Polyphyllin VI induces apoptosis and autophagy in human osteosarcoma cells by modulation of ROS/JNK activation

Abstract

Purpose

Polyphyllin VI, a main active saponin isolated from traditional medicinal plant Paris polyphylla, has exhibited antitumor activities in several cancer cell lines. In the present study, we investigated the antitumor effect of Polyphyllin VI against human osteosarcoma cells (U2OS) and the underlying molecular mechanisms.

Methods

The U2OS cell lines were used to determine the antiproliferative effect of Polyphyllin VI by CCK8 assay. Cell cycle was analyzed by flow cytometry. The Polyphyllin VI-induced apoptosis was determined by Annexin V-APC/7-AAD apoptosis detection kit and JC-1 staining. Meanwhile, the autophagy was determined by acridine orange staining. The apoptosis and autophagy-related proteins were monitored by Western blot assay. Subsequently, intracellular hydrogen peroxide (H₂O₂) and the activation of ROS/JNK pathway were detected.

Results

Polyphyllin VI could potently inhibit cell proliferation by causing G2/M phase arrest. Polyphyllin VI induced mitochondria-mediated apoptosis with the upregulation of proapoptotic proteins Bax and poly ADP-ribose polymerase, and downregulation of antiapoptotic protein Bcl-2 in U2OS cells. Concomitantly, Polyphyllin VI provoked autophagy with the upregulation of critical Atg proteins and accumulation of LC3B-II. Intracellular H₂O₂ production was triggered upon exposure to Polyphyllin VI, which could be blocked by ROS scavenger. Polyphyllin VI dramatically promoted JNK phosphorylation, whereas it decreased the levels of phospho-p38 and ERK.

Conclusion

Our results reveal that Polyphyllin VI may effectively induce apoptosis and autophagy to suppress cell growth via ROS/JNK activation in U2OS cells, suggesting that Polyphyllin VI is a potential drug candidate for the treatment of osteosarcomas.

Keywords:

• Polyphyllin VI
• osteosarcoma
• apoptosis
• autophagy
• JNK activation

Acknowledgments

The authors gratefully acknowledge the financial supports by the Program of the National Natural Science Foundation of China (grant numbers 81903874, 81603287 and 81703331), as well as the Program of Natural Science Foundation of Guangxi Province of China (grant number 2017GXNSFAA198077) and Key Research Projects of Henan Higher Education Institutions(20B360018, 20A350010).

Disclosure

The authors report no conflicts of interest in this work.

Supplementary materials

Figure S1 Polyphyllin VI induces G2/M cell cycle arrest. (A) The alterations in cell cycle distribution of U2OS cells were determined by flow cytometry after treatment with control and Polyphyllin VI (2.5, 5, and 7.5 μM) for 24 h. (B) Ratios of cells in different cell cycle phases in U2OS cells. The percentage of cells in each phase is shown as mean ± SD from three independent experiments. *P<0.05 and **P<0.01, significantly different compared with control.

Figure S2 U2OS cells were incubated with control and Polyphyllin VI (2.5, 5, and 7.5 μM) for 24 h. Cell lysates were prepared and analyzed by Western blotting for Caspase-3 and Caspase-7. Results are presented as the mean ± SD from three independent experiments. *P<0.05 and **P<0.01, significantly different compared with control.