Concentrations Of Flavopiridol Research Articles

Flavopiridol induces cell cycle arrest and apoptosis by interfering with CDK1 signaling pathway in human ovarian granulosa cells

Several clinical trials have been conducted to evaluate the use of flavopiridol (FP) to treat a variety of cancers, and almost all cancer drugs were found to be associated with toxicity and side effects. It is not clear whether the use of FP will affect the female reproductive system. Granulosa cells, as the important cells that constitute the follicle, play a crucial role in determining the reproductive ability of females. In this study, we investigated whether different concentrations of FP have a toxic effect on the growth of immortalized human ovarian granulosa cells. The results showed that FP had an inhibitory effect on cell proliferation at a level of nanomole concentration. FP reduced cell proliferation and induced apoptosis by inducing mitochondrial dysfunction and oxidative stress, as well as increasing BAX/BCL2 and pCDK1 levels. These results suggest that toxicity to the reproductive system should be considered when FP is used in clinical applications.

Potent inhibition of rhabdoid tumor cells by combination of flavopiridol and 4OH-tamoxifen

BackgroundRhabdoid Tumors (RTs) are highly aggressive pediatric malignancies with poor prognosis. There are currently no standard or effective treatments for RTs in part because treatments are not designed to specifically target these tumors. Our previous studies indicated that targeting the cyclin/cdk pathway is a novel therapeutic strategy for RTs and that a pan-cdk inhibitor, flavopiridol, inhibits RT growth. Since the toxicities and narrow window of activity associated with flavopiridol may limit its clinical use, we tested the effect of combining flavopiridol with 4-hydroxy-Tamoxifen (4OH-Tam) in order to reduce the concentration of flavopiridol needed for inhibition of RTs.MethodsThe effects of flavopiridol, 4OH-Tam, and their combination on RT cell cycle regulation and apoptosis were assessed by: i) cell survival assays, ii) FACS analysis, iii) caspase activity assays, and iv) immunoblot analysis. Furthermore, the role of p53 in flavopiridol- and 4OH-Tam-mediated induction of cell cycle arrest and apoptosis was characterized using RNA interference (siRNA) analysis. The effect of p53 on flavopiridol-mediated induction of caspases 2, 3, 8 and 9 was also determined.ResultsWe found that the combination of flavopiridol and 4OH-Tam potently inhibited the growth of RT cells. Low nanomolar concentrations of flavopiridol induced G2 arrest, which was correlated to down-modulation of cyclin B1 and up-regulation of p53. Addition of 4OH-Tam did not affect flavopiridol-mediated G2 arrest, but enhanced caspase 3,7-mediated apoptosis induced by the drug. Abrogation of p53 by siRNA abolished flavopiridol-induced G2 arrest, but enhanced flavopiridol- (but not 4OH-Tam-) mediated apoptosis, by enhancing caspase 2 and 3 activities.ConclusionsCombining flavopiridol with 4OH-Tam potently inhibited the growth of RT cells by increasing the ability of either drug alone to induce caspases 2 and 3 thereby causing apoptosis. The potency of flavopiridol was enhanced by abrogation of p53. Our results warrant further studies investigating the combinatorial effects of flavopiridol and 4OH-Tam as a novel therapeutic strategy for RTs and other tumors that have been shown to respond to flavopiridol.

Abstract 667: Mechanisms underlying synergistic interactions between the CDK inhibitor flavopiridol (Alvocidib) and the BH3 mimetic GX15-070 (Obatoclax) in human multiple myeloma cells

