Synergistic cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea and Rana catesbeiana ribonuclease-6 in hepatoma cells

Chyou-Wei Wei^a, †, Pei-Lun Chou^b, †, Yu-Ting Hung^a, †, Giou-Teng Yiang^{c, d}

^a Institute of Biomedical Nutrition, Hung Kuang University, Sha Lu, Taichung, Taiwan
^b Division of Allergy, Immunology, and Rheumatology, Department of Internal Medicine, Taipei Medical University, Shuang Ho Hospital, Taipei, Taiwan
^c Department of Emergency Medicine, Buddhist Tzu Chi General Hospital, Taipei Branch, New Taipei City, Taiwan
^d Department of Emergency Medicine, Buddhist Tzu Chi University, Hualien, Taiwan

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Abstract
Objective

To demonstrate that a combination of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and Rana catesbeiana ribonuclease-6 (RC6) exerts synergistic cytotoxic effects on human hepatoma cells.

Materials and Methods

Human hepatoma cells (J5 and HepG2) were treated with various concentrations of BCNU or RC6. The survival rate was determined by XTT assay. Apoptosis was determined by fluorescence-activated cell sorting analysis with propidium iodide/annexin-V double stain. Caspase activation was determined by Western blot assay.

Results

BCNU and RC6 are able to inhibit the cell growth of hepatoma cells in a dose-dependent manner. BCNU combined with RC6 exerts a synergistic cytotoxic effect on hepatoma cells. Normal cells had less cytotoxicity than on hepatoma cells with BCNU/RC6 treatment. In addition, apoptosis was observed in hepatoma cells with BCNU treatment, RC6 treatment, and combination treatment. Our data also showed that combination treatment can activate the caspase-9/caspase-3 cascade obviously in hepatoma cells.

Conclusion

Combination treatment with BCNU and RC6 exerts a synergistic cytotoxic effect on hepatoma cells.

Keywords
1,3-Bis(2-chloroethyl)-1-nitrosourea; Apoptosis; Caspase; Hepatoma cells; Synergistic effect