Department of Pathology, The Third Hospital, Affiliated to The Second Military Medical University, Shanghai 201805, P.R. China.
Department of General Surgery, The Third Hospital, Affiliated to The Second Military Medical University, Shanghai 201805, P.R. China.
Oncol Rep. 2018 Jun;39(6):2931-2941. doi: 10.3892/or.2018.6365. Epub 2018 Apr 12.
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 12 h, 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 24 h, 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 48 h, 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.
自噬是调节细胞生长和死亡过程的重要机制。蟾毒灵通过调控自噬来影响肝癌细胞增殖的生物学作用尚不清楚。本研究旨在探讨蟾毒灵对肝癌细胞自噬的影响。采用吖啶橙荧光染色、流式细胞术和透射电镜观察蟾毒灵对肝癌细胞自噬的影响。结合自噬抑制剂（3-MA 和 CQ），采用 CCK8 染色和 Western blot 分析检测蟾毒灵在不同时间点对 HCC-LM3 细胞增殖及自噬相关蛋白表达的影响。结果表明，与自噬抑制剂 3-MA 和 CQ 联合应用后，蟾毒灵对 HCC-LM3 细胞增殖的抑制作用明显增强。与自噬抑制剂 3-MA 或 CQ 联合应用后，蟾毒灵明显减少 HCC-LM3 细胞中的自噬体和酸性囊泡。与自噬抑制剂 3-MA 和/或 CQ 联合应用 12 h 后，蟾毒灵明显抑制 HCC-LM3 细胞中 LC3-I 和 Beclin-1 的表达，上调 HCC-LM3 细胞中 LC3-II 和 P62 的表达。与自噬抑制剂 3-MA 和/或 CQ 联合应用 24 h 后，蟾毒灵明显抑制 HCC-LM3 细胞中 LC3-II 的表达，上调 HCC-LM3 细胞中 LC3-I、P62 和 Beclin-1 的表达。与自噬抑制剂 3-MA 和/或 CQ 联合应用 48 h 后，蟾毒灵明显抑制 HCC-LM3 细胞中 LC3-II 和 Beclin-1 的表达，上调 HCC-LM3 细胞中 LC3-I 和 P62 的表达。这些结果表明，蟾毒灵通过影响肝癌细胞自噬相关蛋白 LC3-I、LC3-II、P62 和 Beclin-1 的表达诱导细胞自噬，并抑制肝癌细胞增殖。肝癌细胞的自噬状态影响蟾毒灵对肝癌细胞增殖的抑制作用。