Abstract To establish a mechanistic basis for a strategy combining CDK inhibitors and Bcl-2 antagonists, in vitro and in vivo interactions between flavopiridol (FP) and GX15-070 (GX) were investigated in human multiple myeloma (MM) cells. The activity of this regimen was also examined in primary CD138+ MM cells and in a murine MM xenograft model. Co-administration of sub-toxic concentrations of FP interacted synergistically with GX to induce apoptosis in U266, RPMI8226, H929, OPM-2, and MM.1S, as well as IL-6-dependent ANBL6 and KAS 6/1 MM cells. Combination treatment also effectively killed MM cells in the presence of stromal cells or growth factors. The regimen was active against multiple drug-resistant MM cell lines, including bortezomib/Velcade- (U266/VR and OPM-2/VR), Revlimid- (8226/R10R), dexamethasone- (MM.1R), melphalan- (8226/LR5), and doxorubicin-resistant (8226/Dox40) MM cells. FP also sharply promoted GX lethality in primary CD138+ MM cells, while sparing normal CD138- bone marrow cells. GX induced clear declines in Mcl-1 and Bcl-xL levels which were potentiated by FP, whereas Bcl-2 expression did not change. Significantly, FP/GX overcame cytoprotective effects mediated by ectopic Mcl-1, Bcl-xL, or Bcl-2 expression. Notably, BH3-only protein expression profiling revealed multiple changes: a) FP, in the presence or absence of GX, triggered a clear increase in Bim expression (both BimEL and BimL isoforms); b) FP/GX co-treatment sharply induced Bik (∼35kDa species) and Noxa (∼27 kDa species),; c) co-exposure (24hr) to FP and GX induced a clear reduction in Puma expression; and c) no changes in the expression of other BH3-only proteins occurred. These findings were confirmed in various MM cell lines, including multiple drug-resistant cells, and were associated with Bak/Bax activation, Bax translocation, cytochrome C release, and caspase activation. Importantly, Bim, Bik, or Noxa knock-down by stable transfection with shRNA dramatically diminished FP/GX lethality. Lastly, co-administration of FP and GX significantly suppressed tumor growth, reflected by diminished tumor size and bioluminescence intensity, in athymic nude mice inoculated with 8226 cells stably expressing luciferase. These events were associated with pronounced apoptosis, manifested by caspase 3 activation and PARP degradation. Western blot analysis also revealed down-regulation of Mcl-1, as well as up-regulation of Bik and Noxa in tumor tissues following FP/GX co-administration. Collectively, these findings provide a mechanistic framework for a strategy combining CDK inhibitors with Bcl-2 antagonists in which down-regulation of anti-apoptotic proteins and induction of BH3-only proteins cooperate to trigger synergistic killing of MM cells, including those resistant to either conventional or novel anti-MM agents, both in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 667.

A phase I clinical trial of FOLFIRI in combination with the pan-cyclin-dependent kinase (CDK) inhibitor flavopiridol

The cyclin-dependent kinase inhibitor flavopiridol increases irinotecan- and fluorouracil-induced apoptosis. We conducted a phase I trial of FOLFIRI + flavopiridol in patients with advanced solid tumors. FOLFIRI + flavopiridol were administered every 2 weeks. Based on sequence-dependent inhibition, flavopiridol was given 3 h after irinotecan but before 5-FU. Two maximum tolerated doses were determined, one with flavopiridol administered over 1 h, and one with flavopiridol split as a 30-min bolus followed by a 4-h infusion. A total of 74 patients were enrolled and 63 were evaluable. The MTD with FOLFIRI was flavopiridol 80 mg/m(2) over 1 h or 35 mg/m(2) bolus + 35 mg/m(2) over 4 h. Dose-limiting toxicities were diarrhea, fatigue, neutropenia, and neuropathy. Clinical activity included 2 partial responses in small bowel cancer and bladder cancer and 1 complete response in mucosal melanoma. Stable disease was seen in 22 patients. Pharmacokinetic studies showed increasing C(max) with increasing flavopiridol dose. Clinical benefit was correlated with the presence of wild-type p53. Of 25 patients with colorectal cancer, 11 had as best response SD for >3 m (median 6 m, range 4.2-15.4 m), despite failing ≥1 irinotecan-containing regimen. Treatment with flavopiridol and FOLFIRI is a safe and effective regimen. Concentrations of flavopiridol that enhance the effects of FOLFIRI can be achieved. Clinical activity is encouraging and includes prolonged stable disease in patients with irinotecan-refractory colorectal cancer.

