Anticancer effect of tetrandrine on primary cancer cells isolated from ascites and pleural fluids

Objective

The present study was designed to examine the effect of tetrandrine on primary cancer cells isolated from the ascites or pleural fluids of patients with metastatic cancers.

Methods

Primary cancer cells were isolated from the pleural fluids (n = 13) or ascites (n = 21) of patients. Compared with culture cell lines, the response of these cancer cells to tetrandrine and chemotherapeutic agents commonly used in clinical practice were determined by WST-8 assay. Tetrandrine-induced apoptosis in primary cancer cells was determined by Annexin V-FITC assay. Quantitative RT-PCR was used to examine the role of apoptotic associated genes in the anticancer effect of tetrandrine.

Results

The primary cancer cells isolated from effusions showed sensitivity to tetrandrine with IC₅₀ values of 38.23 ± 25.77 μM, similar to the IC₅₀ in established cell lines. Patients with gastric cancers were more sensitive to tetrandrine than patients with lung cancers (P = 0.04). Four cancer cells isolated from effusions were resistant to tetrandrine, which also had increased tolerance to docetaxel, cisplatin and 5-fluorouracil. We also observed a weak but significant correlation between sensitivity to tetrandrine and cellular expression of bcl-2 (P = 0.035, r = −0.364).

Conclusions

Using cancer cells isolated from the ascites or pleural fluids, this study shows the potential anticancer effect of tetrandrine against primary cancer cells.     

灵芝孢子油对肺腺癌癌性胸水中原代肿瘤细胞的抗肿瘤作用

目的:研究灵芝孢子油对肺腺癌的癌性胸水中原代肿瘤细胞的抗癌作用。方法:体外培养肺腺癌细胞系A549,用CCK-8法检测灵芝孢子油对A549细胞的体外抗肿瘤作用。收集12例经病理确诊的肺腺癌患者的癌性胸水分离原代肿瘤细胞,用CCK-8法检测灵芝孢子油及临床常用化疗药物对原代肿瘤细胞的药物敏感性;流式细胞仪检测灵芝孢子油对原代肿瘤细胞的凋亡诱导作用,RT-PCR检测灵芝孢子油对凋亡相关蛋白基因bax及bc l-2 mRNA表达的影响。结果:灵芝孢子油对5例癌性胸水中原代肿瘤细胞的IC50平均值为0.28±0.04μl/m l,与对A549细胞的IC500.46±0.08μl/m l无显著差异(P>0.05)。在各测试药物浓度(TDC)下,灵芝孢子油、多西紫杉醇、顺铂和健择对原代肿瘤细胞的抑制率分别为48.7%±3.3%,31.6%±14.1%,30.2%±11.6%,51.4%±5.6%,灵芝孢子油与这些化疗药物的抗肿瘤作用无显著差异(P>0.05)。灵芝孢子油显著促进原代肿瘤细胞凋亡,对原代肿瘤细胞中凋亡相关蛋白bax mRNA有明显上调作用,对bc l-2 mRNA有明显下调作用。结论:灵芝孢子油对肺腺癌的癌性胸水原代肿瘤细胞有较好的抗癌作用。     

