alvocidib and Carcinoma--Small-Cell

Small-cell lung cancers (SCLC) are defective in many regulatory mechanisms that control cell cycle progression, i.e., functional retinoblastoma protein (pRb). Flavopiridol inhibits proliferation and induces apoptosis in SCLC cell lines. We hypothesized that the sequence flavopiridol followed by doxorubicin would be synergistic in pRb-deficient SCLC cells.. A H69 pRb-deficient SCLC cell line, H865, with functional pRb and H865 pRb small interfering RNA (siRNA) knockdown cells were used for in vitro and in vivo experiments. The in vivo efficiencies of various sequential combinations were tested using nude/nude athymic mice and human SCLC xenograft models.. Flavopiridol then doxorubicin sequential treatment was synergistic in the pRB-negative H69 cell line. By knocking down pRb with specific siRNA, H865 clones with complete pRb knockdown became sensitive to flavopiridol and doxorubicin combinations. pRb-deficient SCLC cell lines were highly sensitive to flavopiridol-induced apoptosis. pRb-positive H865 cells arrested in G0-G1 with flavopiridol exposure, whereas doxorubicin and all flavopiridol/doxorubicin combinations caused a G2-M block. In contrast, pRb-negative SCLC cells did not arrest in G0-G1 with flavopiridol exposure. Flavopiridol treatment alone did not have an in vivo antitumor effect, but sequential flavopiridol followed by doxorubicin treatment provided tumor growth control and a survival advantage in Rb-negative xenograft models, compared with the other sequential treatments.. Flavopiridol and doxorubicin sequential treatment induces potent in vitro and in vivo synergism in pRb-negative SCLC cells and should be clinically tested in tumors lacking functional pRB.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Small Cell; Cell Cycle; Cell Line, Tumor; Doxorubicin; Flavonoids; Genes, Retinoblastoma; Humans; Lung Neoplasms; Male; Mice; Piperidines; RNA, Small Interfering; Xenograft Model Antitumor Assays