Rhabdoid Tumor Growth is Inhibited by Flavopiridol

Rhabdoid tumors are aggressive and incurable pediatric malignancies. INI1/hSNF5, a tumor suppressor biallelically deleted/inactivated in rhabdoid tumors, directly represses cyclin D1. Rhabdoid tumors and cells are exquisitely dependent on cyclin D1 for genesis and survival, suggesting that targeting the cyclin/cyclin-dependent kinase (cdk) axis may be an effective therapeutic strategy for these tumors. Because cdk inhibitors have not been used for preclinical or clinical testing on rhabdoid tumors, we investigated the effect of flavopiridol, a pan-cdk inhibitor with promising clinical activity, on rhabdoid tumors. The effect of flavopiridol on rhabdoid cells was tested in vitro using survival, cell cycle, and apoptosis assays. Its effect was assessed in vivo using xenografted rhabdoid tumor models. Immunoblot and immunohistochemical analysis was used to assess the effect of flavopiridol on cyclin D1 and p21 expression in vitro and in vivo, respectively. Nanomolar concentrations of flavopiridol inhibited rhabdoid cell growth (IC(50) approximately 200 nmol/L), induced G(1) and G(2) arrest, and apoptosis in vitro in a concentration-dependent manner. These effects were correlated with the down-modulation of cyclin D1, up-regulation of p21, and induction of caspase 3/7 activities. Flavopiridol (at 7.5 mg/kg) significantly inhibited the growth of xenografted rhabdoid tumors, and its effect was correlated with the induction of p21 and down-modulation of cyclin D1. Flavopiridol is effective in inducing cell cycle arrest and cytotoxicity in rhabdoid tumors. Its effects are correlated with the down-regulation of cyclin D1 and the up-regulation of p21. Flavopiridol is potentially a novel chemotherapeutic agent for rhabdoid tumors.

Phase I Dose-Finding Study of Weekly Docetaxel Followed by Flavopiridol for Patients with Advanced Solid Tumors

Flavopiridol is a cyclin-dependent kinase inhibitor that enhances docetaxel-induced apoptosis in a sequence-specific manner. In vivo, docetaxel must precede flavopiridol by at least 4 h to induce this effect. We conducted a phase I trial of weekly, sequential docetaxel followed 4 h later by flavopiridol in patients with advanced solid tumors. Docetaxel at a fixed dose of 35 mg/m2 was administered over 30 min, followed 4 h later by escalating doses of flavopiridol, ranging from 20 to 80 mg/m2 in successive cohorts, administered weekly over 1 h. This schedule was repeated for 3 weeks of each 4-week cycle. Twenty-seven evaluable patients were enrolled. The combination was well tolerated, with one dose-limiting toxicity occurring at flavopiridol 70 mg/m2 (grade 3 mucositis) and one dose-limiting toxicity at 80 mg/m2 (grade 4 neutropenia). We observed 1 complete response in a patient with pancreatic carcinoma and 4 partial responses in pancreatic (1), breast (2), and ovarian (1) cancer patients. Stable disease was seen in 10 patients. Pharmacokinetic studies showed Cmax ranging from 1.49 +/- 0.69 micromol/L (flavopiridol 20 mg/m2) to 4.54 +/- 0.08 micromol/L (flavopiridol 60 mg/m2) in cycle 1. Treatment with weekly, sequential docetaxel followed by flavopiridol is an effective and safe regimen at all flavopiridol dose levels. The pharmacokinetic data indicate that concentrations of flavopiridol that enhance the effects of docetaxel both in vitro and in vivo can be achieved. Clinical activity is encouraging, even in patients who have received a prior taxane and in patients with gemcitabine-refractory metastatic pancreatic cancer.

