed that the apoptosis rates in the control, low-dose α-hederin (5 μmol/L), mid-dose α-hederin (10 μmol/L) and high-dose α-hederin (20 μmol/L) groups were 0.90% ± 0.26%, 12% ± 2.05, 21% ± 2.15 and 37% ± 3.8%, respectively. In comparison to the control, after treatment with α-hederin, ROS increased significantly, while the ATP levels decreased. When SMMC-7721 cells were pretreated with BSO (2 mmol/L), compared with the mid-dose α-hederin group, the apoptosis rate increased to 27% ± 3.5% (P < 0.05); what’s more, the increase of ROS and the decrease of ATP were both enhanced. However, NAC pretreatment had a protective effect on SMMC-7721 cells and could alleviate the change of ROS and ATP. The proteins involving in the mitochondria-mediated pathway were detected by western blotting. The results showed α-hederin increased the levels of Bax, cleaved caspase-3 and cleaved caspase-9, and decreased Bcl-2 expression levels. Meanwhile, AIF and Cyt C in cytoplasm were up-regulated, but AIF and Cyt C in mitochondria were down-regulated. Subcutaneous xenografts were successfully constructed in 24 nude mice. After treatment with α-hederin for 3 wk, the weight of xenograft tumor was significantly reduced (P < 0.05). Compared to the control group, TUNEL staining showed a gradual increase in the proportion of apoptotic cells with the increase of α-hederin concentration (P < 0.05). There was no difference between the control mice and α-hederin-treated mic for the hepatic and renal functions. This research indicated that α-hederin could induce HCC cell apoptosis via mitochondria-mediated pathway by depleting GSH and accumulating ROS. But it did not explain how α-hederin changed the expression of GSH and ROS, and the effect of α-hederin on HCC cell invasion was not studied either. In addition, apoptosis involves multiple factors and multiple links, making it necessary to conduct in-depth research to clarify the specific mechanism.
The α-hederin saponin induces apoptosis of HCC cells via the mitochondrial pathway mediated by increased intracellular ROS and may be an effective treatment for human HCC.
It is of great value to discover natural anticancer compounds which have high efficacy and low toxicity in the treatment of HCC. In our study, we show that α-hederin could induce HCC cell apoptosis via mitochondria-mediated pathway by depleting GSH and accumulating ROS, which identifies α-hederin as a potential highly effective natural medicine with limited toxicity for HCC treatment. But some points remain unclear. How does α-hederin change the expression of ATP? The effect of α-hederin on HCC cell migration and invasion was not studied, either. In addition, apoptosis involves multiple factors and multiple links, and it’s necessary to conduct in-depth research to clarify specific mechanism. These results will facilitate the development of treatment for HCC.