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Background: Flavopiridol is a flavonoid with antiproliferative effects mediated, in part, by inhibition of cyclin-dependent kinases. Clinical manifestations in a previous Phase I trial in patients with refractory malignancies treated with a 72-h flavopiridol infusion included a proinflammatory syndrome consisting of fever, fatigue, and "local" tumor pain with concomitant alterations in plasma acute-phase reactant proteins., Purpose: The aim of this study was to determine whether the proinflammatory syndrome observed in this trial was associated with modulation of plasma cytokines., Methods: Patients receiving flavopiridol (n = 76) had serial plasma samples drawn preinfusion and during the infusion for evaluation of interleukin (IL)-6, IL-10, IL-12, granulocyte macrophage colony-stimulating factor, basic-fibroblast growth factor, transforming growth factor-beta, and tumor necrosis factor-alpha levels by standard ELISA assays. The Wilcoxon signed rank test was used to test the significance of the difference between the baseline (time 0) plasma cytokine levels compared with the values of each subsequent data collection time points (8, 24, 48, and 72 h)., Results: There was a significant and sustained increase in plasma IL-6 levels at all time points when compared with baseline values. Paired values were used in the statistical analysis. Median plasma (interquartile range) values of IL-6 were elevated from 15.5 (9-52) pg/ml at baseline to 23 (4-48) pg/ml (P < 0.01) at 8 h; from 15 (2-48) pg/ml at baseline to 46 (21-105) pg/ml (P < 0.001) at 24 h; from 16 (9-52) pg/ml at baseline to 61 (32-170) pg/ml (P < 0.001) at 48 h; and from 15.5 (6-48) pg/ml to 68 (40-200) pg/ml (P < 0.001) at 72 h. Significance was maintained even when adjusted for multiple comparisons. The relative increase in IL-6 concentration was dose-dependent. Moreover, IL-6 elevation had a direct correlation with flavopiridol peak plasma concentration, flavopiridol area under the curve, and plasma C-Reactive protein levels. A significant decrease in plasma granulocyte macrophage colony-stimulating factor occurred at the 8-h sampling point: 50 pg/ml (interquartile range 10-205 pg/ml, P < 0.01) when compared with baseline plasma levels and 71 pg/ml (interquartile range 5-152 pg/ml, P < 0.01). No changes in the other pro or anti-inflammatory cytokines were observed. Immunohistochemistry studies in bone marrow aspirates from a prospective group of patients in this trial demonstrated approximately 4-fold induction of IL-6 (compared with baseline), mostly in non-T cells., Conclusion: Biochemical analysis of plasma in patients undergoing infusional flavopiridol found a significant dose-dependent induction of IL-6. IL-6 elevation could be a marker for the process leading to the appearance of the proinflammatory syndrome observed in patients treated with infusional flavopiridol. The mechanism(s) underlying IL-6 induction and its significance are still unknown but may influence strategies to modulate flavopiridol's clinical effects.

Altered and deregulated cyclin-dependent kinase (cdk) activity is now believed to play a major role in the pathogenesis of head and neck squamous cell carcinomas (HNSCC), thus providing a suitable cellular target for therapeutic intervention. UCN-01 (7-hydroxy-staurosporine), a known protein kinase C and cdk modulator, demonstrates antiproliferative and antitumor properties in many experimental tumor models and may represent a potential candidate to test in HNSCC. In this study, UCN-01 displayed potent antiproliferative properties (IC50 of approximately 17-80 nM) in HNSCC cells. Cell cycle analysis revealed that UCN-01 treatment of HNSCC cells for 24 h leads to a G1 block with a concomitant loss of cells in S and G2-M and the emerging sub-G1 cell population, confirmed to be apoptotic by terminal deoxynucleotidyl transferase-mediated nick end labeling analysis. Additional in vitro studies demonstrated a G1 arrest that was preceded by depletion in cyclin D3, elevation of p21(WAF1) and p27(KIP1) leading to a loss in activity of G1 cdks (cdk2, cdk4), and reduction in pRb phosphorylation. Antitumor properties of UCN-01 were also assessed in vivo by treating HN12 xenografts (7.5 mg/kg/i.p./daily) with UCN-01 for 5 consecutive days. Total sustained abolition of tumor growth (P < 0.00001) was obtained with only one cycle of UCN-01 treatment. Terminal deoxynucleotidyl transferase-mediated nick end labeling staining of xenograft samples revealed a higher incidence of apoptosis in treated tissues when compared with control. Additional tissue analysis demonstrated that elevated p27(KIP1) with minimal increase in p21(WAF1) and reduced cyclin D3 levels were readily detected in those animals treated with UCN-01, similar to those observed in HNSCC cells. Thus, UCN-01 exhibits both in vitro and in vivo antitumor properties in HNSCC models, and these effects are associated with a decrease in cyclin D3 and an increase in p27(KIP1) protein levels, thus providing appropriate surrogate markers to follow treatment efficacy in vivo and, therefore, a suitable drug candidate for treating HNSCC patients.

We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100 microM. To determine putative mechanisms of resistance to flavopiridol, we exposed the human breast cancer cell line MCF-7 to incrementally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7 FLV1000, is maintained in 1,000 nM flavopiridol and was found to be 24-fold resistant to flavopiridol, as well as highly cross-resistant to mitoxantrone (675-fold), topotecan (423-fold), and SN-38 (950-fold), the active metabolite of irinotecan. Because this cross-resistance pattern is consistent with that reported for ABCG2-overexpressing cells, cytotoxicity studies were repeated in the presence of 5 microM of the ABCG2 inhibitor fumitremorgin C (FTC), and sensitivity of MCF-7 FLV1000 cells to flavopiridol, mitoxantrone, SN-38, and topotecan was restored. Mitoxantrone efflux studies were performed, and high levels of FTC-reversible mitoxantrone efflux were found. Northern blot and PCR analysis revealed overexpression of the ABCG2 gene. Western blot confirmed overexpression of ABCG2; neither P-glycoprotein nor MRP overexpression was detected. These results suggest that ABCG2 plays a role in resistance to flavopiridol.