Mechanism and functional role of XIAP and Mcl-1 down-regulation in flavopiridol/vorinostat antileukemic interactions

The mechanism and functional significance of XIAP and Mcl-1 down-regulation in human leukemia cells exposed to the histone deacetylase inhibitor vorinostat and the cyclin-dependent kinase inhibitor flavopiridol was investigated. Combined exposure of U937 leukemia cells to marginally toxic concentrations of vorinostat and flavopiridol resulted in a marked increase in mitochondrial damage and apoptosis accompanied by pronounced reductions in XIAP and Mcl-1 mRNA and protein. Down-regulation of Mcl-1 and XIAP expression by vorinostat/flavopiridol was associated with enhanced inhibition of phosphorylation of RNA polymerase II and was amplified by caspase-mediated protein degradation. Chromatin immunoprecipitation analysis revealed that XIAP and Mcl-1 down-regulation were also accompanied by both decreased association of nuclear factor-kappaB (XIAP) and increased E2F1 association (Mcl-1) with their promoter regions, respectively. Ectopic expression of Mcl-1 but not XIAP partially protected cells from flavopiridol/vorinostat-mediated mitochondrial injury at 48 h, but both did not significantly restored clonogenic potential. Flavopiridol/vorinostat-mediated transcriptional repression of XIAP, Mcl-1-enhanced apoptosis, and loss of clonogenic potential also occurred in primary acute myelogenous leukemia (AML) blasts. Together, these findings indicate that transcriptional repression of XIAP and Mcl-1 by flavopiridol/vorinostat contributes functionally to apoptosis induction at early exposure intervals and raise the possibility that expression levels may be a useful surrogate marker for activity in current trials.

Updated Results of a Phase I Study of Flavopiridol in Acute Leukemias Using a Novel, Pharmacokinetically Derived Schedule: Clinical Activity Including Hyperacute Tumor Lysis Syndrome (TLS), Pharmacokinetics (PK), and Pharmacodynamics (PD).

Flavopiridol is a cyclin-dependent kinase inhibitor that induces apoptosis in acute leukemia cell lines. In earlier studies with flavopiridol in several malignancies, plasma concentrations of flavopiridol were not reached or maintained at sufficient levels to induce apoptosis due to unexpectedly high levels of bound drug in human serum relative to fetal calf serum used in preclinical studies. PK modeling in chronic lymphocytic leukemia (CLL) cells cultured in human plasma in vitro indicated that administering flavopiridol by 30 minute intravenous (IV) bolus followed by 4 hour continuous IV infusion (CIVI) would achieve sustained in vivo plasma drug concentration and time exposure similar to that necessary to induce apoptosis. We designed a phase I dose escalation trial in acute leukemias of single agent flavopiridol given as a 30 minute bolus followed by a 4 hour CIVI on days 1–3 with the ability to repeat cycles every 21 days; 16 patients (pts) have been enrolled to date. Dose escalation was as follows (bolus dose/4 hr CIVI dose in mg/m2): 20/30 (n=3), 30/35 (n=7), 30/50 (n=3), and 40/60 (n=3). Based on prior experience with flavopiridol at our institution in CLL, aggressive measures for the prevention and management of hyperacute tumor lysis syndrome (TLS) were employed. Pts had relapsed/refractory AML (N=12) and ALL (N=4), and were 25–78 yrs old (median age 64 yrs). Average plasma levels were 1.0–2.5 μM at the first three dose levels during the infusion (N=13) and declined with terminal half-lives comparable to previously reported 72 hr and more recent 4.5 hr infusions. Clinically significant TLS occurred in 2/16 pts with chemical evidence of lysis in 4 additional pts. A dose-limiting toxicity (renal failure) occurred at dose level 4 (40 mg/m2 bolus/60 mg/m2 CIVI), and the level is currently being expanded. Treatment was otherwise well tolerated. Downregulation of Mcl-1 protein by standard immunoblotting at 4 and/or 24 hrs was demonstrated in blood and/or bone marrow cells of 6/10 patients Anti-leukemic activity including transient reductions in WBCs/circulating blasts (n=7), bone marrow blasts (n=2), and platelet transfusion independence (n=1) was observed. Two received a second course of therapy, but no pt experienced an objective response by standard criteria. The current dose level exceeds that previously given in ongoing CLL studies; dose escalation to identify the maximum tolerated dose using this pharmacokinetically derived schedule in acute leukemia continues. Given the activity of this drug as a single agent, combination studies with conventional chemotherapy or other novel agents in acute leukemias should be considered.

Flavopiridol Decreases the Level of p21 and Induces Apoptosis in Human Myeloma Cell Lines.

