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

AIM: To study the effect of red oil A5 on pancreatic cancer cells and its possible mechanisms. METHODS: 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 3H-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 ^3H-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. Fiowcytometric analysis of cellular DNA content in the control and red oil A5 treated AsPC-1, MiaPaCa-2 and $2013 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 $2013 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 polyADP ribose polymerase (PARP) protein in AsPC-1, MiaPaCa2 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. RESULTS: Red oil A5 caused dose- and time-dependent inhibrdon 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 assayFurthermore, Western blotting analysis indicated that cytochrome c was released from mitochondda to cytosol during apoptosis,and caspase-3 was activated following red oil A5 treatment which was measured by procaspase-3 cleavage and PARP cleavage. CONCLUSION: These findings show that red oil A5 has potent anti-proliferative effects on human pancreatic cancer cells with induction of apoptosis in vitro.

AIM:To study the effect of red oil A5 on pancreatic cancer cells and its possible mechanisms. METHODS: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 3H-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 ~3H-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. RESULTS: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,Westem 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. CONCLUSION:These findings show that red oil A5 has potent anti-proliferative effects on human pancreatic cancer cells with induction of apoptosis in vitro.