Red oil A5 inhibits proliferation and induces apoptosis in pancreatic cancer cells.

Abstract

To study the effect of red oil A5 on pancreatic cancer cells and its possible mechanisms. Effect of different concentrations of red oil A5 on proliferation of three pancreatic cancer cell lines, AsPC-1, MiaPaCa-2 and S2013, was measured by (3)H-methyl thymidine incorporation. Time-dependent effects of 1:32 000 red oil A5 on proliferation of three pancreatic cancer cell lines, were also measured by (3)H-methyl thymidine incorporation, and Time-course effects of 1:32 000 red oil A5 on cell number. The cells were counted by Z1-Coulter Counter. Flow-cytometric analysis of cellular DNA content in the control and red oil A5 treated AsPC-1, MiaPaCa-2 and S2013 cells, were stained with propidium iodide. TUNEL assay of red oil A5-induced pancreatic cancer cell apoptosis was performed. Western blotting of the cytochrome c protein in AsPC-1, MiaPaCa-2 and S2013 cells treated 24 hours with 1:32 000 red oil A5 was performed. Proteins in cytosolic fraction and in mitochondria fraction were extracted. Proteins extracted from each sample were electrophoresed on SDS-PAGE gels and then were transferred to nitrocellulose membranes. Cytochrome c was identified using a monoclonal cytochrome c antibody. Western blotting of the caspase-3 protein in AsPC-1, MiaPaCa-2 and S2013 cells treated with 1:32 000 red oil A5 for 24 hours was carried out. Proteins in whole cellular lysates were electrophoresed on SDS-PAGE gels and then transferred to nitrocellulose membranes. Caspase-3 was identified using a specific antibody. Western blotting of poly-ADP ribose polymerase (PARP) protein in AsPC-1, MiaPaCa-2 and S2013 cells treated with 1:32 000 red oil A5 for 24 hours was performed. Proteins in whole cellular lysates were separated by electrophoresis on SDS-PAGE gels and then transferred to nitrocellulose membranes. PARP was identified by using a monoclonal antibody. Red oil A5 caused dose- and time-dependent inhibition of pancreatic cancer cell proliferation. Propidium iodide DNA staining showed an increase of the sub-G0/G1 cell population. The DNA fragmentation induced by red oil A5 in these three cell lines was confirmed by the TUNEL assay. Furthermore, Western blotting analysis indicated that cytochrome c was released from mitochondria to cytosol during apoptosis, and caspase-3 was activated following red oil A5 treatment which was measured by procaspase-3 cleavage and PARP cleavage. These findings show that red oil A5 has potent anti-proliferative effects on human pancreatic cancer cells with induction of apoptosis in vitro.