Flavopiridol is a cyclin dependent kinase (CDK) inhibitor with a broad spectrum of activity both in vitro and in vivo and has been demonstrated to inhibit cell growth and induce apoptosis in cancer cells. We have evaluated the effect of Flavopiridol on a panel of human myeloma cell lines (HMCL)( NCI H929, LP-1, RPMI 8226, OPM-2 and U266) and in an in vivo murine model of multiple myeloma (5T33). Dose response was determined by MTS assays with a range of Flavopiridol doses (0–5mM) for 72 hours. The profiles of the cells after treatment with Flavopiridol were evaluated via FACS and cell cycle analysis. MTS assays showed a dose and time dependent Flavopiridol-induced cell killing in HMCL with effective concentrations of Flavopiridol ranging from 10nM - 5mM. This observation was associated with increasing number of cells in the late apoptotic stage and accumulation of cells in the SubG0 and G0/G1 phases. Western analysis using antibodies against p21, cyclin A, p27, Mcl-1, Bcl-XL, Bcl-2a, pRB, and PARP were performed to help characterize the mechanism(s) of cell killing. Cleavage of PARP in NCI H929 was noted within 24 hours of Flavopiridol treatment indicating early Flavopiridol-induced HMCL apoptosis. Expression of p21 protein, a member of the Cip/Kip family of CDK that can bind and obstruct a broad range of cyclin/Cdk complexes, was found to decrease following 24 hours of exposure to Flavopiridol. A reduction of another CDK member, Cyclin A, was apparent following Flavopiridol treatment whilst p27 expression was unchanged even after 72 hours. Three anti apoptotic proteins, Mcl-1, Bcl-2a and Bcl-XL protein expression were evaluated. Mcl-1 protein expression increased with time in all three cell lines tested while Bcl-2a was unaltered and Bcl-XL decreased following treatment. Retinoblastoma protein (RB), a tumour suppressor which acts as a cell cycle checkpoint between G1 and S phases was evaluated. A decrease of hyperphosphorylated RB (ppRB) was demonstrated in all 3 cell lines indicating a decrease in proliferating cells. Furthermore, silencing of p21 by siRNA enhanced Flavopiridol induced apoptosis indicating cell death occurred via a p21 dependent mechanism. The impact of combination and scheduling with bortezomib was evaluated in NCI-H929 and primary myeloma cells and it was found that addition of bortezomib subsequent to Flavopiridol improved cell killing compared to single-agent treatment. Reversal of drug treatment was found to be not as efficient. 2.5 mg/ml Flavopiridol and 1% DMSO/saline (vehicle) were administered to a 5T33 murine model of multiple myeloma for 5 days intraperitoneally. Hind limp paralysis was observed later in the Flavopiridol treated mice compared to vehicle treated mice with the median survival for vehicle treated mice of 23 days compared to 24.5 days in the AZA treatment (p= 0.018). These data provide a rationale for further evaluation of Flavopiridol as a therapeutic agent in multiple myeloma. Furthermore, we propose that Flavopiridol induced killing of HMCL may be mediated by a novel mode of action involving suppression of p21.

Induction of apoptosis by flavopiridol unrelated to cell cycle arrest in germ cell tumour derived cell lines