灵芝孢子油对肺腺癌癌性胸水中原代肿瘤细胞的抗肿瘤作用

目的：研究灵芝孢子油对肺腺癌的癌性胸水中原代肿瘤细胞的抗癌作用。方法：体外培养肺腺癌细胞系A549,用CCK-8法检测灵芝孢子油对A549细胞的体外抗肿瘤作用。收集12例经病理确诊的肺腺癌患者的癌性胸水分离原代肿瘤细胞,用CCK-8法检测灵芝孢子油及临床常用化疗药物对原代肿瘤细胞的药物敏感性;流式细胞仪检测灵芝孢子油对原代肿瘤细胞的凋亡诱导作用,RT-PCR检测灵芝孢子油对凋亡相关蛋白基因bax及bc l-2 mRNA表达的影响。结果：灵芝孢子油对5例癌性胸水中原代肿瘤细胞的IC50平均值为0.28±0.04μl/m l,与对A549细胞的IC500.46±0.08μl/m l无显著差异（P〉0.05）。在各测试药物浓度（TDC）下,灵芝孢子油、多西紫杉醇、顺铂和健择对原代肿瘤细胞的抑制率分别为48.7%±3.3%,31.6%±14.1%,30.2%±11.6%,51.4%±5.6%,灵芝孢子油与这些化疗药物的抗肿瘤作用无显著差异（P〉0.05）。灵芝孢子油显著促进原代肿瘤细胞凋亡,对原代肿瘤细胞中凋亡相关蛋白bax mRNA有明显上调作用,对bc l-2 mRNA有明显下调作用。结论：灵芝孢子油对肺腺癌的癌性胸水原代肿瘤细胞有较好的抗癌作用。     

The cell cycle as a target for cancer therapy: basic and clinical findings with the small molecule inhibitors flavopiridol and UCN-01

Many tumor types are associated with genetic changes in the retinoblastoma pathway, leading to hyperactivation of cyclin-dependent kinases and incorrect progression through the cell cycle. Small-molecule cyclin-dependent kinase inhibitors are being developed as therapeutic agents. Of these, flavopiridol and UCN-01 are being explored in cancer patients in phase I and phase II clinical trials, both as single agents and in combination with conventional chemotherapeutic agents. The present article discusses the mechanisms of action of flavopiridol and UCN-01 as well as the outcome of clinical trials with these novel agents.     

Induction of apoptosis and inhibition of c-erbB-2 in breast cancer cells by flavopiridol.

Flavopiridol is a flavone that inhibits several cyclin-dependent kinases and exhibits potent growth-inhibitory activity against a number of human tumor cell lines, both in vitro and when grown as xenografts in mice. It is presently being investigated as a novel antineoplastic agent in the primary screen conducted by the Developmental Therapeutics Program, National Cancer Institute. Because breast cancer is the most common cancer and second leading cause of cancer-related deaths in women in the United States, we investigated whether flavopiridol could be an effective agent against a series of isogenic breast- cancer cell lines having different levels of erbB-2 expression and differential invasion and metastatic characteristics. Flavopiridol was found to inhibit the growth of MDA-MB-435 (parental) and 435.eB (stable transfectants) cells that were established by transfecting c-erbB-2 cDNA into MDA-MB-435. Induction of apoptosis was also observed in these cell lines when treated with flavopiridol, as measured by DNA laddering, PARP, and CPP32 cleavages. We also found modest up-regulation of Bax and down-regulation of Bcl-2, but there was a significant down-regulation of c-erbB-2 in flavopiridol-treated cells. Gelatin zymography showed that flavopiridol inhibits the secretion of matrix metalloproteinase (MMP; MMPs 2 and 9) in the breast cancer cells and that the inhibition of c-erbB-2 and MMPs may be responsible for the inhibition of cell invasion observed in flavopiridol-treated cells. Collectively, these molecular effects of flavopiridol, however, were found to be independent of c-erbB-2 overexpression, suggesting that flavopiridol may be effective in all breast cancer. From these results, we conclude that flavopiridol inhibits the growth of MDA-MB-435 breast cancer cells, induces apoptosis, regulates the expression of genes, and inhibits invasion and, thus, may inhibit metastasis of breast cancer cells. These findings suggest that flavopiridol may be an effective chemotherapeutic or preventive agent against breast cancer.     

