Grape seed proanthocyanidin extract induces apoptosis of HL-60/ADR cells via the Bax/Bcl-2 caspase-3/9 signaling pathway

Abstract

Background

Our previous study detailed the direct induction of apoptosis by grape seed proanthocyanidin extract (GSPE) in a multidrug resistant human acute myeloid leukemia (AML) HL-60/adriamycin (HL-60/ADR) cell line, although the mechanism of this effect was not detailed. This study aims to elucidate the mechanism underlying GSPE-induced cell apoptosis in HL-60/ADR cells.

Methods

HL-60/ADR cells were studied to evaluate effects of GSPE (0–100 µg/mL); a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to identify the cytotoxic effect of varying GSPE concentrations. Trypan blue staining was used to observe changes in cell viability; flow cytometry assays were used to verify apoptosis. Expression of Bax and Bcl-2 mRNA was analyzed using real-time polymerase chain reaction (PCR). Activity of caspase-3 and caspase-9 was also detected.

Results

Here, GSPE was found to inhibit HL-60/ADR cell growth and induce cell apoptosis in a dose-dependent manner. Real-time PCR findings revealed that GSPE concentrations above 75 µg/mL significantly increase expression of Bax mRNA (P<0.001). GSPE concentrations above 25 µg/mL were found to significantly decrease expression of Bcl-2 mRNA (P<0.01), while concentrations above 50 µg/mL were found to significantly increase caspase-3 activity after 6, 12 and 24 h (P<0.01). However, only 100 µg/mL GSPE was found to significantly increase caspase-9 activity (P<0.001 at 6 and 12 h; P<0.05 at 24 h).

Conclusions

GSPE inhibits the proliferation of HL-60/ADR cells by the induction of apoptosis in a dose-dependent manner via the Bax/Bcl-2 caspase-3/9 signaling pathway.