Purpose: Flavopiridol is a cyclin-dependent kinase inhibitor currently under development by the National Cancer Institute both as a single agent and in combination with chemotherapy. There have been numerous reports that flavopiridol potently enhances the induction of apoptosis by chemotherapy. However, the effect of flavopiridol on radiotherapy (RT)-induced apoptosis has been largely untested. RT has become the cornerstone of adjuvant treatment of colorectal and gastric cancer. In view of this, we elected to evaluate the effect of flavopiridol on potentiating RT-induced apoptosis in the human colon cancer cell line HCT-116 and the gastric cancer cell line MKN-74.
Experimental design: The efficacy of combination of gamma-irradiation and flavopiridol was tested in vitro in MKN-74 and HCT-116 cells and correlated to changes in p21 expression. HCT-116 cells were also established as tumors in nude mice and treated with gamma-irradiation and flavopiridol either as single agents or in sequential combinations such that flavopiridol was either given 7 h before, concomitantly, or 3 and 7 h after gamma-irradiation.
Results: Flavopiridol significantly enhanced the induction of apoptosis by gamma-irradiation in both cell lines as measured by quantitative fluorescent microscopy, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and cytochrome c release. To achieve the best effect, it was important to expose the tumor cells to gamma-irradiation before the flavopiridol. This sequence dependence was confirmed in vivo. When gamma-irradiation was administered 7 h before flavopiridol, 42% of the tumor-bearing animals were rendered disease free, compared with no animals treated with either gamma-irradiation or flavopiridol alone. Examination of the p21 status of HCT-116 and MKN-74 cells, after treatment with sequential gamma-irradiation and flavopiridol, indicated a loss of p21 protein expression. Loss of p21 was mainly due to cleavage by caspases. HCT-116 cells that lack p21 (p21(-/-)) also exhibited sensitization to gamma-irradiation and showed an even greater enhancement of gamma-irradiation-induced apoptosis by flavopiridol when compared with the parental HCT-116 cells.
Conclusions: These studies indicate that gamma-irradiation followed by flavopiridol enhances apoptosis and yields significantly increased tumor regressions and cures that are not achievable with radiation alone. These results indicate that flavopiridol can potently enhance the effect of gamma-radiation both in vitro and in vivo and may provide a new means to treat patients with locally advanced gastrointestinal cancers.