Selective sensitization of transformed cells to flavopiridol-induced apoptosis following recruitment to S-phase

Flavopiridol is a potent inhibitor of cyclin-dependent kinases (cdks). In a large proportion of solid tumor cell lines, the initial response to flavopiridol is cell cycle arrest. NCI-H661 non-small cell lung cancer cells are representative of a subset of more sensitive cell lines in which apoptosis is observed during the first 24 h of drug exposure. Analysis of the apoptotic population indicates that cells in S-phase are preferentially dying. In addition, cells are sensitized to flavopiridol following recruitment to S-phase, whether accomplished by synchronization or by treatment with noncytotoxic concentrations of chemotherapy agents that impose an S-phase delay. Combinations of gemcitabine or cisplatin, followed by flavopiridol at concentrations that correlate with cdk inhibition, produce sequence-dependent cytotoxic synergy. A survey of paired cell lines, including WI38 diploid fibroblasts or normal human bronchial epithelial cells, along with their SV40-transformed counterparts, demonstrates that treatment with flavopiridol during S-phase is selectively cytotoxic to transformed cells. These data suggest that treatment during S-phase may maximize responses to flavopiridol and that the administration of flavopiridol after chemotherapy agents that cause S-phase accumulation may be an efficacious antitumor strategy.     

Combining flavopiridol with various signal transduction inhibitors

Treatment of human tumors with a combination of chemotherapeutic agents results in improved response as well as the ability to use less toxic concentrations of the drugs. Recent phase I clinical trials with the cyclin-dependent kinase inhibitor, flavopiridol, have shown some promise in the treatment of a variety of human tumors. Because of the severe toxicity, however, the use of less toxic doses in combination with other antiproliferative agents would be desirable. The purpose of this study was to examine the effects of combining flavopiridol with several signal transduction inhibitors: the SC236 COX-2 inhibitor, a PKC kinase inhibitor and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor in a control vector transfected MCF-7 human breast cancer cell line (MCF/neo) and a HER-2/neu transfected MCF-7 cell line (MCF/18). Enhanced (better than that seen with either agent alone but not additive) growth inhibition was observed in both cell lines with the combination of flavopiridol and the PKC kinase inhibitor. The combination of flavopiridol and the SC236 COX-2 inhibitor resulted in an enhanced effect in the MCF/18 cell line and a synergistic effect in the MCF/neo cells. The combination of flavopiridol and LY294002 resulted in a synergistic effect in the MCF/18 cell line and an additive effect in the MCF/neo cells. These data suggest that combinations of flavopiridol and signal transduction inhibitors warrant further studies as treatments for breast tumors, and that HER-2/neu expression may influence the choice of inhibitor to combine with flavopiridol.     

Induction of growth inhibition and apoptosis in prostate cancer cells by flavopiridol

Flavopiridol is an inhibitor of several cyclin-dependent kinases, and exhibits potent growth-inhibitory activity against a number of human tumor cell lines both in vitro, and when grown as xenografts in mice. It has shown promising antineoplastic activity and is currently undergoing clinical phase II testing. Prostate cancer (PCa) remains a leading cause of morbidity and mortality among males in the United States. There are no effective treatments for hormone and/or radiation refractory PCa, suggesting that novel and newer treatment strategy may be useful in the management of PCa. Our previous study showed that flavopiridol induces cell growth inhibition and apoptosis in breast cancer cells. Here, we investigated whether flavopiridol was effective against prostate cancer cells. Flavopiridol was found to inhibit growth of PC3 prostate cancer cells. Induction of apoptosis was also observed in PC3 cells treated with flavopiridol, as measured by DNA laddering and PARP cleavage. We also found a significant down-regulation of Bcl-2 in flavopiridol-treated cells. These findings suggest that down-regulation of Bcl-2 may be one of the molecular mechanisms through which flavopiridol induces apoptosis and inhibits cell growth, suggesting that flavopiridol may be an effective chemotherapeutic agent against prostate cancer.     

