In vitro effects of tanshinone II A on autophagy of A375 melanoma cells and related signaling pathway

Abstract

Objective To investigate in vitro effects of tanshinone ⅡA on the autophagy of A375 melanoma cells and related signaling pathway. Methods Some cultured A375 cells were divided into 5 groups to be treated with tanshinone ⅡA at concentrations of 0.5, 1, 2 and 4 mg/L, and DMEM containing 0.1% dimethyl sulfoxide (DMSO), respectively, for 24, 48, 72 hours. Methyl thiazol tetrazolium (MTT) assay was performed to estimate the proliferative activity of A375 cells. Some cultured A375 cells were divided into 4 groups to be treated with 1, 2 and 4 mg/L tanshinone ⅡA (1-, 2- and 4-mg/L tanshinone group), and DMEM containing 0.1% DMSO (control group), respectively, for 48 hours. Then, flow cytometry was conducted to count autophagosome-positive cells, and Western blot analysis to determine protein expression of autophagy-associated proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3) -Ⅱ, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR) and p70 ribosomal protein S6 kinase 1 (p70S6K1). Results MTT assay showed that 24-, 48-, 72-hour treatments with tanshinone Ⅱ A at concentrations of 0.5, 1, 2 and 4 mg/L all could inhibit the proliferative activity of A375 cells, and the inhibitory effects increased in a dose- and time-dependent manner (F = 2 564.12, 1 235.25, both P < 0.05). The percentage of autophagosome-positive cells and protein expression of Beclin-1 and LC3-Ⅱ increased gradually and significantly in the 1-, 2- and 4-mg/L tanshinone groups (autophagosome-positive cells: 6.91% ± 0.35%, 13.11% ± 0.73%, 25.51% ± 0.83%, respectively; Beclin-1: 0.33 ± 0.01, 0.53 ± 0.04, 0.63 ± 0.02, respectively; LC3-Ⅱ: 0.41 ± 0.01, 0.52 ± 0.02, 0.64 ± 0.02, respectively), after 48-hour treatment, which were significantly different between the tanshinone groups (all P < 0.05), and higher in the tanshinone groups than in the control group (0.41% ± 0.02%; 0.09 ± 0.02; 0.21 ± 0.01, all P < 0.05). However, the protein expression of PI3K, phosphorylated Akt (p-Akt), p-mTOR and p-p70S6K1 in the PI3K-Akt-mTOR-p70S6K1 signaling pathway decreased gradually and significantly with the increase in tanshinone concentrations after 48-hour treatment, and were significantly lower in all the tanshinone groups than in the control group (all P < 0.05). Conclusion Tanshinone Ⅱ A can promote the auophagy of A375 cells, likely by blocking the PI3K-Akt-mtTOR-p70S6K1 signaling pathway. Key words: Melanoma; Tanshinone; Autophagy; A375 cells