Abstract
Autophagy is an important mechanism which regulates the processes of cell growth and death. The biological role of autophagy in regulating and controlling the proliferation of liver cancer cells by bufalin remains unknown. In the present study we investigated the effect of bufalin on autophagy of liver cancer cells. The growth inhibition and autophagy of liver cancer cells were detected using acridine orange fluorescence staining, flow cytometry and transmission electron microscopy. Combined with autophagy inhibitors (3‑MA and CQ), CCK8 staining and western blot analysis were used to detect the effect of bufalin on the proliferation and autophagy‑related protein expression in HCC‑LM3 cells at the indicated time‑points. Results indicated that combined with autophagy inhibitors 3‑MA and CQ, the inhibitive effect of bufalin on the proliferation of HCC‑LM3 cells was significantly enhanced. When combined with autophagy inhibitors 3‑MA or CQ, bufalin significantly reduced the autophagosome and acidic vesicles in HCC‑LM3 cells. When combined with autophagy inhibitors 3‑MA and/or CQ for 12h, bufalin significantly inhibited the expression of LC3‑I and Beclin‑1 in HCC‑LM3 cells, and upregulated the expression of LC3‑II and P62 in HCC‑LM3 cells. When combined with autophagy inhibitors 3‑MA and/or CQ for 24h, bufalin significantly inhibited the LC3‑II expression in HCC‑LM3 cells, and upregulated the LC3‑I, P62 and Beclin‑1 expression in HCC‑LM3 cells. When combined with autophagy inhibitors 3‑MA and/or CQ for 48h, bufalin significantly inhibited the expression of LC3‑II and Beclin‑1 in HCC‑LM3 cells, and upregulated the expression of LC3‑I and P62 in HCC‑LM3 cells. These findings indicated that bufalin induced cell autophagy and inhibited proliferation of liver cancer cells by influencing the expression of autophagy related proteins including LC3‑I, LC3‑II, P62 and Beclin‑1 in liver cancer cells. The autophagic state of liver cancer cells affected the inhibitory effect of bufalin on the proliferation of liver cancer cells.
Highlights
Autophagy is an important mechanism for regulating the process of cell growth and death and is a highly conserved process which can maintain the metabolic balance and preserve stable environmental energy to maintain cell metabolic needs [1]
Compared with the bufalin group, autophagy inhibitors 3‐methyladenine autophagy inhibitor (3‐MA) or Chloroquine autophagy inhibitor (CQ) significantly enhanced the inhibitory effect of bufalin on the growth of hepatocellular carcinoma (HCC)‐LM3 cells
There was no significant difference of the inhibitory effect on the growth of HCC‐LM3 cells between the bufalin + 3‐MA group and the bufalin + CQ group (F=6.58, P>0.05) (Fig. 1)
Summary
Autophagy is an important mechanism for regulating the process of cell growth and death and is a highly conserved process which can maintain the metabolic balance and preserve stable environmental energy to maintain cell metabolic needs [1]. Tumor cells with apoptotic dysfunction can maintain long‐term survival by autophagy which reduces cell necrosis, inflammation and genetic damage. Autophagic abnormality is closely related to the occurrence and development of hepatocellular carcinoma (HCC). Autophagy can inhibit the occurrence of HCC, autophagy occurred in liver cancer cells when they were under hypoxia, drug or toxic chemical damage. The degraded substances in vesicles are digested, hydrolyzed and released into the cytoplasm to be used again and aid liver cancer cells to tolerate hypoxic conditions and resist chemotherapy. The promoting or inhibiting effect of autophagy on the proliferation of tumor cells mainly depends on the cell state and survival environment as well as the stimulating factors [3,4,5]. Bufalin, which is derived from the traditional Chinese medicine named ‘Chan Su’, has strong
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