CDKI-71, a novel CDK9 inhibitor, is preferentially cytotoxic to cancer cells compared to flavopiridol

Cancer cells appear to depend heavily on antiapoptotic proteins for survival and so targeted inhibition of these proteins has therapeutic potential. One innovative strategy is to inhibit the cyclin-dependent kinases (CDKs) responsible for the regulation of RNA polymerase II (RNAPII). In our study, we investigated the detailed cellular mechanism of a novel small-molecule CDK inhibitor (CDKI-71) in cancer cell lines, primary leukemia cells, normal B - & T- cells, and embryonic lung fibroblasts and compared the cellular and molecular responses to the clinical CDK inhibitor, flavopiridol. Like flavopiridol, CDKI-71 displayed potent cytotoxicity and caspase-dependent apoptosis induction that were closely associated with the inhibition of RNAPII phosphorylation at serine-2. This was caused by effective targeting of cyclinT-CDK9 and resulted in the downstream inhibition of Mcl-1. No correlation between apoptosis and inhibition of cell-cycle CDKs 1 and 2 was observed. CDKI-71 showed a 10-fold increase in potency in tumor cell lines when compared to MRC-5 human fibroblast cells. Significantly, CDKI-71 also demonstrated potent anti-chronic lymphocytic leukemia activity with minimal toxicity in normal B- and T-cells. In contrast, flavopiridol showed little selectivity between cancer and normal cells. Here, we provide the first cell-based evidence that flavopiridol induces DNA double-strand breaks: a fact which may explain why flavopiridol has such a narrow therapeutic window in preclinical and clinical settings. Taken together, our data provide a rationale for the development of selective CDK inhibitors as therapeutic agents and CDKI-71 represents a promising lead in this context.     

The effect of flavopiridol on the growth of p16+ and p16- melanoma cell lines

Flavopiridol is the first cyclin-dependent kinase inhibitor to enter clinical trials. Flavopiridol has been shown to mimic, in part, the effect of the cell cycle control gene p16, which is frequently lost or mutated in malignant melanoma, making it an ideal candidate for targeted therapy in this disease. In these studies we investigated the effect of flavopiridol, at various concentrations, on the growth and gene expression of nine human melanoma cell lines with intact, absent or mutated p16. A cytostatic effect of flavopiridol on the growth of six melanoma cell lines with a mutated or non-expressed p16 (p16-) was seen at low concentrations of flavopiridol (mean 50% inhibitory concentration [IC(50)] = 12.5 nM), while the three melanoma cell lines with intact p16 (p16+) required higher concentrations (mean IC(50) = 25 nM) to produce this effect. Apoptotic cell death increased with increasing concentrations of flavopiridol in both p16- and p16+ cells. Exposure of cells to high flavopiridol concentrations (>100 nM) resulted in decreased expression of genes downstream in the normal p16 cell cycle control pathway (Rb and E2F) and the anti-apoptotic gene BCL2. No change in BCL2 expression was found after exposure to IC(50) concentrations of flavopiridol. These data indicate that flavopiridol in low, clinically achievable concentrations may have significant cytostatic effects, particularly in p16- melanoma cells, and may provide new molecular-based therapies for melanoma, particularly when combined with agents that target anti-apoptotic mechanisms.     

Growth inhibition and apoptosis of myeloma cells by the CDK inhibitor flavopiridol.

Although myeloma shows responsiveness in intensive chemotherapy, overall survival remains less than 40% at 2 years. Since myeloma appears to be dependent on cytokines, such as IL-6, we hypothesized that targeting signal transduction molecules could effectively treat myeloma. Two myeloma cell lines U266 and RPMI-8226 and CD38+ myeloma cells were studied by immune complex kinase assay or anti-phosphotyrosine blot for evidence of constitutive activation of tyrosine kinases. Growth arrest and apoptosis were evaluated in these two cell lines following their treatment with specific kinase inhibitors. We found that a variety of Src and Janus kinases were present and constitutively active in U266 and RPMI-8226 cells. Inhibitors of both Src and Janus kinases were inferior to the cyclin-dependent kinase inhibitor, flavopiridol, in inducing both growth arrest with GI50 of 100 nM and apoptosis in both cell lines and CD38+ myeloma cells. Although, flavopiridol did not affect cyclin D1 and cyclin A levels, it inhibited Mcl-1 and Bcl-2 protein levels and cyclin-dependent kinase 2 activity. Flavopiridol is a well-tolerated drug, currently in phase I-II trials for a variety of tumors. A clinical trial using flavopiridol should be performed in patients with myeloma. Its mechanism of action may involve targets other than the cyclin-dependent kinases.     

