Carcinogenesis Advance Access published online on June 23, 2009
Carcinogenesis, doi:10.1093/carcin/bgp157
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Benzyl isothiocyanate mediated generation of reactive oxygen species causes cell cycle arrest and induces apoptosis via activation of MAPK in human pancreatic cancer cells
Department of Biomedical and Pharmaceutical Sciences and Cancer Biology Center, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas, USA
2 To whom requests for reprints should be addressed: Sanjay K. Srivastava, Ph.D. Department of Biomedical Pharmaceutical Sciences, Texas Tech University Health Sciences Center, School of Pharmacy, Suite 1103, 1406 Coulter Drive, Amarillo, Texas. Phone: 806-356-4750, Ext. 224; Fax: 806-356-4770; E-mail: Sanjay.Srivastava{at}TTUHSC.edu
In our previous studies, we have shown that BITC inhibits the growth of human pancreatic cancer cells by inducing apoptosis. In the present study, we demonstrate the activation of all the three MAPK family members (ERK, JNK and P38) in response to BITC treatment. Exposure of Capan-2 cells with varying concentrations of BITC for 24h resulted in the phosphorylation (activation) of ERK at Thr202/Tyr204, JNK at Thr183/Tyr185 and P38 at Thr180/Tyr182, leading to the induction of apoptosis. Similar MAPK activation was also observed in Mia-Paca-2 cells in response to BITC treatment. However, normal human pancreatic ductal epithelial cells did not show the activation of MAPK's and remained unaffected by BITC treatment. To confirm the role of ERK, JNK and P38 in BITC-induced G2/M arrest and apoptosis, Capan-2 cells were pretreated with MAPK specific inhibitors or MAPK8-shRNA prior to BITC treatment. Significant protection from BITC induced G2/M arrest was observed in the cells pretreated with MEK-1 but not JNK or P38 inhibitors. On the other hand, BITC induced apoptosis was almost completely abrogated in the cells pretreated with MEK-1, JNK or P38 inhibitors. Similarly, MAPK8-shRNA also offered almost complete protection against BITC-induced G2/M arrest and apoptosis. Furthermore, we observed that BITC treatment leads to the generation of ROS in Capan-2 and MiaPaca-2 cells, which in part was orchestrated by depletion of reduced glutathione level. Blocking ROS generation with NAC significantly prevented glutathione depletion and activation of ERK and JNK but not P38. Further, NAC or tiron prevented G2/M arrest by blocking G2/M regulatory proteins and completely protected the cells from BITC-induced apoptosis. Taken together our results suggest that BITC mediated G2/M arrest is mediated through ERK activation whereas apoptosis is via ERK, JNK and P38.
Key Words: Benzyl isothiocyanate • ROS • MAPK • G2/M cell cycle arrest • pancreatic cancer
1 Grant support: Supported in part by RO1 grant CA106953 (S.K.S.) awarded by the National Cancer Institute. The funds from Texas Tech University Health Sciences Center, School of Pharmacy (S.K.S.) are also acknowledged.
Received March 24, 2009; revised June 15, 2009; accepted June 16, 2009.