Oxocrebanine inhibits the proliferation of hepatocellular carcinoma cells by promoting apoptosis and autophagy

Abstract

BACKGROUND

Finding active lead anti-hepatocellular carcinoma compounds from traditional Chinese medicine has important research value.

AIM

To assess the detailed mechanism of oxocrebanine, a compound separated from the traditional Chinese medicinal plant Stephania hainanensis H.S. Lo et Y. Tsoong, and to evaluate its inhibition of the proliferation of human hepatocellular carcinoma cells via apoptosis and autophagy.

METHODS

MTT, BrdU labeling, and colony formation assays were used to assess the inhibitory effect of oxocrebanine on the growth and proliferation of human hepatocellular carcinoma Hep3B2.1-7 cells. Flow cytometry was used to detect the effect of oxocrebanine on the apoptosis of Hep3B2.1-7 cells. Western blotting was used to assess the expression of apoptosis-related proteins in Hep3B2.1-7 cells. The aforementioned methods were also used to evaluate the effects of oxocrebanine on cell proliferation, autophagy markers, and autophagy-related protein expression levels after adding autophagy inhibitor 3-mA. Furthermore, to verify the anti-hepatocellular carcinoma effect of oxocrebanine in vivo, a nude mouse model was used to investigate the inhibitory effect of oxocrebanine treatment and its mechanism. Apoptosis was detected using a TUNEL assay and the expression of microtubule-associated protein 1 LC3 in tumor specimens was assessed using immunohistochemistry.

RESULTS

Oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells, whilst upregulating the protein expression of cleaved caspase-3, downregulating poly(ADP-ribose) polymerase 1 protein expression, increasing the levels of Bax and Bcl-2 antagonist/killer 1 protein expression, and decreasing the levels of Bcl-2 and myeloid cell leukemia 1 protein expression, which could promote apoptosis in Hep3B2.1-7 cells. Oxocrebanine promoted the transformation of LC3-I to LC3-II in Hep3B2.1-7 cells, suggesting the occurrence of autophagy, whilst the autophagy inhibitor 3-MA could reverse this process. Oxocrebanine was also shown to reduce the phosphorylation levels of the eukaryotic translation initiation factor 4EBP1 and ribosomal protein S6 kinase B1 (P70S6K), two downstream effector molecules in the PI3K/Akt/mTOR pathway, inducing autophagy in Hep3B2.1-7 cells. Moreover, the tumor-bearing nude mouse experiment indicated that oxocrebanine effectively inhibited the growth of Hep3B2.1-7 cells in vivo. The results of the TUNEL assay and immunohistochemistry also revealed that oxocrebanine induced apoptosis in vivo and increased the expression level of LC3, an autophagy marker.

CONCLUSION

Oxocrebanine can inhibit the proliferation of human hepatocellular carcinoma cells by promoting apoptosis and inducing autophagy in vitro and in vivo.

Keywords: Stephania hainanensis H.S. Lo et Y. Tsoong; Oxocrebanine; Hep3B2.1-7 cells; Apoptosis; Autophagy

Core Tip: The present study revealed that oxocrebanine can inhibit the proliferation of hepatocellular carcinoma cells by promoting apoptosis and inducing autophagy both in vitro and in vivo. Oxocrebanine can induce apoptosis in Hep3b2.1-7 cells by upregulating cleaved caspase-3, Bax, and Bak protein expression levels and downregulating PARP1, Bcl-2, and Mcl-1 protein expression levels. The inhibition of Hep3b2.1-7 cell proliferation by oxocrebanine may be related to the induction of protective autophagy. The results of the TUNEL assay and immunohistochemistry also revealed that oxocrebanine induced apoptosis in vivo and increased the expression level of LC3, an autophagy marker.