Flavopiridol induces cellular FLICE-inhibitory protein degradation by the proteasome and promotes TRAIL-induced early signaling and apoptosis in breast tumor cells

The cyclin-dependent kinase inhibitor flavopiridol is undergoing clinical trials as an antitumor drug. We show here that pretreatment of different human breast cancer cell lines with flavopiridol facilitates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. In breast tumor cells, apoptosis induction by TRAIL is blocked at the level of apical caspase-8 activation. Flavopiridol treatment enhances TRAIL-induced formation of death-inducing signaling complex and early processing of procaspase-8. Subsequently, a TRAIL-induced, mitochondria-operated pathway of apoptosis is activated in cells treated with flavopiridol. Down-regulation of cellular FLICE-inhibitory proteins (c-FLIP; c-FLIP(L) and c-FLIP(S)) is observed on flavopiridol treatment. c-FLIP loss and apoptosis sensitization by flavopiridol are both prevented in cells treated with an inhibitor of the ubiquitin-proteasome system. Furthermore, targeting c-FLIP directly with small interfering RNA oligonucleotides also sensitizes various human breast tumor cell lines to TRAIL-induced apoptosis. Our results indicate that flavopiridol sensitizes breast cancer cells to TRAIL-induced apoptosis by facilitating early events in the apoptotic pathway, and this combination treatment could be regarded as a potential therapeutic tool against breast tumors.     

Seventy-two hour continuous infusion flavopiridol in relapsed and refractory mantle cell lymphoma

The cell cycle regulatory protein cyclin D1, which is over-expressed in 95-100% of mantle cell lymphomas (MCL), is a potential therapeutic target. Flavopiridol inhibits the cyclin-dependent kinase (CDK)4-cyclin D1 complex and induces apoptosis in lymphoma cell lines. Previous phase I clinical studies had demonstrated that this drug could be safely administered in humans, prompting further evaluation of flavopiridol as a single agent in MCL. Ten patients with relapsed or refractory MCL, who had received one prior chemotherapy regimen, were treated with flavopiridol 50 mg/m2/day given as a 72 h continuous intravenous infusion every 14 days. Treatment was well tolerated, and only one patient developed grade III-IV non-hematologic toxicity. However, there were no clinical responses; despite therapy, three patients maintained stable disease, and seven patients demonstrated progressive disease within two months. In relapsed and refractory MCL, flavopiridol is ineffective as a single agent given by 72 h continuous infusion at 50 mg/m2/day. Recent in vitro studies using human plasma suggest that higher plasma drug levels may be necessary to achieve clinical efficacy. In vitro studies of flavopiridol indicate that the agent is synergistic with DNA-damaging compounds. Further investigation into flavopiridol as a clinical agent should focus on alternative dosing schedules and the compound's potential use in combination chemotherapeutic regimens.     

Rhabdoid tumor growth is inhibited by flavopiridol.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.     

ONYX-015 works synergistically with chemotherapy in lung cancer cell lines and primary cultures freshly made from lung cancer patients

