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AIM:To study the effect of red oil A5 on pancreatic cancercells and its possible mechanisms.METHODS:Effect of different concentrations of red oil A5on proliferation of three pancreatic cancer cell lines,AsPC-1,MiaPaCa-2 and S2013,was measured by 3H-methyl thymidineincorporation.Time-dependent effects of 1:32 000 red oil A5on proliferation of three pancreatic cancer cell lines,werealso measured by ~3H-methyl thymidine incorporation,andTime-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 controland red oil A5 treated AsPC-1,MiaPaCa-2 and S2013 cells,were stained with propidium iodide.TUNEL assay of red oilA5-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 000red oil A5 was performed.Proteins in cytosolic fraction andin mitochondria fraction were extracted.Proteins extractedfrom each sample were electrophoresed on SDS-PAGE gelsand then were transferred to nitrocellulose membranes.Cytochrome c was identified using a monoclonal cytochromec antibody.Western blotting of the caspase-3 protein inAsPC-1,MiaPaCa-2 and S2013 cells treated with 1:32 000red oil A5 for 24 hours was carried out.Proteins in wholecellular lysates were electrophoresed on SDS-PAGE gels andthen transferred to nitrocellulose membranes.Caspase-3 wasidentified 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 24hours was performed.Proteins in whole cellular lysates wereseparated by electrophoresis on SDS-PAGE gels and thentransferred to nitrocellulose membranes.PARP was identifiedby using a monoclonal antibody.RESULTS:Red oil A5 caused dose-and time-dependentinhibition of pancreatic cancer cell proliferation.Propidium iodideDNA staining showed an increase of the sub-G0/G1 cellpopulation.The DNA fragmentation induced by red oil A5 inthese three cell lines was confirmed by the TUNEL assay.Furthermore,Westem blotting analysis indicated that cytochromec was released from mitochondria to cytosol during apoptosis,and caspase-3 was activated following red oil A5 treatment whichwas measured by procaspase-3 cleavage and PARP cleavage. CONCLUSION:These findings show that red oil A5 haspotent anti-proliferative effects on human pancreatic cancercells 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 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 A5on 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 controland 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 for 24 hours with 1:32 000red oil A5 was performed. Proteins in cytosolic fraction and in mitochondria fraction were extracted. Proteins extrac Cytochrome c was identified using a monoclonal cytochromec antibody. Western blotting of the caspase-3 protein in AsPC-1, MiaPaCa-2 and S2013 cells treated with 1:32 000red oil A5 for 24 hours was carried out. Proteins in wholecellular lysates were electrophoresed on SDS-PAGE gels and then transferred to nitrocellulose membranes. Caspase-3 wasidentified 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 24hours was performed. Proteins in whole cellular lysates were separated by electrophoresis on SDS-PAGE gels and the ntransferred 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 cellpopulation.The DNA fragmentation induced by red oil A5 inthese three cell lines was confirmed by the TUNEL assay.Furthermore, Westem blotting analysis indicated that cytochromec was released from mitochondria to cytosol during apoptosis, and caspase-3 was activated following red oil A5 treatment whichwas measured by procaspase-3 cleavage and PARP cleavage. CONCLUSION: These findings show that red oil A5 haspotent anti-proliferative effects on human pancreatic cancer cells with induction of apoptosis in vitro.