nitrophenols and Lung-Neoplasms

Lung cancer is the leading cause of cancer deaths throughout the world. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. In traditional anti-cancer therapy, promotion of apoptosis in NSCLC is an important part of treatment, but anticancer drugs have the toxic side effects, resistance and other problems. Therefore, the search for new targets of anticancer drugs becomes one of the foci in the treatment of NSCLC. The BH3-only protein plays an important role in activation and communication in apoptosis pathways. BIM is the core member in BH3-only protein family. The target at BIM in the treatment of NSCLC has an irreplaceable role. This paper briefly describes the BCL-2 family and BH3-only pro-apoptotic protein, elaborates the important role of BIM and BH3-only protein in targeted therapy of NSCLC.. 肺癌死亡率居全球恶性肿瘤死亡之首，非小细胞肺癌是肺癌中最常见的类型。在传统的抗肺癌治疗中，促进细胞凋亡是非小细胞肺癌治疗的一个重要组成部分，但抗肿瘤药物存在毒副作用大和耐药性等问题。因此，寻找新的抗肿瘤药物作用靶点成为非小细胞肺癌治疗的重点之一。BH3-only蛋白在凋亡的启动及凋亡通路的沟通中发挥极其重要的作用。BIM是BH3-only蛋白家族中的核心成员。以BIM为靶点的治疗在非小细胞肺癌的治疗中具有不可取代的作用。本文简单介绍了BCL-2家族和其中的BH3-only促凋亡蛋白，并且阐述了BIM、BH3-only蛋白在非小细胞肺癌靶向治疗中的重要作用。

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Carcinoma, Non-Small-Cell Lung; Humans; Lung Neoplasms; Mitochondrial Membrane Transport Proteins; Models, Biological; Molecular Targeted Therapy; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Tumor radioresistance and dose-limiting toxicity restrict the curative potential of radiotherapy, requiring novel approaches to overcome the limitations and augment the efficacy. Here, we investigated the effects of signal transducer and activator of transcription 3 (STAT3) activation and autophagy induction by irradiation on antiapoptotic proteins and the effectiveness of the BH3 mimetic ABT-737 as a radiosensitizer using K-ras mutant non-small cell lung cancer (NSCLC) cells and a Kras. A549 and H460 cells were irradiated, and the expression of Bcl-2 family proteins, JAK/STAT transcriptional pathway, and autophagic pathway were evaluated by immunoblotting. The radiosensitizing effects of ABT-737 were evaluated using A549 and H460 cell lines with clonogenic assays and also by a KP mouse model with microcomputed tomography and immunohistochemistry.. In A549 and H460 cells and mouse lung tissue, irradiation-induced overexpression of the antiapoptotic molecules Bcl-xL, Bcl-2, Bcl-w, and Mcl-1 through JAK/STAT transcriptional signaling induced dysfunction of the autophagic pathway. After treatment with ABT-737 and exposure to irradiation, the number of surviving clones in the cotreatment group was significantly lower than that in the group treated with radiation or ABT-737 alone. In the KP mouse lung cancer model, cotreatment with ABT-737 and radiation-induced significant tumor regression; however, body weight changes in the combination group were not significantly different, suggesting that combination treatment did not cause systemic toxicity.. These findings supported the radiosensitizing activity of ABT-737 in preclinical models, and suggested that clinical trials using this strategy may be beneficial in K-ras mutant NSCLC.

Topics: Animals; Apoptosis; Biphenyl Compounds; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Lung Neoplasms; Mice; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Radiation, Ionizing; Sulfonamides; X-Ray Microtomography; Xenograft Model Antitumor Assays

Despite the dramatic efficacy of ABT-737, a large percentage of cancer cells ultimately become resistance to this drug. Evidences show that over-expression of Mcl-1 is linked to ABT-737 resistance in NSCLC cells. The aim of this study was to investigate the effect of miRNA-101 on Mcl-1 expression and sensitivity of the A549 NSCLC cells to ABT-737.. After miRNA-101 transfection, the Mcl-1 mRNA expression levels were quantified by RT-qPCR. Trypan blue staining was used to explore the effect of miRNA-101 on cell growth. The cytotoxic effects of miRNA-101 and ABT-737, alone and in combination, were measured using MTT assay. The effect of drugs combination was determined using the method of Chou-Talalay. Cell death was assessed using cell death detection ELISA assay kit.. Results showed that miRNA-101 markedly suppressed the expression of Mcl-1 mRNA in a time dependent manner, which led to A549 cell proliferation inhibition and enhancement of apoptosis (p < 0.05, relative to blank control). Pretreatment with miRNA-101 synergistically decreased the cell survival rate and lowered the IC50 value of ABT-737. Furthermore, miRNA-101 dramatically enhanced the apoptotic effect of ABT-737. Negative control miRNA had no remarkable effect on cellular parameters.. Our findings propose that suppression of Mcl-1 by miRNA-101 can effectively inhibit the cell growth and sensitize A549 cells to ABT-737. Therefore, miRNA-101 can be considered as a potential therapeutic target in patients with non-small cell lung cancer.
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Topics: A549 Cells; Biphenyl Compounds; Carcinoma, Non-Small-Cell Lung; Genetic Therapy; Humans; Lung Neoplasms; MicroRNAs; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Sulfonamides

