Flavopiridol, a synthetic analog of a natural alkaloid, was shown to have potent CDK4-blocking activity [115]. Initial studies with this flavonoid demonstrated its ability to induce G1/S as well as G2/M cell cycle arrest due to loss of CDK1 and CDK2 activity [116-118]. Flavopiridol was shown to bind to the ATP-binding pocket of CDK2 and, thus, competes with ATP for binding [115] (Fig. 9.7). Unlike olomou-cine and roscovitine which show some CDK specificity, flavopiridol inhibits all CDKs as well as other protein kinases [118,119] with the greatest activity towards CDKs (reviewed in [120]).

Administration of flavopiridol has been shown to have multiple effects on cells including transcrip-tional inhibition, induction of apoptosis, and antian-giogenesis [121-123]. Direct cell cycle arrest at both the G1/S and G2/M boundaries is induced by flavopiridol's direct inhibition of CDKs 1, 2, and 4 [124,125]. Its effects on RNA polymerase II transcription are via inhibition of CDK9/cyclin T [121]. Of interest, is flavopiridol's inhibition of CDK9 activity, which is non-competitive with ATP binding unlike its activity towards other CDKs [122]. Microarray data suggest that the broad-range effects of flavopir-idol-induced transcriptional arrest are similar to those of actinomycin D and DRB (5,6-dichloro-beta-D-ribofuranosylbenzimidazole), global inhibitors of transcription [126]. It has been demonstrated that flavopiridol is capable of inhibiting transcription at concentrations much lower than those required of other CDK inhibitors even in the presence of ATP. One target of interest with respect to transcriptional downregulation is cyclin D1. The transcriptional inhibition of cyclin D1 has been associated with cell cycle arrest and, in some cases, cell death following treatment with flavopiridol. These results are consistent with observations that flavopiridol can delay disease progression in 84 per cent of patients with mantle cell lymphoma, a tumor associated with overexpressed cyclin D1 in 95 per cent of cases [127,128].

flavopiridol flavopiridol

figure 9.7 Inactivation of CDK/cyclin kinase activity by flavopiridol. By binding to the ATP-binding site of CDKs, flavopiridol is capable of inhibiting various CDKs throughout the cell cycle. Inhibition of ATP binding prevents kinase complex activation. See Plate 9.7 in Color Plate Section.

A variety of tumor cell lines have been found to be sensitive to flavopiridol-induced apoptosis in vitro (reviewed in [120]). Glioma cell lines were shown to undergo apoptosis in response to flavopiridol regardless of pRB or p53 status [129]. Despite the fact that flavopiridol is able to inhibit the expression of bcl-2 (an anti-apoptotic factor), downregulation of bcl-2 does not play a role in flavopiridol-mediated apoptosis [130,131]. It is still unclear whether or not inhibition of CDK activity is required for apoptosis.

Several clinical trials combining flavopiridol and various chemotherapeutic agents have been initiated [132,133]. It has been shown that coadministration of flavopiridol with cytostatic drugs, particularly taxanes, significantly enhanced the cytotoxic effects in a variety of tumor cells. The enhanced synergistic effects of flavopiridol and cytostatic drugs appeared to depend on the order of administration. In lung cancer cells, pre-treament with gemcitabine or cisplatin sensitizes cells to the cytotoxicity of flavopiridol whereas in leukemic cells, pre-treatment with flavo-piridol enhances the cytotoxicity of nucleoside analogs such as ara-C [134-136]. Cells with defective pRB signaling undergo enhanced doxorubicin-mediated cell death in conjunction with flavopiridol [131]. Since a large percentage of glial tumors harbour defects in the pRB pathway, treatment with flavoripirdol or flavopiridol/combination chemotherapy might be a therapeutic possibility.

Additional synergism between flavopiridol and signaling modulators has been detected. Co-treatment of leukemic cells with flavopiridol and the phorbol ester PMA, a potent inducer of differentiation, was found to induce apoptosis and overcome resistance mediated by bcl-2 expression [137]. Studies of flavopiridol synergism with additional chemothera-peutic agents have been extended to include a range of additional differentiation-inducing agents including Histone DeACetylase (HDAC) inhibitors. The downstream effects of flavopiridol and PMA or HDAC co-treatments include downregulation of cyclin D1, cleavage of pRB and bcl-2 and activation of E2F1. The identification of cytotoxic synergy between flavopiridol and other agents could produce more effective chemotherapeutic strategies in the future. The fact that flavopiridol does not induce resistance to most conventional cytotoxic agents including etoposide, doxorubicin, vinblastine, pacli-taxel, cisplatin, topotecan, and 5-Fluorouracil (5FU) is promising. In addition, overexpression of the multi-drug resistance proteins mdr-1 or mrp-1 did not cause appreciable resistance to flavopiridol [138-140].

Both phase I and phase II trials for flavopiridol have been completed. Clinical doses higher than those type ii inhibitors: inhibitors of cdk regulatory mechanisms

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