Aim: To explore the mechanisms of effects of oil A on apoptosis of human pancreatic cancer cells.
Methods: Cellular DNA content was analyzed by flow cytometry. Western blotting was used for caspase-3 and PARP, caspase-7, caspase-9, cytochrome c, Bcl-2, Bax, Mcl-1, cyclinA, cyclin B1, cyclin D1, cyclin E, CDK2, CDK4, CDK6, P21, P27, GADD45, GADD153.
Results: The caspase-3, caspase-7, and caspase-9 activities were significantly increased as well as the cleavage of caspase-3, downstream substrate poly-ADP ribose polymerase (PARP) was induced. The amount of cytochrome c in the cytosolic fraction was increased, while the amount of cytochrome c in the mitochondrial fraction was decreased after oil A treatment. The anti-apoptosis proteins Bcl-2 and Mcl-1 were decreased in parallel and Bax increased, indicating that Bcl-2 family proteins-mitochondria-caspase cascade was responsible for oil-induced apoptosis. The proportion of cells in the G0/G1 decreased in MiaPaCa-2 and AsPC-1 cells after the treatment of oil A for 24 hours. The number of cells in S phase was increased in two cancer cell lines at 24 hours. Therefore, cells were significantly accumulated in G2/M phase. The cells with a sub-G0/G1 DNA content, a hallmark of apoptosis, were seen at 24 hours both in MiaPaCa-2 and AsPC-1 cells following exposure to oil A. The expression of cyclin A and cyclin B1 was slightly decreased and cyclin D1 levels were markedly lowered in MiaPaCa-2 cells. The expression of cyclin A and cyclin B1 was markedly decreased and cyclin D1 levels were slightly lowered in AsPC-1 cells, while cyclin E was not affected and the levels of CDK2, CDK4, and CDK6 were unchanged in MiaPaCa-2 and AsPC-1 cells. In response to oil A, P21 expression was increased, but P27 expression was not affected. The expression of both GADD45 and GADD153 was increased in two cell lines following oil A treatment.
Conclusion: Oil A induces apoptosis of pancreatic cancer cells via activating caspase cascade, modifying cell cycle progress and changing cell cycle-regulating proteins and GADD expression.