A subset of non-small cell lung cancer (NSCLC), which does not have a druggable driver mutation, is treated with platinum-based cytotoxic chemotherapy, but it develops resistance triggered by DNA damage responses. Here, we investigated the effect of activation of STAT3 by cisplatin on anti-apoptotic proteins and the effectiveness of a co-treatment with cisplatin and a BH3 mimetic, ABT-737. We analyzed the relationship between cisplatin and STAT3 pathway and effect of ABT-737, when combined with cisplatin in NSCLC cells and K-ras mutant mouse models. The synergism of this combination was evaluated by the Chou-Talalay Combination Index method. In vivo activity was evaluated by micro-CT. In NSCLC cells, there was a time and dose-dependent phosphorylation of SRC-JAK2-STAT3 by cisplatin, followed by increased expression of anti-apoptotic molecules. When the expression of the BCL-2 protein family members was evaluated in clinical samples, BCL-xL was most frequently overexpressed. Dominant negative STAT3 suppressed their expression, suggesting that STAT3 mediates cisplatin mediated overexpression of the anti-apoptotic molecules. ABT-737 displaced BCL-xL from mitochondria and induced oligomerization of BAK. ABT-737 itself showed cytotoxic effects and a combination of ABT-737 with cisplatin showed strong synergistic cytotoxicity. In a murine lung cancer model, co-treatment with ABT-737 and cisplatin induced significant tumor regression. These findings reveal a synergistic cytotoxic and anti-tumor activity of ABT-737 and cisplatin co-treatment in preclinical models, and suggest that clinical trials using this strategy may be beneficial in advanced NSCLC.

Topics: Aged; Animals; Apoptosis; Biomarkers; Biphenyl Compounds; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cisplatin; Disease Models, Animal; Drug Synergism; Female; Gene Expression; Humans; Immunohistochemistry; Lung Neoplasms; Male; Mice; Middle Aged; Neoplasm Grading; Neoplasm Staging; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; STAT3 Transcription Factor; Sulfonamides; Tumor Burden; Xenograft Model Antitumor Assays

Mesenchymal-type cancers after epithelial mesenchymal transition (EMT) were recently shown to acquire chemoresistance through expressing EMT specific transcription factors. However, druggable (or actionable) target(s) for chemoresistance in mesenchymal-type lung cancers remain unidentified. Here, we used a public clinical genomic database and mesenchymal lung cancer cells (MLCC) model derived from the A549 lung adenocarcinoma cell line to demonstrate that BCL2 expression, which is highly induced in mesenchymal-type lung cancers, as a predictor of poor prognosis in mesenchymal lung cancer patients and association with acquired chemoradioresistance. Thereby, combination treatment with BH3 mimetics, such as ABT-263 and ABT-737, clearly attenuated chemoresistance in MLCCs. BCL2 expression in MLCCs was induced by ERK1 activity through the upregulation of the MEK1/ERK1 scaffold protein MEK partner-1 (MP1). Interfering with the MEK1/MP1/ERK1 axis using a MEK1 inhibitor or MP1 depletion repressed BCL2 expression and sensitized MLCCs to chemoradiotherapy. Taken together, our results suggest that targeting druggable proteins in the MEK1/MP1/ERK1/BCL2 axis, such as MEK1 or BCL2, with currently available FDA approved drugs is a currently feasible approach to improve clinical outcomes of mesenchymal lung cancer patients.

Topics: A549 Cells; Adaptor Proteins, Signal Transducing; Adenocarcinoma; Adenocarcinoma of Lung; Aniline Compounds; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Biphenyl Compounds; Cell Proliferation; Cell Survival; Chemoradiotherapy; Diphenylamine; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Etoposide; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molecular Mimicry; Nitrophenols; Peptide Fragments; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Radiation Tolerance; RNA Interference; Signal Transduction; Sulfonamides; Transfection; Up-Regulation

Platinum based drugs are considered as effective agents against various types of carcinoma; however, the severe toxicity associated with the chemically prepared platinum complexes limit their practical applications. Similarly, water pollution caused by various organic moieties is another serious health problem worldwide. Hence, an intense need exists to develop new, effective and biocompatible materials with catalytic and biomedical applications. In the present contribution, we prepared platinum nanoparticles (PtNPs) by a green route using phytochemicals as a source of reducing and stabilizing agents. Well dispersed and crystalline PtNPs of spherical shapes were prepared and characterized. The bio-fabricated PtNPs were used as catalyst and anticancer agents. Catalytic performance of the PtNPs showed that 84% of the methylene blue can be reduced in 32min under visible light irradiation (K=0.078min

Topics: Antineoplastic Agents; Biological Transport; Catalysis; Cell Line, Tumor; Drug Stability; Humans; Lung Neoplasms; Metal Nanoparticles; Nitrophenols; Oxidation-Reduction; Particle Size; Platinum

Small cell lung cancer (SCLC) cannot be efficiently controlled using existing chemotherapy and radiotherapy approaches, indicating the need for new therapeutic strategies. Although ABT-737, a B-cell lymphoma-2 (BCL-2) inhibitor, exerts anticancer effects against BCL-2-expressing SCLC, monotherapy with ABT-737 is associated with limited clinical activity because of the development of resistance and toxicity. Here, we examined whether combination therapy with ABT-737 and heat shock protein 90 (HSP90) inhibitor NVP-AUY922 exerted synergistic anticancer effects on SCLC. We found that the combination of ABT-737 and NVP-AUY922 synergistically induced the apoptosis of BCL-2-expressing SCLC cells. NVP-AUY922 downregulated the expression of AKT and ERK, which activate MCL-1 to induce resistance against ABT-737. The synergistic effect was also partly due to blocking NF-κB activation, which induces anti-apoptosis protein expressions. However, interestingly, targeting BCL-2 and MCL-1 or BCL2 and NF-κB did not induce the cytotoxicity. In conclusion, our study showed that combination of BCL2 inhibitor with HSP90 inhibitor increased activity in in vitro and in vivo study in only BCL-2 expressing SCLC compared to either single BCL2 inhibitor or HSP inhibitor. The enhanced activity might be led by blocking several apoptotic pathways simultaneously rather than a specific pathway.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Biphenyl Compounds; Cell Line, Tumor; Drug Synergism; HSP90 Heat-Shock Proteins; Humans; Isoxazoles; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Random Allocation; Resorcinols; Signal Transduction; Small Cell Lung Carcinoma; Sulfonamides; Xenograft Model Antitumor Assays