p53 mutations and loss of heterozygosity (LOH) have been detected in >50% of lung cancers. Wild-type p53 can prevent replication of damaged DNA and promote apoptosis of cells with abnormal DNA. A human adenovirus, ONYX-015, which has a deletion in the E1B region, has shown tumor-specific cytolytic effect in tumor cells with nonfunctional p53 and antitumor efficacy that can be augmented by chemotherapeutic agents. A recent report from an independent group, however, indicates that wild-type p53 is necessary for the infection of this replicating virus, and it is in direct contradiction to previous observations of the ONYX group. In this study, we carried out cytopathic effect (CPE) assays using ONYX-015 on five human lung cancer cell lines with known p53 status. Two of these cell lines, NCI-H522 and NCI-H1703, have mutations and LOH in their p53 gene. Both lines were lysed in a dose-dependent manner and showed 100% cytolysis at a multiplicity of infection of 0.1. Two additional cell lines, NCI-H2347 and NCI-H838, both of which have wild-type p53 gene, showed near complete lysis at a multiplicity of infection of 1. We demonstrate here that the lung cancer cells with nonfunctional p53 are at least 10 times more sensitive to ONYX-015 cytolysis than the lung cancer cells with wild-type p53. In addition, standard chemotherapeutic agents (paclitaxol and cisplatin) showed a synergistic effect when combined with ONYX-015, and this effect was p53 mutant dependent. Furthermore, we tested the cytolytic effect of ONYX-015 on a panel (n = 7) of primary first-passage cultures made from freshly resected lung cancers. ONYX-015 lysed primary lung cancer cells in six of seven (86 %) primary cultures. Two of four primary cultures treated with chemotherapeutic agents had a synergistic effect with ONYX-015. Our data indicate that wild-type p53 is not required for the infection of this replicating virus, and also we demonstrate that ONYX-015 is effective alone and works synergistically with chemotherapeutic agents in lung cancer cell lines and primary cultures. This study suggests that ONYX-015 may be effective, especially in combination with conventional chemotherapy, in the treatment of patients with lung cancer.     

Effect of P-glycoprotein on flavopiridol sensitivity

Flavopiridol is the first potent inhibitor of cyclin-dependent kinases (CDKs) to enter clinical trials. Little is known about mechanisms of resistance to this agent. In order to determine whether P-glycoprotein (Pgp) might play a role in flavopiridol resistance, we examined flavopiridol sensitivity in a pair of Chinese hamster ovary cell lines differing with respect to level of Pgp expression. The IC(50)s of flavopiridol in parental AuxB1 (lower Pgp) and colchicine-selected CH(R)C5 (higher Pgp) cells were 90.2 +/- 6.6 nM and 117 +/- 2.3 nM, respectively (P< 0.01), suggesting that Pgp might have a modest effect on flavopiridol action. Consistent with this hypothesis, pretreatment with either quinidine or verapamil (inhibitors of Pgp-mediated transport) sensitized CH(R)C5 cells to the antiproliferative effects of flavopiridol. Because of concern that colony forming assays might not accurately reflect cytotoxicity, we also examined flavopiridol-treated cells by trypan blue staining and flow cytometry. These assays confirmed that flavopiridol was less toxic to cells expressing higher levels of Pgp. Further experiments revealed that flavopiridol inhibited the binding of [3H]-azidopine to Pgp in isolated membrane vesicles, but only at high concentrations. Collectively, these results identify flavopiridol as a weak substrate for Pgp.     

Sensitivity of 5-fluorouracil-resistant cancer cells to adenovirus suicide gene therapy

A promising approach for cancer gene therapy is the combination of adenovirus vectors (AdV) with the suicide gene cytosine deaminase and uracil phosphoribosyl transferase (CDColon, two colonsUPRT). While such vectors have been tested in tumor cell lines and xenograft models, it is not clear how these therapeutic vectors would perform in primary human tumors. We, thus, examined the effect of the combination of a recombinant adenovirus expressing the CDColon, two colonsUPRT (AdCU) with 5-fluorocytosine (5-FC) on primary cancer cells isolated from the ascites or pleural fluids of patients with metastatic cancers. In such models, we have found a direct correlation between the patients' response to 5-FU and the response shown by the cancer cells in vitro, confirming the clinical relevance of this methodology. Our findings demonstrated that this combination was able to kill primary tumor cells, including those that had developed resistance to 5-FU. Furthermore, while proliferating cells were more susceptible to 5-FU, the combination was effective in both rapid and slow proliferating samples. Our study demonstrated that this gene therapy approach could provide an effective therapeutic option for cancers and is not affected by acquired 5-FU resistance. Also of importance is the effectiveness of this gene therapy approach on slower proliferating cells that is typical of the majority of cancers in vivo. This suggests a greater likelihood that it will be effective in a clinical setting.     

Flavopiridol potently induces small cell lung cancer apoptosis during S phase in a manner that involves early mitochondrial dysfunction.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.