Effects of matrine on the proliferation and apoptosis of vincristine-resistant retinoblastoma cells

Abstract

Matrine is an active component of Leguminosae plants and is thought to exhibit anti-tumor effects. However, the effects of matrine on drug-resistant cancer have not been fully elucidated. The present study aimed to investigate the effects of matrine on vincristine (VCR)-resistant retinoblastoma (RB) cells and to assess the underlying mechanisms governing this effect. The drug-resistant cell line SO-Rb50/VCR was established by incubation with VCR at increasing concentrations. The effects of matrine on SO-Rb50 and SO-RB50/VCR cell growth and proliferation were evaluated using light microscopy and Cell-Counting Kit-8 assay. In addition, the effects of matrine on cell apoptosis, proliferation and cell cycle staging together with its potential underlying mechanisms were investigated. Matrine inhibited the proliferation of SO-Rb50 and SO-RB50/VCR cells in a concentration-dependent manner (0.2-1.1 mg/ml). However, matrine at the half-maximal inhibitory concentration (IC₅₀) appeared to trigger apoptosis of these cells and had a tendency to arrest the cell cycle at the G0/G1 phase. Matrine treatment also promoted the expression of Bax and reduced the expression of Bcl-2 and cyclin D1 compared with the control. However, matrine was not able to increase the sensitivity of cells to VCR. The results of the present study suggested that matrine has the potential to promote the apoptosis of SO-Rb50/VCR cells and arrest cell cycling, indicating a possible benefit of matrine for the treatment of drug-resistant RB.

Keywords: SO-Rb50; drug resistance; matrine; vincristine-resistant retinoblastoma.

Figure 1

Features of SO-Rb50/VCR cells. (A) IC50 value of VCR in SO-Rb50 was significantly increased in SO-Rb50/VCR cells. (B) Western blot analysis was used to determine the expression levels of MDR1/P-gp protein. The level of MDR1/P-gp was increased in SO-Rb50/VCR cells compared with SO-Rb50 cells. ^*P<0.05 vs. SO-Rb50. IC₅₀, half-maximal inhibitory concentration; MDR1/P-gp, multi-drug resistance protein 1/p-glycoprotein; SO-RB50/VCR, vincristine-resistant SO-RB50 cells; VCR, vincristine.

Figure 2

Matrine inhibits the proliferation of SO-RB50 and SO-RB50/VCR. (A) Proliferation inhibition curves of two RB cell lines treated with different concentrations of matrine for 24 h. Matrine (0.3-1.3 mg/ml; 24 h) exhibited significant suppressive effects on cellular growth of SO-Rb50 and SO-Rb50/VCR cells in a dose-dependent manner. The IC₅₀ values of matrine on SO-Rb50 and SO-Rb50/VCR cells for 24 h were 0.96±0.04 mg/ml and 0.97±0.08 mg/ml, respectively. (B) Cell morphology before and after matrine treatment in SO-RB50/VCR cells. SO-Rb50/VCR cells grew as suspended aggregates in a uniform pattern with large nuclei and small cytoplasms. After treatment with matrine (IC₅₀) for 24 h, the cell mass was reduced, uneven in size (a) and cytoplasmic debris (b) were observed. IC₅₀, half-maximal inhibitory concentration; RB, retinoblastoma; SO-RB50/VCR, vincristine-resistant SO-RB50 cells; VCR, vincristine

Figure 3

Matrine triggers apoptosis in SO-RB50/VCR cells. (A) Representative electron microscope images of cell morphology. The cells became uneven in size and cytoplasmic debris (arrow) and vacuolated cells (Triangle) were observed. (B) Apoptosis was detected using flow cytometry in cells treated with matrine for 0 (control), 12, 24 or 48 h. SO-RB50/VCR, vincristine-resistant SO-RB50 cells.

Figure 4

Matrine arrests the cell cycle at G0/G1 phase. After treatment with matrine for 0, 12, 24 and 48 h, cell cycle was arrested at G0/G1 phase. Arrows indicate cells at the G1 and G2 phase, respectively.

Figure 5

Matrine downregulates Bcl-2 expression and promotes Bax expression in SO-RB50/VCR cells. (A) Representative blots for Bax, Bcl-2 and cyclin D1. (B) Bax expression was promoted after matrine treatment for 0 (control), 12, 24 and 48 h. (C) Bcl-2 expression was reduced after matrine treatment for 0 (control), 12, 24 and 48 h. (D) cyclinD1 expression was reduced after matrine treatment for 0 (control), 12, 24 and 48 h. ^**P<0.01 vs. control.

Figure 6

Matrine does not affect drug resistance of SO-Rb50/VCR cells to VCR. SO-RB50/VCR, vincristine-resistant SO-RB50 cells; VCR, vincristine.