Although activating apoptosis in cancer cells by targeting the mitochondria is an effective strategy for cancer therapy, insufficient targeting of the mitochondria in cancer cells restricts the availability in clinical treatment. Here, we report on a polyethylene glycol-coated carbon nanotube (CNT)-ABT737 nanodrug that improves the mitochondrial targeting of lung cancer cells. The polyethylene glycol-coated CNT-ABT737 nanodrug internalized into the early endosomes via macropinocytosis and clathrin-mediated endocytosis in advance of early endosomal escape and delivered into the mitochondria. Cytosol release of the nanodrug led to apoptosis of lung cancer cells by abruption of the mitochondrial membrane potential, inducing Bcl-2-mediated apoptosis and generating intracellular reactive oxygen species. As such, this study provides an effective strategy for increasing the anti-lung cancer efficacy by increasing mitochondria accumulation rate of cytosol released anticancer nanodrugs.

Topics: A549 Cells; Biphenyl Compounds; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Endosomes; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Nanotubes, Carbon; Nitrophenols; Piperazines; Polyethylene Glycols; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Sulfonamides

Inhibition of Notch by γ-secretase inhibitor (GSI) has been shown to have an antitumor effect in Notch-expressing non-small cell lung cancer (NSCLC) and to induce apoptosis through modulation of Bcl-2 family proteins. In particular, Bim, a BH3-only member of the Bcl-2 family of proteins, has an important role in the induction of apoptosis in NSCLC when cells are treated with GSI. ABT-737, a BH3-only mimetic, targets the pro-survival Bcl-2 family and also induces apoptosis.. The Notch-expressing NSCLC cell lines H460, A549, H1793, and HCC2429 were used. The combined antitumor effect of GSI and ABT-737 was evaluated using the MTT proliferation assay in vitro and in xenograft mouse models. The expression of the Notch pathway and Bcl-2 family was analyzed using Western blotting analysis when cells were treated with a single drug treatment or a combination treatment.. GSI XX or ABT-737 alone inhibited cell proliferation in a dose-dependent manner, and combination drug treatment showed a synergistic antitumor effect in vitro. In vivo, this drug combination significantly suppressed tumor proliferation compared to the single drug treatment. Phospho-Bcl-2 was downregulated and Bax was upregulated by both the single and combination drug treatments. Bim was induced by a single drug treatment and was enhanced by combination treatment. Combination treatment-induced apoptosis was decreased by Bim inhibition, suggesting that the antitumor effect of the drug combination was dependent on Bim.. Based on our data, we propose that the combination treatment is a promising strategy for NSCLC therapy.

Topics: Amyloid Precursor Protein Secretases; Animals; Apoptosis; Biphenyl Compounds; Carcinoma, Non-Small-Cell Lung; Dibenzazepines; Drug Therapy, Combination; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Nitrophenols; Piperazines; Receptors, Notch; Signal Transduction; Sulfonamides; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Here we explored the potential synergism between the novel Bcl-2 antagonist ABT-737 and the AKT inhibitor perifosine in lung cancer cells. Our in vitro results showed that perifosine and ABT-737 synergistically induced growth inhibition and apoptosis in both established (A549 and H460 lines) and patient-derived lung cancer cells. The combined activity was dramatically more potent than either single agent. For the molecular study, we showed that perifosine downregulated Mcl-1 expression, thus potentiating ABT-737 lethality against lung cancer cells. Exogenous over-expression of Mcl-1 remarkably attenuated perifosine plus ABT-737 combo-induced lung cancer cell apoptosis. In vivo, perifosine and ABT-737 co-administration strikingly inhibited A549 lung cancer xenograft growth in nude mice. The combined treatment in vivo was again superior than single treatment establishing a synergistic activity. Mcl-1 expression was also downregulated in combo-treated A549 tumors. The results of this preclinical study support the feasibility of further investigation of the perifosine plus ABT-737 regimen in future lung cancer clinical tests.

Topics: A549 Cells; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Nitrophenols; Phosphorylcholine; Piperazines; Sulfonamides; Treatment Outcome

Most small cell lung cancer (SCLC) patients are initially responsive to cytotoxic chemotherapy, but almost all undergo fatal relapse with progressive disease, highlighting an urgent need for improved therapies and better patient outcomes in this disease. The proapoptotic BH3 mimetic ABT-737 that targets BCL-2 family proteins demonstrated good single-agent efficacy in preclinical SCLC models. However, so far clinical trials of the BH3 mimetic Navitoclax have been disappointing. We previously demonstrated that inhibition of a PI3K/BMX cell survival signaling pathway sensitized colorectal cancer cells to ABT-737. Here, we show that SCLC cell lines, which express high levels of BMX, become sensitized to ABT-737 upon inhibition of PI3K in vitro, and this is dependent on inhibition of the PI3K-BMX-AKT/mTOR signaling pathway. Consistent with these cell line data, when combined with Navitoclax, PI3K inhibition suppressed tumor growth in both an established SCLC xenograft model and in a newly established circulating tumor cell-derived explant (CDX) model generated from a blood sample obtained at presentation from a chemorefractory SCLC patient. These data show for the first time that a PI3K/BMX signaling pathway plays a role in SCLC cell survival and that a BH3 mimetic plus PI3K inhibition causes prolonged tumor regression in a chemorefractory SCLC patient-derived model in vivo These data add to a body of evidence that this combination should move toward the clinic. Mol Cancer Ther; 15(6); 1248-60. ©2016 AACR.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biphenyl Compounds; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; Humans; Lung Neoplasms; Mice; Nitrophenols; Phosphatidylinositol 3-Kinases; Piperazines; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Signal Transduction; Small Cell Lung Carcinoma; Sulfonamides; Xenograft Model Antitumor Assays