Germ cell tumours (GCTs) are highly sensitive to cisplatin-based chemotherapy. The inability to arrest the cell cycle at the G1/S-check-point due to a lack of retinoblastoma gene product RB has been suggested as one potential explanation for this feature. Flavopiridol (FP), an inhibitor of cyclin dependent kinases, causes cell cycle arrest or apoptosis depending on the relation of the transcription factor E2F1 and RB. The effect of FP was evaluated in GCT-derived cell lines NT2, 2102 EP and NCCIT in comparison to cell lines derived from ovarian cancer (SKOV), breast cancer (MCF7), and cervical cancer (HeLa) using the MTT-assay. Cell cycle progression and induction of apoptosis were assessed by flow cytometry and immunoblot analysis of PARP-cleavage. FP did not affect cell cycle progression and proliferation of GCT cell lines at sublethal doses. At higher concentrations, cell death occurred independent of cell cycle progression. The IC50 was approximately fivefold lower for the three GCT cell lines (60/60/70 nM) than for the other tumour cell lines tested (350/280/300 nM). Lethal doses in vitro were markedly lower than plasma concentrations of FP achieved in clinical studies. In vitro sensitivity to FP did not correlate with that to cisplatin. The cell lines NTera2 and NCCIT showed comparable responses to FP despite differing in their IC50 to cisplatin by factor 4. Flow cytometry and immunoblot for PARP indicated apoptotic cell death induced by FP. Synergism between either cisplatin or paclitaxel and FP was not observed. However, at low concentrations, cytotoxicity of FP and cisplatin appeared to be additive. These prelinical investigations suggest a significant antitumour activity of FP in GCT. GCT derived cell lines were far more responsive to FP than cell lines derived from other solid tumours. In contrast to other models, FP did not induce cell cycle arrest in the GCT-derived cell lines tested, possibly due to the known lack of RB-expression in GCTs. However, apoptosis was induced unrelated to cell cycle progression already at low concentrations. No cross resistance between FP and cisplatin was observed. A clinical trial evaluating the activity of FP in patients with cisplatin-refractory GCTs appears to be warranted.

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

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. 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. 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.

Potent antileukemic interactions between flavopiridol and TRAIL/Apo2L involve flavopiridol-mediated XIAP downregulation.

Interactions between the cyclin-dependent kinase inhibitor flavopiridol (FP) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L), were examined in human leukemia cells (U937 and Jurkat). Coexposure of cells to marginally toxic concentrations of TRAIL and FP (24 h) synergistically increased mitochondrial injury (eg, cytochrome c, AIF, Smac/DIABLO release), cytoplasmic depletion of Bax, activation of Bid as well as caspase-8 and -3, PARP cleavage, and apoptosis. Coadministration of TRAIL markedly increased FP-induced apoptosis in leukemic cells ectopically expressing Bcl-2, Bcl-x(L), or a phosphorylation loop-deleted form of Bcl-2 (DeltaBcl-2), whereas lethality was substantially attenuated in cells ectopically expressing CrmA, dominant-negative-FADD, or dominant-negative-caspase-8. TRAIL/FP induced no discernible changes in FLIP, DR4, DR5, Mcl-1, or survivin expression, modest declines in levels of DcR2 and c-IAP, but resulted in the marked transcriptional downregulation of XIAP. Moreover, cells stably expressing an XIAP-antisense construct exhibited a pronounced increase in TRAIL sensitivity comparable to degrees of apoptosis achieved with TRAIL/FP. Conversely, enforced XIAP expression significantly attenuated caspase activation and TRAIL/FP lethality. Together, these findings suggest that simultaneous activation of the intrinsic and extrinsic apoptotic pathways by TRAIL and FP synergistically induces apoptosis in human leukemia cells through a mechanism that involves FP-mediated XIAP downregulation.

Flavopiridol inhibits soft tissue sarcoma growth with preferential sensitivity in a CDK4 gene amplified subtype

Abstract Introduction: Current chemotherapy for advanced soft tissue sarcoma (STS) has low overall response rates. It has been shown that the CDK4 gene is frequently amplified in some STS subtypes. Here we tested flavopiridol, a pan cyclin dependent kinase inhibitor, with STS subtypes that have amplified (dedifferentiated liposarcoma, DDLS) or non-amplified (malignant peripheral nerve sheath tumor, MPNST) CDK4. Methods: DDLS and MPNST cell lines were evaluated for cellular proliferation using CyQuant fluorescence assay, cell cycle distribution by FACS analysis and apoptosis using AnnexinV/7-AAD staining following 24h of treatment with flavopiridol. CDK4 amplification was confirmed via FISH analysis. Results: Flavopiridol inhibited proliferation of DDLS and MPNST cells with IC50s of 100 and 150nM, respectively. DDLS cells, which possess amplified CDK4, demonstrated a dose dependent increase in apoptosis with 15% and 27% cells undergoing apoptosis at 150 and 300nM flavopiridol respectively, after 24h of treatment. In contrast, the MPNST cells demonstrated a 3-fold lower apoptotic percentage compared to DDLS cells at these concentrations of flavopiridol. Lastly, the DDLS cells underwent G1 cell cycle arrest with as little as 150nM flavopiridol whereas the cell cycle distribution of the MPNST cells remained unchanged with up to 300nM flavopiridol. Conclusions: Flavopiridol has antiproliferative activity in STS cell lines. CDK4 gene amplification in dedifferentiated liposarcoma renders these cells more sensitive to flavopiridol mediated G1 cell cycle arrest and apoptosis. Our findings suggest flavopiridol to be a novel therapeutic agent for the treatment of STS, preferentially with amplified CDK4.

