Synergistic Effect of Theophylline and Cisplatin on Inhibition of Cell Growth and Induction of Apoptosis in Human Epithelial Ovarian Cancer Cells

Kang, Kyoung Hwa; Huh, Chu Yeop; Suh, Byung Sun

Abstract

OBJECTIVE
Cisplatin is a chemotherapeutic drug which is widely used for cancer therapy including ovarian cancer. The purpose of this study is to elucidate synergistic effect of Cispaltin and Theophylline on the apoptosis of OC-109.
METHODS
Human ovarian cancer cell lines of epithelial origin (OC-109, OC-238) were used for experiments. Antibody of Bcl-2 gene was purchased by Santa Cruz Biotechnology, Inc. (Heidelberg, Germany) and p53 antibody and Mdm-2 antibody were generous gift from M. Oren at the Weizmann Institute (Rehovot, Israel). Apoptosis was measured by FACS analysis and TUNEL method. Western blot for protein expression and biochemical analysis for measuring protein was done by Bradford method.
RESULTS
The inhibitory effect of Cisplatin on the cell growth began at the concentration of 0.3 microM and increased to 9 microM, while Gemzar began to inhibit cell growth at the concentration of 0.0013 microM and increased to 0.12 microM. Theophylline did not show significant inhibitory effect until the concentration of 80 microgram/mL, but the cell growth was inhibited significantly at the concentration of 320 microgram/mL. Synergistic effect of Cisplatin and Theophylline was observed at the concentration of 1 microM and 80 microgram/mL, respectively. Apoptosis was measured by the shifting to sub-G1 phase of cell cycle by FACS analysis. Synergistic effect of Cispatin, Gemzar and Theophylline was observed. TUNEL assay also showed clear synergistic effect of Cisplatin and Theophylline. Bcl-2 gene product which inhibits the apoptosis was down-regulated after Cisplatin, Cisplatin and Theophylline co-treatment. Mdm-2 gene product was up-regulated while p53 protein was not changed significantly.
CONCLUSION
Synergistic effect of Cisplatin and Theophylline on the inhibition of cell growth by apoptosis was clearly observed. This effect suggest the possibility lowering the concentration of chemotherapeutic drugs, which alleviate the side effect of drugs. Bcl-2 gene expression was down-regulated after Cisplatin and Theophylline co-treatment while Mdm-2 gene expression was up-regulated. p53 gene expression was not significantly changed after stimulation of various combination. Apoptosis was measured by FACS analysis and TUNNEL assay, which showed clearly synergistic effect of Cisplatin and Theophylline.