Small cell lung carcinoma (SCLC) is an aggressive, recalcitrant cancer, often metastatic at diagnosis and unresponsive to chemotherapy upon recurrence, thus it is challenging to treat.. Sixty-three human SCLC lines and three NSCLC lines were screened for response to 103 US Food and Drug Administration-approved oncology agents and 423 investigational agents. The investigational agents library was a diverse set of small molecules that included multiple compounds targeting the same molecular entity. The compounds were screened in triplicate at nine concentrations with a 96-hour exposure time using an ATP Lite endpoint. Gene expression was assessed by exon array, and microRNA expression was derived by direct digital detection. Activity across the SCLC lines was associated with molecular characteristics using pair-wise Pearson correlations.. Results are presented for inhibitors of targets: BCL2, PARP1, mTOR, IGF1R, KSP/Eg5, PLK-1, AURK, and FGFR1. A relational map identified compounds with similar patterns of response. Unsupervised microRNA clustering resulted in three distinct SCLC subgroups. Associating drug response with micro-RNA expression indicated that lines most sensitive to etoposide and topotecan expressed high miR-200c-3p and low miR-140-5p and miR-9-5p. The BCL-2/BCL-XL inhibitors produced similar response patterns. Sensitivity to ABT-737 correlated with higher ASCL1 and BCL2. Several classes of compounds targeting nuclear proteins regulating mitosis produced a response pattern distinct from the etoposide response pattern.. Agents targeting nuclear kinases appear to be effective in SCLC lines. Confirmation of SCLC line findings in xenografts is needed. The drug and compound response, gene expression, and microRNA expression data are publicly available at http://sclccelllines.cancer.gov.

Topics: Antineoplastic Agents; Biphenyl Compounds; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Line, Tumor; Drug Screening Assays, Antitumor; Drugs, Investigational; Gene Expression; Humans; Kinesins; Lung Neoplasms; MicroRNAs; Nitrophenols; Piperazines; Polo-Like Kinase 1; Poly (ADP-Ribose) Polymerase-1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptor, Fibroblast Growth Factor, Type 1; Receptor, IGF Type 1; Receptors, Somatomedin; Small Cell Lung Carcinoma; Sulfonamides; TOR Serine-Threonine Kinases

High-grade conventional osteosarcoma is the most common primary bone tumor. Prognosis for osteosarcoma patients is poor and resistance to chemotherapy is common. We performed an siRNA screen targeting members of the Bcl-2 family in human osteosarcoma cell lines to identify critical regulators of osteosarcoma cell survival. Silencing the anti-apoptotic family member Bcl-xL but also the pro-apoptotic member Bak using a SMARTpool of siRNAs as well as 4/4 individual siRNAs caused loss of viability. Loss of Bak impaired cell cycle progression and triggered autophagy. Instead, silencing Bcl-xL induced apoptotic cell death. Bcl-xL was expressed in clinical osteosarcoma samples but mRNA or protein levels did not significantly correlate with therapy response or survival. Nevertheless, pharmacological inhibition of a range of Bcl-2 family members showed that inhibitors targeting Bcl-xL synergistically enhanced the response to the chemotherapeutic agent, doxorubicin. Indeed, in osteosarcoma cells strongly expressing Bcl-xL, the Bcl-xL-selective BH3 mimetic, WEHI-539 potently enhanced apoptosis in the presence of low doses of doxorubicin. Our results identify Bcl-xL as a candidate drug target for sensitization to chemotherapy in patients with osteosarcoma.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-X Protein; Benzopyrans; Biphenyl Compounds; Bone Neoplasms; Cell Line, Tumor; Cohort Studies; Doxorubicin; Drug Synergism; Enzyme Inhibitors; Humans; Immunohistochemistry; Lung Neoplasms; Nitriles; Nitrophenols; Osteosarcoma; Piperazines; Sulfonamides; Tissue Array Analysis; Transfection

(-)-Epigallocatechin-3-gallate (EGCG) has exhibited been studied for lung cancer inhibitory activity in vitro and in animal models, but it is rapidly methylated and inactivated by catechol-O-methyltransferase (COMT). Entacapone and tolcapone, COMT inhibitors, are used to mitigate the symptoms of Parkinson's disease. We investigated the synergistic effects of entacapone/tolcapone and EGCG against lung cancer cell lines in culture. EGCG, entacapone and tolcapone inhibited the growth of H1299 human lung cancer cells (IC50 = 174.9, 76.8 and 29.3 µM, respectively) and CL-13 murine lung cancer cells (IC50 = 181.5, 50.7 and 19.7 µM, respectively) as single agents following treatment for 72h. Treatment with 1:10, 1:5, 1:2.5 and 1:1 combinations of EGCG and tolcapone or entacapone resulted in synergistically enhanced growth inhibition. The growth inhibitory effect of the combinations was mediated by induction of intracellular oxidative stress, cell cycle arrest and decreased nuclear translocation of nuclear factor-κΒ. Methylation of EGCG was dose dependently inhibited by entacapone and tolcapone (IC50 = 10 and 20 µM, respectively) in a cell-free system, and both compounds increased the intracellular levels of unmethylated EGCG. Treatment of mice with EGCG in combination with tolcapone increased the bioavailability of EGCG and decreased the methylation of plasma norepinephrine: no apparent liver or behavioral toxicity was observed. In conclusion, the combination of EGCG and entacapone/tolcapone synergistically inhibited the growth of lung cancer cells in culture, and the mechanistic basis for this synergy is likely due in part to inhibition of COMT with resultant increase in the levels of unmetabolized EGCG.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; Benzophenones; Blotting, Western; Catechin; Catechol O-Methyltransferase Inhibitors; Catechols; Cell Cycle; Cell Proliferation; Cell-Free System; DNA Methylation; Drug Synergism; Enzyme Inhibitors; Humans; Lung Neoplasms; Male; Mice; Nitriles; Nitro Compounds; Nitrophenols; Oxidative Stress; Reactive Oxygen Species; Tolcapone; Tumor Cells, Cultured

