Effects of polyphyllinIon the proliferation and apoptosis of human melanoma cell line A375

Abstract

Objective To investigate effects of polyphylin Ⅰon the proliferation and apoptosis of human melanoma cell line A375, and to explore their mechanisms. Methods Normal human melanocytes isolated from healthy human foreskin were divided into 6 groups to be treated with 0, 1.5, 3.0, 6.0, 9.0, 12.0 mg/L polyphyllinⅠrespectively. A375 melanoma cells were divided into 4 groups, i.e., control group, 1.5-, 3.0-, 6.0- mg/L polyphyllin Ⅰ groups, to be treated with 0, 1.5, 3.0, 6.0 mg/L polyphyllin Ⅰ, respectively. Cell counting kit-8 (CCK8) assay was performed to evaluate the effect of polyphyllinⅠ on the proliferation of normal human melanocytes and A375 cells. Hoechst 33258 fluorescent staining was conducted to observe the morphology of apoptotic cells, flow cytometry to estimate cell cycle phase distribution and apoptosis rate, dichloro-dihydro-fluorescein diacetate (DCFH-DA) fluorescent probe assay to detect the level of reactive oxygen species (ROS) , rhodamine - 123 staining to evaluate changes of mitochondrial membrane potential, spectrophotography to detect the level of ATP in A375 cells, as well as levels of lactic acid and glucose in the culture supernatant of A375 cells, and Western blot analysis to determine the protein expression of Bcl-2, Bcl-2-related X protein (Bax) , cleaved-caspase-3, cyclin D1 and pyruvate kinase isozyme type M2 (PKM2) . Statistical analysis was carried out by using one-way analysis of variance (ANOVA) for comparisons among groups and Student-Newman-Keuls-q (SNK-q) test for multiple comparisons. Results CCK8 assay showed that the treatment with polyphyllin Ⅰat concentrations of 1.5, 3.0, 6.0 mg/L for 48 hours had no effects on the proliferation of normal human melanocytes, but significantly inhibited the proliferation of A375 cells. The survival rate of A375 cells was significantly lower in the 1.5-, 3.0-, 6.0-mg/L polyphyllinⅠgroups than in the control group (P < 0.01) . After the treatment with polyphyllin Ⅰ, distinct apoptotic morphology of A375 cells was observed under fluorescence microscope. Additionally, along with the increase of polyphyllin Ⅰconcentrations (0, 1.5, 3.0, 6.0 mg/L) , there were gradual increasing trends in the apoptosis rate of A375 cells (4.25% ± 1.27%, 10.03% ± 1.49%, 36.62% ± 1.97%, 44.11% ± 2.47% respectively, F = 665.7, P < 0.01) , the percentage of A375 cells at G0/G1 phase (54.13% ± 2.57%, 67.35% ± 3.79%, 74.39% ± 3.29%, 82.29% ± 3.99% respectively, F= 71.81, P < 0.01) , the level of ROS in A375 cells (P < 0.01) , the level of glucose in the culture supernatant (P < 0.01) , and the protein expression of Bax and cleaved-caspase-3 (both P < 0.01) , while gradual decreasing trends were found in the levels of mitochondrial membrane potential and ATP in A375 cells (both P < 0.01) , the level of lactic acid in the culture supernatant (P < 0.01) , and the protein expression of Cyclin D1, Bcl-2 and PKM2 (all P < 0.01) . Conclusion PolyphyllinⅠcan effectively induce A375 cell apoptosis by promoting the production of ROS in A375 cells and decreasing the mitochondrial membrane potential, and arrest A375 cells at G0/G1 phase by inhibiting the expression of PKM2 and Cyclin D1. Key words: Melanoma; Cell line, tumor; Apoptosis; Reactive oxygen species; Cell proliferation; Polyphyllin Ⅰ