Nonpeptidic small-molecule inhibitor of Bcl-2 and Bcl-XL, (-)-gossypol, enhances biological effect of genistein against BxPC-3 human pancreatic cancer cell line

Objectives: In pancreatic cancer, several important survival molecules such as EGFR, NF-kappa B, and Bcl-2 or Bcl-X-L are highly activated. Thus, agents that inhibit NF-kappa B activation, together with agents that directly inhibit Bcl-2 or Bcl-X-L protein function, may lead to enhanced cell killing. ( 2)- Gossypol, a natural polyphenolic compound isolated from cottonseeds, is a dual and potent small-molecule inhibitor of Bcl-2 and Bcl-X-L proteins, with a K-i value in the 300 - 600 nM range for both proteins. Methods: The BxPC-3 human pancreatic cell line was used in this study. ( 2)- Gossypol was dissolved in DMSO at 20 mmol/L as stock solution, and genistein was dissolved in 0.1 M Na2CO3 to make a 10 mM stock solution. For cell viability, apoptosis, and NF-kappa B studies, MTT assay, histone/DNA ELISA, and Electrophoretic Mobility Shift Assay ( EMSA) were used, respectively. Coimmunoprecipitation experiments were designed to study Bcl-X-L/Bim heterodimerization, and Western blots to study cytochrome c release. Results: ( 2)- Gossypol showed a concentration-dependent growth inhibition effect against BxPC-3 pancreatic cancer cell line and induced apoptosis with no effect on normal peripheral blood lymphocytes. Results from coimmunoprecipitation experiments indicate that the effect of ( 2)- gossypol is mediated, at least, in part via disrupting the heterodimerization of Bcl-X-L with Bim in BxPC-3 pancreatic cancer cells. (-)- Gossypol completely disrupts Bcl-X-L/Bim heterodimerization with no change in the total Bcl-X-L or Bim protein, indicating that (-)- gossypol treatment does not affect the levels of Bcl-X-L and Bim proteins. We have previously shown that genistein, a prominent soy isoflavone, transcriptionally down-regulates Bcl-2, Bcl-X-L, VEGF, MMP-9, and uPAR via inhibiting NF-kappa B activity. In this study, genistein down-regulated NF-kappa B DNA binding activity and inhibited the growth of BxPC-3 pancreatic cancer cells. In addition, the combination of (-)- gossypol with genistein showed significantly greater growth inhibition compared with either agent alone. Conclusion: From these results, we conclude that inhibition of NF-kappa B activity and direct inhibition of Bcl-2 or Bcl-X-L function should serve as a novel strategy for pancreatic cancer therapy.