Flavopiridol is one of the first cyclin-dependent kinase inhibitors undergoing clinical tests. We found that the combination treatment of flavopiridol (100-500 nM) with tumor necrosis factor (TNF)-alpha (10 ng/ml) induced a rapid and eminent apoptosis, 20 +/- 5% in 6-h treatment, in a human non-small cell lung carcinoma cell line, A549, as determined by the increase of sub-G(1) fraction in flow cytometry. A similar observation was also made in human colon cancer cell lines, HCT-116 and HCT-15, but not in Rat2, a rat fibroblast cell line. In A549 cells, the cytotoxic synergy by the combination treatment involved the activation of caspase-1, caspase-3, and caspase-8 and generated huge chromosomal degradation. The treatment schedules were so important that only the treatments of flavopiridol concomitantly with or followed by TNF-alpha showed the pronounced apoptosis in A549 cells. Prior treatment of TNF-alpha inhibited the apoptosis by the following combination treatment, leading to little cell death. Yet, such inhibition was reversed when 100 microM of 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, a transcription inhibitor, was present during the TNF-alpha pretreatment, suggesting that the inhibitory pretreatment of TNF-alpha might involve antiapoptotic gene expression at the transcriptional level. TNF-alpha treatment resulted in nuclear factor (NF)-kappa B activation, revealed by NF-kappa B activity reporter assay. In contrast, flavopiridol was found to inhibit the NF-kappa B-dependent gene transcription, which might give an explanation for the synergistic effect of flavopiridol with TNF-alpha. TNF-related apoptosis-inducing ligand (TRAIL; 100 ng/ml) also caused a rapid and strong cytotoxic synergy with flavopiridol. In contrast to TNF-alpha, however, all of the treatment sequences supported the synergy by TRAIL and flavopiridol. The combination of flavopiridol with TNF-alpha or TRAIL may be of use for the development in cancer therapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Small Cell; Caspase Inhibitors; Caspases; Colonic Neoplasms; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Activation; Fibroblasts; Flavonoids; Humans; Lung Neoplasms; Membrane Glycoproteins; NF-kappa B; Piperidines; Rats; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Transcriptional Activation; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Accumulating evidence indicates that small cell lung cancer (SCLC) is defective in many of the regulatory mechanisms that control cell cycle progression. The purpose of this study was to determine the effects of flavopiridol, a pan-cyclin-dependent kinase inhibitor, on growth and apoptosis of SCLC cell lines.. Cell growth was monitored using 3-(4,5dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) and clonogenic assays. Induction of apoptosis was assessed using multiple assays, including flow cytometric determination of DNA content and mitochondrial membrane potential, terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL), and Western blot analysis of procaspase 3 and poly(ADP-ribose) polymerase cleavage.. Flavopiridol induced growth inhibition and cytotoxicity in multiple SCLC cell lines, with an IC(50) of 50-100 nM and an LD(50) of 150-200 nM in 72-h MTT assays. The cytotoxicity seen in the MTT assay proved to be apoptosis by several criteria. Interestingly, inhibition of caspase activation with the caspase inhibitor Boc-Asp(OMe)-CH(2)F reduced TUNEL labeling by 40% but did not have any effect on the loss of mitochondrial membrane potential (detected as early as 4 h after drug exposure) or cytotoxicity in MTT assays. These results suggest that the primary event in flavopiridol-induced apoptosis involves induction of mitochondrial dysfunction. Cells synchronized with aphidicolin at the G(1)-S border and treated with flavopiridol during S phase showed a marked increase in apoptosis compared with an asynchronous population or a population treated during G(2)-M. Despite the increased apoptosis, a significant proportion of synchronized cells proceeded through S, G(2)-M, and into G(1) phase in the presence of flavopiridol, demonstrating that a high-grade cell cycle arrest is not required for apoptosis. Cells synchronized at the G(1)-S border treated with a short exposure to flavopiridol also showed more than a 10-fold decrease in clonogenicity compared with asynchronous cells treated identically.. Taken together, these data demonstrate that flavopiridol potently and selectively induces SCLC apoptosis preferentially during S phase, in a manner that involves early mitochondrial dysfunction without a requirement for a high-grade block to cell cycle progression. Furthermore, clonogenicity data suggests that prior S phase synchronization could be a highly effective way of enhancing the efficacy of bolus or short infusions of flavopiridol in the clinical setting.

Topics: Aphidicolin; Apoptosis; Blotting, Western; Carcinoma, Small Cell; Caspase 3; Caspases; Cell Division; Enzyme Inhibitors; Flavonoids; Flow Cytometry; Humans; In Situ Nick-End Labeling; Lung Neoplasms; Membrane Potentials; Mitochondria; Piperidines; Poly(ADP-ribose) Polymerases; S Phase; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured; Tumor Stem Cell Assay

The multidrug resistance protein (MRP) is an ATP-dependent transport protein for organic anions, as well as neutral or positively charged anticancer agents. In this study we report that dinitrophenyl-S-glutathione increases ATPase activity in plasma membrane vesicles prepared from the MRP-overexpressing cell line GLC4/ADR. This ATPase stimulation parallels the uptake of DNP-SG in these vesicles. We also show that the (iso)flavonoids genistein, kaempferol and flavopiridol stimulate the ATPase activity of GLC4/ADR membranes, whereas genistin has no effect. The present data are consistent with the hypothesis that certain (iso)flavonoids affect MRP-mediated transport of anticancer drugs by a direct interaction with MRP.

Topics: Adenosine Triphosphatases; ATP-Binding Cassette Transporters; Carcinoma, Small Cell; Cell Membrane; Drug Resistance, Multiple; Flavonoids; Genistein; Glutathione; Humans; Isoflavones; Kaempferols; Lung Neoplasms; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Piperidines; Quercetin; Tumor Cells, Cultured