Activation of checkpoint kinase 1 by curcumin causes cell cycle arrest and apoptosis in human pancreatic cancer cells

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Although, curcumin has been shown to inhibit the growth of pancreatic cancer cells, the exact mechanism by which it prevents the cell growth is not well understood. In the present investigation, we observed that treatment of BxPC-3 human pancreatic cancer cells with 2.5µM curcumin for 24h resulted in the significant arrest of cells in G2/M phase. Our studies also revealed that curcumin treatment caused extensive apoptosis in this cell line. In immunoblot studies, cells treated with 2.5µM curcumin for 24h showed marked phosphorylation (activation) of H2A.X proteins at Ser-139 and down-regulation of DNA polymerase β, as compared to control. Phosphorylation of H2A.X proteins is an indicator of DNA double strand breaks whereas DNA polymerase β plays role in the repair of DNA strand breaks. Further, curcumin treatment resulted in the phosphorylation of checkpoint kinase 1 (Chk-1) at Ser-280 but not at Ser-296 or Ser-317. Interestingly, activation of Chk-1 at Ser-280 has been shown to be involved in DNA damage. In addition, curcumin treatment also caused phosphorylation of Chk-1 at Ser-345 and Cdc25C at Ser-216, whereas we observed subtle phosphorylation of ATM at Ser-1981. The expression of other G2/M cell cycle regulatory proteins such as cyclin B1, Cdc-2 and Chk-2 however were down-regulated in response to curcumin treatment. In order to confirm the role of Chk-1 in curcumin mediated cell cycle arrest and apoptosis, cells were transfected with Chk-1-siRNA followed by treatment with 2.5µM curcumin for 24h. Activation of Chk-1 at Ser-280/345 and Cdc25C at Ser-216 by curcumin was significantly blocked in the cells transiently transfected with Chk-1-siRNA. Further, the apoptosis-inducing effects of curcumin were also abrogated in these cells. To the best of our knowledge, the results of our present study for the first time indicates the critical role of Chk-1 in curcumin-induced DNA damage leading to G2/M cell cycle arrest and apoptosis in pancreatic cancer cells. [Supported in part by RO1 grant CA 106953 (to S.K.S.) awarded by the National Cancer Institute].