Alpha-lipoic acid modulates ovarian surface epithelial cell growth.

OBJECTIVE: The intracellular redox state plays an important role in controlling inflammation. Clinical and laboratory data suggest that inflammation can lead to tumor progression. We hypothesized that restoring intracellular redox control would inhibit inflammation and subsequently tumor progression. Our studies were designed to investigate the effect of alpha-lipoic acid (ALA), a naturally occurring antioxidant, on a key inflammatory signaling pathway and cell proliferation in normal and tumorigenic ovarian surface epithelial cells. METHODS: Normal and tumorigenic ovarian surface epithelial cells were isolated as described by Roby and coworkers [Roby KF, Taylor CC, Sweetwood JP, Cheng Y, Pace JL, Tawpik O, Persons DL, Smith PG, Terranova PF, Development of a syngeneic mouse model for events related to ovarian cancer. Carcinogen 2000;21 (4):585. [1]]. The effect of ALA on cellular function was measured in cell proliferation and apoptosis assays. p27(kip1) protein levels were measured by Western analysis. Activation of NF-kappaB dependent transcription was assessed in cell cultures transiently transfected with NF-kappaB controlled reporter constructs. RESULTS: Our results reveal that ALA selectively inhibits the growth of tumorigenic as compared to non-tumorigenic ovarian surface epithelial cells. The growth inhibitory effect of ALA is not due to induction of apoptosis but instead is associated with an increase in the half-life of the cyclin-dependent kinase inhibitor, p27(kip1). In parallel to the growth inhibitory effect, ALA also affects a key inflammatory signaling pathway by inhibiting TNFalpha-induced NF-kappaB signaling activity. CONCLUSIONS: Our studies are the first to show that ALA treatment has a growth inhibitory effect on malignant surface epithelial cells of ovarian origin. We have also confirmed the reproducibility of the immunocompetent mouse ovarian cancer model originally described by Roby and coworkers [Roby KF, Taylor CC, Sweetwood JP, Cheng Y, Pace JL, Tawpik O, Persons DL, Smith PG, Terranova PF, Development of a syngeneic mouse model for events related to ovarian cancer. Carcinogen 2000;21 (4):585].

Overexpression of a stabilized mutant form of the cyclin-dependent kinase inhibitor p27(Kip1) inhibits cell growth.

OBJECTIVE: The purpose of this study was to test the hypothesis that the expression of the mutant p27(Kip1) protein enhances cell growth inhibition and is more stable than that of the wild-type p27(Kip1). METHODS: Site-directed mutagenesis was used to mutate threonine 187 to an alanine residue, generating the mutant p27(Kip1). To study the effects of the p27(Kip1) mutant on cell growth, luciferase assays were performed. Cells were transiently transfected with the Renilla luciferase reporter construct and empty vector, wild-type p27(Kip1), or mutant p27(Kip1) using Fugene 6. The transfected cells were lysed and assayed for luciferase activity 24 h later with a dual-luciferase reporter assay system. To further assess the effects of the p27(Kip1) mutant on cell growth, colony count assays were performed. The experiments were repeated in duplicate and a standard two-tailed Student t test was use to analyze the data. RESULTS: Wild-type p27(Kip1) protein has a half-life of approximately 2 h while the p27(Kip1) mutant has a half-life of greater than 12 h. Furthermore, the p27(Kip1) mutant retained the ability to inhibit CDK2-associated H1 kinase activity. Cells expressing the p27(Kip1) mutant had an 88% reduction in luciferase activity compared to cells expressing the wild-type p27(Kip1) (P = 0.001). Colony assays revealed that cells expressing the p27(Kip1) mutant had fewer colonies compared to cells expressing the wild-type p27(Kip1) (P = 0.04). CONCLUSIONS: These data are consistent with the hypothesis that the mutated form of p27(Kip1) is more effective in cell growth inhibition than the wild-type p27(Kip1) protein.