The sensitivity to ABT-737, a prototype BH3 mimetic drug, varies in a broad range in small cell lung cancer (SCLC) cells. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is the critical determinant of ABT-737 sensitivity. We show here that Noxa regulates the localization and stability of MCL-1, an anti-apoptotic member, which results in modulating ABT-737 sensitivity. Mutations in Noxa within the BH3 domain, the carboxyl terminus mitochondrial targeting domain, or of ubiquitinated lysines not only change the localization and stability of Noxa itself but also affect the mitochondrial localization and phosphorylation/ubiquitination status of MCL-1 and consequently modulate sensitivity to ABT-737. Results of studies utilizing these mutant proteins indicate that Noxa recruits MCL-1 from the cytosol to the mitochondria. Translocation of MCL-1 initiates its phosphorylation and subsequent ubiquitination, which triggers proteasome-mediated degradation. The precise regulatory mechanisms of Noxa/MCL-1 expression and stability could provide alternative targets to modulate apoptosis induced by BH3 mimetic drugs or other chemotherapeutic reagents.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Cell Survival; Dose-Response Relationship, Drug; HEK293 Cells; HeLa Cells; Humans; Lung Neoplasms; Mice, Knockout; Mitochondria; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Phosphorylation; Piperazines; Proteasome Endopeptidase Complex; Protein Stability; Protein Transport; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Small Cell Lung Carcinoma; Sulfonamides; Transfection; Ubiquitination

Overexpression of the antiapoptotic protein Bcl-2 is observed in the majority of small cell lung cancer (SCLC) cases and is associated with resistance to chemotherapy. While targeting Bcl-2 in hematologic malignancies continues to show signs of promise, translating the BH3 mimetic ABT-737 (or ABT-263; navitoclax) to the clinic for solid tumors has remained problematic, with limited single-agent activity in early-phase clinical trials. Here, we used patient-derived xenograft (PDX) models of SCLC to study ABT-737 resistance and demonstrated that responses to ABT-737 are short lived and coincide with decreases in HIF-1α-regulated transcripts. Combining the mTOR inhibitor rapamycin with ABT-737 rescued this resistance mechanism, was highly synergistic in vitro, and provided durable tumor regressions in vivo without notable hematologic suppression. In comparison, tumor regressions did not occur when ABT-737 was combined with etoposide, a gold-standard cytotoxic for SCLC therapy. Rapamycin exposure was consistently associated with an increase in the proapoptotic protein BAX, whereas ABT-737 caused dose-dependent decreases in BAX. As ABT-737 triggers programmed cell death in a BAX/BAK-dependent manner, we provide preclinical evidence that the efficacy of ABT-737 as a single agent is self-limiting in SCLC, but the addition of rapamycin can maintain or increase levels of BAX protein and markedly enhance the anticancer efficacy of ABT-737. These data have direct translational implications for SCLC clinical trials.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Biphenyl Compounds; Cell Line, Tumor; Drug Synergism; Etoposide; Female; Humans; Lung Neoplasms; Mice; Mice, SCID; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Random Allocation; Sirolimus; Small Cell Lung Carcinoma; Sulfonamides; Xenograft Model Antitumor Assays

Lung cancer is the most common cause of cancer-related mortality worldwide, urging the discovery of novel molecular targets and therapeutic strategies. Stem cells have been recently isolated from non-small cell lung cancer (NSCLC), thus allowing the investigation of molecular pathways specifically active in the tumorigenic population. We have found that Bcl-XL is constantly expressed by lung cancer stem cells (LCSCs) and has a prominent role in regulating LCSC survival. Whereas chemotherapeutic agents were scarcely effective against LCSC, the small molecule Bcl-2/Bcl-XL inhibitor ABT-737, but not the selective Bcl-2 inhibitor ABT-199, induced LCSC death at nanomolar concentrations. Differently from gemcitabine, which preferentially eliminated proliferating LCSC, ABT-737 had an increased cytotoxic activity in vitro towards quiescent/slow-proliferating LCSC, which expressed high levels of Bcl-XL. In vivo, ABT-737 as a single agent was able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors. Altogether, these results indicate that quiescent/slow-proliferating LCSC strongly depend on Bcl-XL for their survival and indicate Bcl-XL inhibition as a potential therapeutic avenue in NSCLC.

Topics: Animals; Antineoplastic Agents; bcl-X Protein; Biphenyl Compounds; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Humans; Lung Neoplasms; Mice, Inbred NOD; Mice, SCID; Neoplastic Stem Cells; Nitrophenols; Piperazines; Sulfonamides; Tumor Burden; Xenograft Model Antitumor Assays

