Anti-proliferation and apoptosis-inducing effects of sodium aescinate on retinoblastoma Y79 cells

Abstract

Aim: To investigate the anti-proliferation and apoptosis-inducing effects of sodium aescinate (SA) on retinoblastoma Y79 cells and its mechanism.

Methods: Y79 cells were cultured at different drug concentrations for different periods of time (24, 48, and 72h). The inhibitory effect of SA on proliferation of Y79 cells was detected by the cell counting kit-8 (CCK-8) assay, and the morphology of Y79 cells in each group was observed under an inverted microscope. An IC50 of 48h was selected for subsequent experiments. After pretreatment with SA for 24 and 48h, cellular DNA distribution and apoptosis were detected by flow cytometry. Real-time qunatitative polymerase chain reaction (RT-qPCR) and Western blot were used to assess changes in related genes (CDK1, CyclinB1, Bax, Bcl-2, caspase-9, caspase-8, and caspase-3).

Results: SA inhibited proliferation and induced apoptosis of Y79 cells in a time-dependent and concentration-dependent manner. Following its intervention in the cell cycle pathway, SA can inhibit the expression of CDK1 and CyclinB1 at the mRNA and protein levels, and block cells in the G2/M phase. In caspase-related apoptotic pathways, up-regulation of Bax and down-regulation of Bcl-2 caused caspase-9 to self-cleave and further activate caspase-3. What's more, the caspase-8-mediated extrinsic apoptosis pathway was activated, and the activated caspase-8 was released into the cytoplasm to activate caspase-3, which as a member of the downstream apoptotic effect group, initiates a caspase-cascade reaction that induces cell apoptosis.

Conclusion: SA inhibits the proliferation of Y79 cells by arresting the cell cycle at the G2/M phase, and induces apoptosis via the caspase-related apoptosis pathway, indicating that SA may have promising potential as a chemotherapeutic drug.

Keywords: cell cycle arrest; extrinsic apoptosis pathway; intrinsic apoptosis pathway; retinoblastoma; sodium aescinate.

Figure 1. Morphology of Y79 cells after treatment with different concentrations of SA for 24, 48, and 72h.

Figure 2. SA inhibits the proliferation of Y79 cells by preventing the cell cycle in G2/M phase

A: Cell cycle distribution in Y79 cells following their incubation in the control and the SA groups for 24h and 48h was assessed by flow cytometry; B, C: Histogram showing cell cycle distribution after 24 and 48h, respectively; D: Expression levels of CDK1 and CyclinB1 in Y79 cells, detected by qRT-PCR assay after treatment with SA (35 µg/mL) for 48h; E: Expression levels of CDK1 and CyclinB1 in Y79 cells, detected by Western blot assay; F: Histogram compares relative protein expression levels of CDK1 and CyclinB1. ^bP<0.01 and ^cP<0.001 vs control.

Figure 3. Intrinsic pathway involved in SA-induced apoptosis in Y79 cells

A: Flow cytometry demonstrates changes over time (24 and 48h) in Y79-cell apoptosis in the control and the SA group; B: Changes in Y79-cell apoptosis rate in the control group and after SA treatment for 24 and 48h; C: The expression levels of Bax, Bcl-2, caspase-3, and caspase-9 in Y79 cells, detected by RT-qPCR assay after treatment with SA (35 µg/mL) for 48h; D: In Western blot assay, the protein expression levels of caspase-3, cleaved-caspase 3, caspase-9, cleaved-caspase-9, Bcl-2, and Bax in Y79 cells; E: Comparison of Bax and Bcl-2 protein expression and Bax/Bcl-2 ratio; F: Comparison of cleaved-caspase-3 and cleaved-caspase-9 protein expression. The expression level of β-actin is considered as one. ^bP<0.01 and ^cP<0.001 vs control.

Figure 4. Extrinsic pathway involved in SA-induced apoptosis in Y79 cells

A: mRNA expression levels of caspase-3 and caspase-8 in Y79 cells after treatment with SA (35 µg/mL) for 48h; B: Protein expression levels of caspase-3, cleaved-caspase-3, caspase-8, and cleaved-caspase-8 in Y79 cells; C: Comparison of cleaved-caspase-3 and cleaved-caspase-8 protein expression. ^cP<0.001 vs control.