Xihan Wu

In vitro Effects of the BH3 Mimetic, (−)-Gossypol, on Head and Neck Squamous Cell Carcinoma Cells

Purpose: Bcl-xL overexpression is common in head and neck squamous cell carcinomas (HNSCC) and correlates with resistance to chemotherapy. Thus, a nonpeptidic, cell-permeable small molecule that mimics the BH3 domain of proapoptotic proteins may inhibit Bcl-xL function and have therapeutic potential for HNSCC by overcoming drug-resistance. (−)-Gossypol, the levorotatory isomer of a natural product isolated from cottonseeds and roots, was recently discovered to bind to the BH3 binding groove of Bcl-xL and Bcl-2. Experimental Design: We investigated the in vitro effects of (−)-gossypol on HNSCC cell lines as well as on fibroblast and keratinocyte cultures by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell survival assays and assessed the results with respect to Bcl-2 family protein expression. Results: We observed dose-dependent growth inhibition of 10 HNSCC cell lines at biologically achievable doses (2.5–10 μmol/L). (−)-Gossypol doses required to inhibit the growth of human fibroblast cell lines by 50% were 2- to 10-fold higher than for HNSCC cell lines. To inhibit human oral keratinocyte growth by 50%, (−)-gossypol concentrations were 2-to 3-fold higher than for HNSCC cell lines. Conclusions: There is a direct correlation between Bcl-xL-to-Bcl-xS ratios and sensitivity to (−)-gossypol. This agent induced apoptosis in a much higher proportion of cells with wild-type p53. Importantly, cell lines resistant to cisplatin were very sensitive to (−)-gossypol. These results demonstrate that (−)-gossypol has potent antitumor activity in HNSCC in vitro. This agent may be developed as a novel therapeutic agent for HNSCC, either alone or in combination with existing chemotherapeutic agents.

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-κB, and Bcl-2 or Bcl-XL are highly activated. Thus, agents that inhibit NF-κB activation, together with agents that directly inhibit Bcl-2 or Bcl-XL protein function, may lead to enhanced cell killing. (−)-Gossypol, a natural polyphenolic compound isolated from cottonseeds, is a dual and potent small-molecule inhibitor of Bcl-2 and Bcl-XL proteins, with a Ki value in the 300-600 nM range for both proteins. Methods: The BxPC-3 human pancreatic cell line was used in this study. (−)-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-κB studies, MTT assay, histone/DNA ELISA, and Electrophoretic Mobility Shift Assay (EMSA) were used, respectively. Coimmunoprecipitation experiments were designed to study Bcl-XL/Bim heterodimerization, and Western blots to study cytochrome c release. Results: (−)-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 (−)-gossypol is mediated, at least, in part via disrupting the heterodimerization of Bcl-XL with Bim in BxPC-3 pancreatic cancer cells. (−)-Gossypol completely disrupts Bcl-XL/Bim heterodimerization with no change in the total Bcl-XL or Bim protein, indicating that (−)-gossypol treatment does not affect the levels of Bcl-XL and Bim proteins. We have previously shown that genistein, a prominent soy isoflavone, transcriptionally down-regulates Bcl-2, Bcl-XL, VEGF, MMP-9, and uPAR via inhibiting NF-κB activity. In this study, genistein down-regulated NF-κB 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-κB activity and direct inhibition of Bcl-2 or Bcl-XL function should serve as a novel strategy for pancreatic cancer therapy.