Publishing ID: 2546
Mitogen-Activated Protein Kinase Activation is Required for Induction of MGI 114-Mediated Apoptosis In Human Pancreatic Carcinoma Cells
Weixin Wang, Shealandra Lakhanpal, James W Freeman, Stephen J Waters, John R MacDonald, Alexander R Miller, Univ of Texas Health Sci Ctr at San Antonio, San Antonio, TX; MGI Pharma, Inc, Bloomington, MN.
Pancreatic carcinoma is a malignancy characterized by chemoresistance to conventional agents. We previously observed that MGI 114 (irofulven), a novel analogue of mushroom toxin illudin S, induced caspase-mediated apoptosis and cytotoxicity in pancreatic carcinoma cell lines. One path by which tumor cells may be induced to undergo apoptosis is through the activation of mitogen-activated protein kinases (MAPKs). In an effort to determine the mechanism of apoptotic induction active in this pancreatic carcinoma model, we assessed whether MGI 114 was capable of activating members of the MAPK family. Immunoprecipitation and in vitro kinase assay was performed for JNK 1 (substrate GST-cJun), Erk 1/2 (substrate antibody against phosphorylated Erk 1/2) and p38 (substrate antibody against phosphorylated p38) in the MiaPaCa-2 human pancreatic cancer cell line treated with 5mM MGI 114 for 1-12 hours, alone and in the presence of PD98059, a specific MAPKK inhibitor. Kinase activity was determined, and protein levels were assessed by Western blot analysis. Our data demonstrate sustained JNK 1 and Erk 1/2 activatation by MGI 114 treatment over a 12 hour period. Erk activation is blocked by the addition of PD98059. Further, treatment with this compound abrogates MGI 114-induced caspase-mediated apoptosis, as evidenced by the absence of caspase-8 and -9, as well as poly-ATP ribose polymerase cleavage bands in protein lysates of pancreatic cancer cells. These results indicate that one potential mechanism of MGI 114-induced cytotoxicity and apoptosis may involve the activation of multiple subfamilies of the MAPKs. |