New therapies are urgently needed for patients with small cell lung cancer (SCLC). Chemotherapy and targeted therapies, including the Bcl-2 inhibitor ABT-737, may induce tumor cell autophagy. Autophagy can promote survival of cancer cells under stress and comprise a pathway of escape from cytotoxic therapies.. We explored the combination of ABT-737 and chloroquine, an inhibitor of autophagy, in preclinical models of SCLC. These included cell culture analyses of viability and of autophagic and apoptotic pathway induction, as well as in vivo analyses of efficacy in multiple xenograft models.. Combination treatment of SCLC lines with ABT-737 and chloroquine decreased viability and increased caspase-3 activation over treatment with either single agent. ABT-737 induced several hallmarks of autophagy. However, knockdown of beclin-1, a key regulator of entry into autophagy, diminished the efficacy of ABT-737, suggesting either that the effects of chloroquine were nonspecific or that induction but not completion of autophagy is necessary for the combined effect of ABT-737 and chloroquine. ABT-737 and chloroquine in SCLC cell lines downregulated Mcl-1 and upregulated NOXA, both of which may promote apoptosis. Treatment of tumor-bearing mice demonstrated that chloroquine could enhance ABT-737-mediated tumor growth inhibition against NCI-H209 xenografts, but did not alter ABT-737 response in three primary patient-derived xenograft models.. These data suggest that although ABT-737 can induce autophagy in SCLC, autophagic inhibition by choroquine does not markedly alter in vivo response to ABT-737 in relevant preclinical models, arguing against this as a treatment strategy for SCLC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Biphenyl Compounds; Cell Proliferation; Cell Survival; Chloroquine; Drug Resistance, Neoplasm; Female; Gene Knockdown Techniques; Humans; Lung Neoplasms; Membrane Proteins; Mice; Mice, Nude; Nitrophenols; Piperazines; RNA, Small Interfering; Small Cell Lung Carcinoma; Sulfonamides; Tumor Burden; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The myeloid cell leukemia-1 (MCL1) gene encodes antiapoptotic Mcl-1(L) and proapoptotic Mcl-1(S) proteins. In cancer, the Mcl-1(L)/Mcl-1(S) ratio is very high, accounting for the antiapoptotic nature of cancer cells. As such, reducing this ratio can render the cancer cells prone to apoptosis. The Mcl-1(L)/Mcl-1(S) ratio is determined in the alternative pre-mRNA splicing step that is regulated by splicing factor 3B1 (SF3B1). Here, we report that meayamycin B, a potent inhibitor of SF3B1, reversed the dominant isoform from Mcl-1(L) to Mcl-1(S) at the mRNA and protein levels. The resulting proapoptotic cellular environment was further exploited; when meayamycin B was combined with Bcl-x(L) inhibitor ABT-737, the combination treatment triggered apoptosis in nonsmall cell lung cancer A549 and H1299 cells that were otherwise resistant to ABT-737. These results demonstrate that perturbation of the MCL1 splicing with small molecule inhibitors of SF3B1 provides a means to sensitize cancer cells toward Bcl-x(L) inhibitors.

Topics: Alternative Splicing; Apoptosis; bcl-X Protein; Biphenyl Compounds; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Dose-Response Relationship, Drug; Epoxy Compounds; Humans; Lung Neoplasms; Morpholines; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Phosphoproteins; Piperazines; Protein Isoforms; Pyrans; Ribonucleoprotein, U2 Small Nuclear; RNA Splicing Factors; Sulfonamides

Bcl-XL is a major antiapoptotic protein in the Bcl-2 family whose overexpression is more widely observed in human lung cancer cells than that of Bcl-2, suggesting that Bcl-XL is more biologically relevant and therefore a better therapeutic target for lung cancer. Here, we screened small molecules that selectively target the BH3 domain (aa 90-98) binding pocket of Bcl-XL using the UCSF DOCK 6.1 program suite and the NCI chemical library database. We identified two new Bcl-XL inhibitors (BXI-61 and BXI-72) that exhibit selective toxicity against lung cancer cells compared with normal human bronchial epithelial cells. Fluorescence polarization assay reveals that BXI-61 and BXI-72 preferentially bind to Bcl-XL protein but not Bcl2, Bcl-w, Bfl-1/A1, or Mcl-1 in vitro with high binding affinities. Treatment of cells with BXI-72 results in disruption of Bcl-XL/Bak or Bcl-XL/Bax interaction, oligomerization of Bak, and cytochrome c release from mitochondria. Importantly, BXI-61 and BXI-72 exhibit more potent efficacy against human lung cancer than ABT-737 but less degree in platelet reduction in vivo. BXI-72 overcomes acquired radioresistance of lung cancer. On the basis of our findings, the development of BXI(s) as a new class of anticancer agents is warranted and represents a novel strategy for improving lung cancer outcome.

Topics: Acridines; Aminopyridines; Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-X Protein; Benzimidazoles; Biphenyl Compounds; Blotting, Western; Cell Proliferation; Cytochromes c; Female; Humans; Lung; Lung Neoplasms; Mice; Mice, Nude; Nitrophenols; Piperazines; Protein Multimerization; Proto-Oncogene Proteins c-bcl-2; Radiation Tolerance; Radiation, Ionizing; Small Molecule Libraries; Sulfonamides; Survival Rate

While numerous cell signaling pathways are known to play decisive roles in chemotherapeutic response, relatively little is known about the impact of the Smad-dependent transforming growth factor β pathway on the therapeutic outcome. Previous reports suggested that patients with lung cancer who continue to smoke while receiving chemotherapy have a poorer outcome than their nonsmoking counterparts do. In our previous study, we showed that long-term cigarette smoke condensate (CSC)-mediated down-regulation of Smad3 induces tumorigenesis. The objective of this study was to determine the mechanism of function of Smad3 in chemoresistance induced by CSC in human lung cell lines, namely, A549 and HPL1A. Long-term CSC treatment increases the half-maximal inhibitory concentration (IC(50)) of carboplatin and makes cells resistant to carboplatin. The increase in IC(50) of long-term CSC-treated cells is due to the reduced induction in apoptosis by carboplatin. The increase in IC(50) and decrease in apoptosis in long-term CSC-treated cells is correlated with the expression of Bcl2. We have determined that Bcl2 is both necessary and sufficient to make the cells resistant to carboplatin. We have also shown that Smad3 acts upstream to regulate the expression of Bcl2 specifically and, thus, sensitivity of the cells to carboplatin. This is supported by the inverse correlation between the expressions of Smad3 and Bcl2 in human lung tumors. Collectively, these data suggest that loss of Smad3 expression in CSC-treated cells induces resistance to carboplatin by upregulating the expression of Bcl2. This study explains, at least in part, the higher chemoresistance rate observed in smokers.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Carboplatin; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Smad3 Protein; Smoking; Sulfonamides