Transcriptional signature of flavopiridol-induced tumor cell death

Flavopiridol has been shown to inhibit the proliferation of a variety of human tumor cells and is currently undergoing clinical evaluation in cancer treatment. Although the antiproliferative effect of flavopiridol has been attributed to the inhibition of cyclin-dependent kinases 2 and 4, recent reports indicate that the mechanism responsible for the cell death induced by this agent is more complex. To provide insight into the molecular processes mediating flavopiridol-induced cytotoxicity and to investigate the availability of markers indicative of its activity, we have applied cDNA microarray technology. Gene expression profiles were determined for four human tumor cell lines (prostate carcinomas PC3 and DU145 and gliomas SF359 and U251) following exposure to selected concentrations of flavopiridol. Treatment of these cell lines with a concentration of flavopiridol sufficient to reduce survival to 10% resulted in the identification of a set of 209 genes, the expression of which were altered in each of the cell lines. This common set of 209 gene expression changes suggested that flavopiridol-induced cell death can be defined in terms of a specific transcriptome. The flavopiridol death transcriptome consisted primarily of down-regulated genes; however, there were also a significant number of genes with increased expression. Whereas causal relationships were not established, these data suggest molecular events/processes that may be associated with flavopiridol-induced tumor cell death. Moreover, the identification of a set of gene expression changes in four human tumor cell lines suggests that such a transcriptome may be applicable to investigations of flavopiridol pharmacodynamics.

Gene profiling and the cyclin-dependent kinase inhibitor flavopiridol: What's in a name?

Gene profiling and the cyclin-dependent kinase inhibitor flavopiridol: What's in a name?

Androgen-induced recruitment of RNA polymerase II to a nuclear receptor-p160 coactivator complex.

The androgen receptor, like other nuclear receptors, activates target genes by binding to hormone-responsive enhancers. Here we demonstrate that androgen induces robust recruitment of androgen receptor, members of the p160 coactivator family, and CREB-binding protein p300 specifically at the distant enhancer of prostate-specific antigen (PSA) gene. Unexpectedly, we found that RNA polymerase II (Pol II) is directly recruited to the enhancer in a hormone-dependent manner, independent of the proximal promoter, and that the isolated PSA enhancer can mediate efficient androgen induction of transcription. Inhibition of the Pol II carboxyl-terminal domain kinase activity with low concentrations of flavopiridol blocks Pol II transfer from the enhancer to the promoter and selectively abolishes PSA induction by androgen. Moreover, elevated levels of the p160 coactivator ACTR/AIB1 increase both androgen-dependent and -independent PSA expression, by facilitating Pol II recruitment to the enhancer. These results support a model in which nuclear receptors and their coactivators mediate hormone induction by serving as a staging platform for Pol II recruitment.

