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19 May 2020 : Laboratory Research  

Effect of Oleanolic Acid on Apoptosis and Autophagy of SMMC-7721 Hepatoma Cells

Weipeng Zhou1ABCE, Xianjun Zeng2BE, Xiaoping Wu2ACE*

DOI: 10.12659/MSM.921606

Med Sci Monit 2020; 26:e921606

Abstract

BACKGROUND: Liver cancer is a common cancer with high morbidity and mortality. Due to the large toxic side effects of chemotherapeutic drugs and the overexpression of multidrug resistance genes in liver cancer, no effective chemotherapeutic drug has yet been found. Therefore, the search for a highly effective, low-toxic, and safe natural anticancer therapy is a hot issue.

MATERIAL AND METHODS: SMMC-7721 cells (a hepatocellular carcinoma cell line) were treated with different concentrations of oleanolic acid (OA) plus autophagy inhibitor 3-methyladenine (3-MA) (3-MA+OA) or chloroquine (CQ) plus OA (CQ+OA). We used MTT and Hoechst 33258 staining methods to determine the proliferation and apoptotic effect of OA on cells. Flow cytometry was used to detect apoptosis. Mitochondrial function was assessed by measuring mitochondrial membrane potential and adenosine triphosphate (ATP) concentration. To evaluate the ability of OA on apoptosis and autophagy mechanisms on SMMC 7721 cells, the related protein expression for apoptosis, autophagy, and the autophagic pathway were detected and analyzed by western blot.

RESULTS: OA can inhibit and induce apoptosis of SMMC-7721 in a dose-dependent manner. Compared with the control group, OA significantly reduced the intracellular mitochondrial membrane potential, and the intracellular ATP concentration was also significantly reduced. Moreover, OA reduced the expression of p-Akt and p-mTOR. The expression of p62 was decreased, and LC3-II and Beclin-1 protein expression levels increased. After inhibiting autophagy with 3-MA or CQ, compared with OA alone, cell mitochondrial membrane potential and ATP concentration were significantly reduced, cell p62 expression was reduced, and LC3-II expression was increased, apoptosis-related protein Bax protein was increased, and Bcl-2 protein was decreased, which suggested that 3-MA or CQ treatment increased OA-induced apoptosis of SMMC-7721 cells. This suggested that OA activated autophagy of SMMC-7721 cells in a protective autophagic manner.

CONCLUSIONS: The study findings suggest that OA combined with autophagy inhibitor 3-MA can better exert its anticancer effect.

Keywords: Autophagy, Carcinoma, Hepatocellular, Oleanolic Acid, Adenine, Adenosine Triphosphate, Apoptosis, Chloroquine, Liver Neoplasms, Membrane Potential, Mitochondrial

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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750