Sequence dependent potentiation of gemcitabine by flavopiridol in human breast cancer cells

Purpose. Flavopiridol is a novel cyclin dependent kinase (cdk) inhibitor currently in Phase I and II clinical trials. We investigated the interaction between flavopiridol and gemcitabine in two human breast cancer cell lines. Experimental design. MCF-7 [Estrogen receptor (ER) positive] and MDA-MB-231 cells (ER negative) were treated with sub-cytotoxic concentrations of gemcitabine (G), flavopiridol (F), and flavopiridol followed by gemcitabine (F-G), or gemcitabine followed by flavopiridol (G-F) and assayed for biological activity. Results. Growth inhibition assessed by serial cell counting and MTT assay was highest in the G-F group. Significant increase in apoptosis assessed by flow cytometry, poly-ADP-ribose polymerase (PARP) and Caspase-3 degradation was also highest in the G-F group. Expression of pro-apoptotic Bax was up-regulated and anti-apoptotic Bcl-2 was down-regulated in only the G-F treated cells. Significant up regulation of p21^WAF-1 was demonstrated in the G-F group but not in the reverse regimen treated cells. No effect on protein kinase C (PKC) expression was seen in any of the treated cells. Conclusions. In conclusion, similar to the results in the gastrointestinal cell lines, a sequence dependent potentiation of the effect of gemcitabine by flavopiridol was demonstrated in breast cancer cell lines and it was independent of ER status. This was accompanied by enhanced apoptosis and the up regulation of p21 ^WAF-1 protein. These results provide rationale for pre-clinical evaluation of this treatment strategy using animal models and in the design of clinical trials of this drug in combination with cytotoxic therapy.