Flavopiridol Inversely Affects p21WAF1/CIP1 and p53 and Protects p21-Sensitive Cells from Paclitaxel

Resting cells are relatively resistant to microtubule-active drugs including paclitaxel (PTX). By causing p53-mediated arrest, pretreatment with low concentrations of doxorubicin (DOX) protected HCT116 cells from the cytotoxicity caused by PTX. Unlike DOX, flavopiridol (FL) did not protect HCT116 cells. Low concentrations of FL (50 nM) induced p21 but not p53. High concentrations of FL (500 nM) decreased levels of p21 and Mdm-2 but dramatically induced p53. Thus, FL reciprocally affects p21 and p53. In LNCaP, a prostate cancer cell line which is highly sensitive to p21-induced growth arrest (p21-sensitive), low concentrations of FL (50 nM) induced p21 (without induction of p53) and caused G1 and G2 arrest. This precluded mitotic arrest, Bcl-2 and Raf-1 phosphorylation, and diminished cell death caused by PTX. In contrast, FL did not protect PC3M, arrest-resitant and highly aggressive prostate cancer cells. Like LNCaP, HL60 and SKBr3 cells are known to be p21-sensitive. As predicted, low concentrations of FL antagonized PTX-mediated cytotoxicity in HL60 and SKBr3 cell lines. In summary, only low concentrations of FL can induce p21, and, in turn, only p21-sensitive cells are protected from PTX.

Flavopiridol Induces Apoptosis and Caspase-3 Activation of a Newly Characterized Burkitt's Lymphoma Cell Line Containing Mutant P53 Genes

ABSTRACTBurkitt's lymphoma cell lines have been important in vitro models for studying the pathogenesis of Burkitt's lymphoma (BL) and for exploring new treatment strategies. A new EBV(−) Burkitt's lymphoma cell line (GA-10) was established from a patient with a clinically aggressive, chemorefractory BL and characterized. Although functional p-glycoprotein could not be demonstrated by dye-efflux assays, both p53 genes were mutated in the GA-10 cells, perhaps contributing to the resistant phenotype of the original neoplasm. Two properties of BL cells which may be useful targets for novel cytotoxic therapeutics are their surface expression of CD77, the receptor for Shiga toxin (Stx), and their high rate of proliferation. Expression of CD77 on the GA-10 cells was heterogeneous in that certain subclones expressed high levels of CD77 and correspondingly exhibited strong growth inhibition by Stx while others showed low levels of CD77 expression and weak Stx-induced growth inhibition. Flavopiridol, a potent inhibitor of cell cycle progression through G1 and G2, induced cytotoxicity of the GA-10 cells with an LC50 of approximately 40 nM vs 70 nM for HL-60 cells (P < 0.05). The concentrations of flavopiridol at which only 10% of the cells were viable (LC10) were approximately 280 nM for the GA-10 cells and 520 nM for the HL-60 cells (P < 0.05). Dose-related induction of apoptosis in response to flavopiridol was demonstrated in the GA-10 cells by morphology, TUNEL assay, and activation of caspase-3. Flavopiridol was also cytotoxic to seven other BL cell lines tested. These data suggest that flavopiridol may have therapeutic value in the treatment of Burkitt's lymphoma.

Flavopiridol: the first cyclin-dependent kinase inhibitor in human clinical trials.

The discovery and cloning of the cyclin-dependent kinases (cdks), main regulators of cell cycle progression, allowed several investigators to design novel modulators of cdk activity. Flavopiridol (HMR 1275, L86-8275), a flavonoid derived from an indigenous plant from India, demonstrated potent and specific in vitro inhibition of all cdks tested (cdks 1, 2, 4 and 7) with clear block in cell cycle progression at the G1/S and G2/M boundaries. Moreover, preclinical studies demonstrated the capacity of flavopiridol to induce programmed cell death, promote differentiation, inhibit angiogenic processes and modulate transcriptional events. The relationship between the latter effects and cdk inhibition is still unclear. Initial testing in early clinical human trials with infusional flavopiridol showed activity in some patients with non-Hodgkin's lymphoma, renal, prostate, colon and gastric carcinomas. Main side effects were secretory diarrhea and a pro-inflammatory syndrome associated with hypotension. Biologically active plasma concentrations of flavopiridol (approximately 300-500 nM) are easily achievable in patients receiving infusional flavopiridol. Phase 2 trials with infusional flavopiridol in several tumor types, other schedules and combination with standard chemotherapies are being assessed. In conclusion, flavopiridol is the first cdk inhibitor to be tested in clinical trials. Although important questions remain to be answered, this positive experience will stimulate the development of novel cdk modulators for cancer therapy.