Targeted therapies for cancer are inherently limited by the inevitable recurrence of resistant disease after initial responses. To define early molecular changes within residual tumor cells that persist after treatment, we analyzed drug-sensitive lung adenocarcinoma cell lines exposed to reversible or irreversible epidermal growth factor receptor (EGFR) inhibitors, alone or in combination with MET-kinase inhibitors, to characterize the adaptive response that engenders drug resistance. Tumor cells displaying early resistance exhibited dependence on MET-independent activation of BCL-2/BCL-XL survival signaling. Further, such cells displayed a quiescence-like state associated with greatly retarded cell proliferation and cytoskeletal functions that were readily reversed after withdrawal of targeted inhibitors. Findings were validated in a xenograft model, showing BCL-2 induction and p-STAT3[Y705] activation within the residual tumor cells surviving the initial antitumor response to targeted therapies. Disrupting the mitochondrial BCL-2/BCL-XL antiapoptotic machinery in early survivor cells using BCL-2 Homology Domain 3 (BH3) mimetic agents such as ABT-737, or by dual RNAi-mediated knockdown of BCL-2/BCL-XL, was sufficient to eradicate the early-resistant lung-tumor-cells evading targeted inhibitors. Similarly, in a xenograft model the preemptive cotreatment of lung tumor cells with an EGFR inhibitor and a BH3 mimetic eradicated early TKI-resistant evaders and ultimately achieved a more durable response with prolonged remission. Our findings prompt prospective clinical investigations using BH3-mimetics combined with targeted receptor kinase inhibitors to optimize and improve clinical outcomes in lung-cancer treatment.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; bcl-X Protein; Biphenyl Compounds; Cell Line, Tumor; Drug Resistance, Neoplasm; ErbB Receptors; Erlotinib Hydrochloride; Humans; Indoles; Lung Neoplasms; Mice; Nitrophenols; Peptide Fragments; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-met; Quinazolines; Signal Transduction; STAT3 Transcription Factor; Sulfonamides; Transplantation, Heterologous

Topics: Aniline Compounds; Animals; Apoptosis; Biphenyl Compounds; HL-60 Cells; Humans; Lung Neoplasms; Lymphoma; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

ABT-737, which blocks the function of Bcl-2 and Bcl-X(L) but not Mcl-1, has shown single-agent activity in preclinical models of small cell lung cancer (SCLC). Elevated expression of Mcl-1 induces resistance to ABT-737 in SCLC. Based on the short half-life of Mcl-1 mRNA and protein, we hypothesized that the actinomycin D could reverse Mcl-1-induced resistance to ABT-737.. The dose-response of multiple SCLC cell lines to actinomycin D in the absence and presence of ABT-737 was followed by the assessment of Bcl-2 family expression and poly ADP ribose polymerase cleavage by Western blot, viability by tetrazolium dye reduction and clonogenic assay, and cell cycle kinetics by flow cytometry.. Actinomycin D decreased Mcl-1 expression and resulted in a cell line-dependent increase in Noxa expression. Clinically relevant concentrations of actinomycin D from 0.4 to 4 ng/mL showed single-agent activity across a panel of SCLC cell lines. When combined with low micromolar doses of ABT-737, near complete loss of viability was seen with synergistic combination indices of 0.5 to 0.7. Exposure to 4 ng/mL actinomycin was only required for the first 24 hours of the combined incubation, mimicking a clinically achievable area under the curve, but the presence of ABT-737 was required for an additional 48 hours to obtain maximal effect.. Clinically relevant concentrations of actinomycin D act synergistically with ABT-737 to induce SCLC apoptosis, which can be at least partially attributed to the actinomycin D-induced decrease in Mcl-1 and increase in Noxa expression. Taken together, these data suggest the feasibility of combining actinomycin D with BH3-mimetic drugs in the clinical setting.

Topics: Antibiotics, Antineoplastic; Apoptosis; Biphenyl Compounds; Blotting, Western; Cell Division; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dactinomycin; Dose-Response Relationship, Drug; Drug Synergism; Flow Cytometry; G2 Phase; Humans; Lung Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Small Cell Lung Carcinoma; Sulfonamides; Time Factors

Combinatorial therapies using the proteasome inhibitor, bortezomib, have been found to induce synergistic apoptosis in cancer cells grown as monolayers; however, three-dimensional spheroid culture may be a better model for the multicellular resistance found in solid tumors, such as lung cancer. We tested the combinatorial apoptotic strategy of using bortezomib together with TNF-related apoptosis-inducing ligand (TRAIL), both in monolayers and in spheroids of A549 lung cancer cells. Indeed, bortezomib plus TRAIL induced synergistic apoptosis in A549 cells grown as monolayers, but had little effect on A549 cells grown as three-dimensional multicellular spheroids. The acquired resistance of spheroids was not due to a limitation of diffusion, to survival pathways, such as NF-kappaB or PI3K/Akt/mTOR, or to the up-regulation of FLIP(S) (Fas-associated death domain-like IL-1 beta-converting enzyme inhibitory protein, short). We then investigated a role for the Bcl-2 family of anti- and proapoptotic proteins. When cells formed spheroids, antiapoptotic Bcl-2 increased, whereas antiapoptotic Mcl-1 decreased. ABT-737, a small molecule that inhibits Bcl-2, but not Mcl-1, abolished the multicellular resistance of A549 spheroids to bortezomib plus TRAIL. In another lung cancer cell line, H1299, acquisition of multicellular resistance in spheroids was also accompanied by an increase in Bcl-2 and decrease in Mcl-1. In H1299 spheroids compared with those of A549, however, Mcl-1 remained higher, and Mcl-1 knockdown was more effective than ABT-737 in removing multicellular resistance. Our study suggests that the balance of Bcl-2 family proteins contributes to the acquired multicellular resistance of spheroids, and suggests a possible target for improving the response of lung cancer to bortezomib therapies.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Boronic Acids; Bortezomib; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Communication; Cell Line, Tumor; Diffusion; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; NF-kappa B; Nitrophenols; Phosphatidylinositol 3-Kinases; Piperazines; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Recombinant Proteins; Signal Transduction; Spheroids, Cellular; Sulfonamides; Time Factors; TNF-Related Apoptosis-Inducing Ligand; TOR Serine-Threonine Kinases

