Effect of hyperthermia combined with gemcitabine on apoptotic cell death in cultured human pancreatic cancer cell lines.
It is reported that NF-kappaB is activated by chemotherapy in some cancer cell lines and NF-kappaB activation is one of the mechanisms by which tumors are induced to become resistant to chemotherapy. We reported that heat-treatment-induced heat shock protein 70 (Hsp70) could inhibit I-kappa-B kinase, resulting in the inhibition of NF-kappaB activation. Therefore, we speculated that activated NF-kappaB in a pancreatic cell line might be inhibited by heat treatment, resulting in the enhancement of gemcitabine-induced cytotoxicity. We used the human pancreatic carcinoma cell lines AsPC-1 and MIAPaCa-2. Both cell lines were treated with various concentrations (0, 5, 10, 20, and 30 microM) of gemcitabine for 24 h. Heat treatment (43 degrees C, 1 h) was performed at various times relative to gemcitabine treatment. The effect of gemcitabine and heat treatment on cell survival was determined by WST-8 assay. The status of NF-kappaB in carcinoma cells exposed to gemcitabine was investigated by electrophoretic mobility shift assay and immunocytochemistry. We analyzed apoptosis and necrosis in AsPC-1 and MIAPaCa-2 cells by flow cytometry. Furthermore, the levels of Hsp70, cyclin D1, caspase-3, and vascular endothelial growth factor in each treatment group were detected by western blotting. (1) Significant cytotoxicity was observed with gemcitabine. (2) Gemcitabine activated NF-kappaB binding activity in both cell lines. (3) Heat treatment inhibited the gemcitabine-induced activation of NF-kappaB. (4) Heat treatment enhanced the cytotoxicity of gemcitabine, especially when heat treatment was performed 24 h before gemcitabine was given. (5) The levels of Hsp70 were increased by heat treatment. Gemcitabine did not affect the protein level of Hsp70. The levels of pro-caspase-3 were decreased by heat treatment combined with gemcitabine. Heat treatment inhibited gemcitabine-induced activation of NF-kappaB, resulting in the enhancement of the cytotoxicity of gemcitabine.