Nonpeptidic small-molecule inhibitor of Bcl-2 and Bcl-XL, (-)-Gossypol, enhances biological effect of genistein against BxPC-3 human pancreatic cancer cell line.
OBJECTIVE
In pancreatic cancer, several important survival molecules such as EGFR, NF-kappaB, and Bcl-2 or Bcl-XL are highly activated. Thus, agents that inhibit NF-kappaB 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-kappaB 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-kappaB activity. In this study, genistein down-regulated NF-kappaB 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.


CONCLUSIONS
From these results, we conclude that inhibition of NF-kappaB activity and direct inhibition of Bcl-2 or Bcl-XL function should serve as a novel strategy for pancreatic cancer therapy.
Mohammad
Ramzi M
RM
Division of Hematology and Oncology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA. Mohammad@karmanos.org
Wang
Shaomeng
S
Banerjee
Sanjeev
S
Wu
Xihan
X
Chen
Jianyong
J
Sarkar
Fazlul H
FH
D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D004357:Drug Synergism; D019833:Genistein / Q000494:pharmacology*; D006072:Gossypol / Q000494:pharmacology*; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000502:physiology; D051020:bcl-X Protein / Q000037:antagonists & inhibitors* / Q000502:physiology
D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D045304:Cytochromes c; D019833:Genistein; D006072:Gossypol