Sensitivity to OSU03012 is p53/p21 dependent and Rb independent in normal human epithelial cell lines

A133

OSU03012 is novel Celecoxib derivative with potential anticancer activity via a non-COX mechanism. We find that OSU03012 selectively targets premalignant and malignant human oral epithelial cell lines but not normal cells via inducing S phase block and apoptosis. Further deciphering this differential effect will be important. To better understand the role of cell cycle regulation in the effect caused by OSU03012, primary normal human oral epithelial cell line TE1177 was transformed by infecting with retrovirus containing HVP16/E6 and HVPE7. Growth inhibition assay, by methylene blue staining, showed that TE1177/E6 cell lines are more sensitive to the treatment with OSU03012 than its parent TE1177 and TE1177/E7 cell lines. Since the S phase accumulation is important to the toxicity of premalignant and malignant human oral cell lines, cell distribution was analyzed by flow cytometer. The results show that all TE1177/E6 and TE1177/E7 cell lines have a higher S phase population, with 14.0% in TE1177, 22.0% in TE1177/E6, and 18.0% in TE1177/E7. Upon treatment with OSU03012 the S phase population was mildly reduced in TE1177/E7 but not in TE1177/E6 cell lines. For example the S phase population was 2.95% in TE1177, 21.9% in TE1177/E6, and 13.6% in TE1177/E7 cell lines after 48hr treatment with 2 µM OSU03012. These results suggest that S phase accumulation may play a role in OSU03012-decreased cell proliferation. Western blot analyses showed that infection with HVP16/E7 dramatically reduced the protein levels of Rb; infection with HVP16/E7 resulted in mild decrease in the protein levels of P53 but a significant reduction in the levels of p21WAF1, a downstream effecter of p53; whereas TE1177/E7 cell lines expressed much higher levels of p21WAF1 than TE1177 cell line. Treatment with OSU03012 resulted in dramatic reduction in the protein levels of cyclin A and cdk2 in the TE1177 cell line and a slight reduction in the TE1177/E6 or TE1177/E7 cell lines. There was a noticeable increase in the protein levels of p21WAF1 in TE1177, much higher levels of p21WAF1 in TE1177/E7, and undetectable level of p21WAF1 in TE1177/E6 cell lines. To further test if p21WAF1 affects the sensitivity of cells to treatment with OSU03012, MEF cells harboring wild type or deleted p21WAF1 were treated with OSU03012. Western blot analyses showed that MEF cells without p21WAF1 were more sensitive to OSU03012-induced apoptosis as evidenced by PARP cleavage. Our data suggest that S phase accumulation and p21WAF1 status are important to the sensitivity of the epithelial cells to the treatment OSU03012. Supported by NCI R01-109012.