To understand the molecular basis for variable sensitivity to the BH3 mimetic drug ABT-737, the abundance of Bcl-2 family members was assayed in a panel of small cell lung cancer cell lines whose sensitivity varied over a 2-log range. Elevated Noxa and Bcl-2 levels directly correlated with sensitivity to ABT-737, whereas Mcl-1 levels were similar in all cell lines tested regardless of sensitivity. Transgenically enforced expression of Noxa but not Bcl-2 resulted in increased sensitivity to ABT-737 in multiple cell lines. This increase was especially pronounced in the H209 cell line in which expression of Noxa resulted in a proportionate decline in Mcl-1 expression. Although overexpression of Noxa enhanced sensitivity of the H526 and H82 cell lines to ABT-737, it did not result in altered Mcl-1 levels. Similarly, small interfering RNA-mediated knockdown of Noxa expression in the H146 cell line, which increased resistance to ABT-737, did not result in altered Mcl-1 levels. Therefore, three of four cell lines studied failed to show Noxa-mediated regulation of Mcl-1 expression. However, despite failure to regulate Mcl-1 levels, Noxa blocked binding of Bim to Mcl-1 following its release from Bcl-2 by ABT-737. Finally, we observed that a 24-hour incubation of the H526 and WBA cell lines with ABT-737 resulted in increased Noxa expression, suggesting that Noxa may play a direct role in ABT-737-mediated apoptosis. These results indicate that Noxa expression is the critical determinant of ABT-737 sensitivity and loss of Noxa-mediated regulation of Mcl-1 expression may be an important feature of small cell lung cancer biology.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Biphenyl Compounds; Blotting, Western; Cell Proliferation; Drug Resistance, Neoplasm; Humans; Immunoprecipitation; Lung Neoplasms; Membrane Proteins; Molecular Mimicry; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Peptide Fragments; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Small Cell Lung Carcinoma; Sulfonamides; Tumor Cells, Cultured

Radiotherapy has a central role in the treatment of non-small cell lung cancer. Effectiveness of this modality, however, is often limited as resistance results from defects in cell death.. We investigated whether simultaneous up-regulation of apoptosis, via Bcl-2 inhibitor ABT-737, and autophagy, via mammalian target of rapamycin inhibitor rapamycin, can be used to enhance radiosensitivity of H460 cells in vitro and growth delay in a xenograft model.. In vitro studies confirmed that ABT-737 and rapamycin induce apoptosis and autophagy, respectively. ABT-737 induced cleaved caspase-3, a marker of apoptosis, and rapamycin correlated with an increase in punctate localization of green fluorescent protein-LC3, characteristic of autophagy. The combination ABT-737/rapamycin markedly enhanced sensitivity of H460 cells to radiation (dose enhancement ratio = 2.47; P = 0.002) in clonogenic assay. In addition, the combination ABT-737/rapamycin/radiation showed a dramatic tumor growth delay in a mouse xenograft model. In vivo immunohistochemistry staining showed that combination therapy yielded over a 100% increase in caspase-3 activity (apoptosis) and a 6-fold decrease in p62 protein level (indicative of autophagic flux) compared with radiation alone control group. Moreover, cell proliferation (Ki-67 staining) was reduced by 77% (P = 0.001) and vascular density (von Willebrand factor staining) by 67.5% (P = 0.09) compared with radiation alone. Additional in vitro studies in human umbilical vein endothelial cells indicated that combined therapy also significantly decreases tubule formation.. These results suggest that concurrent induction of apoptosis and autophagy enhances radiation therapy both in vitro and in lung cancer xenograft models. Further investigations are warranted to assess the clinical potential of such strategy in lung cancer patients.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Biphenyl Compounds; Carcinoma, Non-Small-Cell Lung; Cells, Cultured; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Nitrophenols; Piperazines; Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Radiation Tolerance; Radiation-Sensitizing Agents; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

This study evaluated epithelial cell death ELISAs that measure circulating cytokeratin 18 in mice bearing small-cell lung cancer xenografts treated with a proapoptotic dose of the BH-3 mimetic ABT-737.. H146 tumor-bearing and non-H146 tumor-bearing severe combined immunodeficient (SCID)/bg mice were treated with ABT-737 or vehicle control. Plasma collected before and 2 to 360 hours after treatment was analyzed by M30 (caspase-cleaved cytokeratin 18) and M65 (intact and cleaved cytokeratin 18) ELISA. In parallel, tumors were interrogated for cleaved caspase-3 and cleaved cytokeratin 18 as biomarkers of apoptosis.. ABT-737-treated tumors regressed by 48 hours (P < 0.01) compared with controls, correlating with increased cleaved cytokeratin 18 (P < 0.01; 6 and 24 hours) and increased intact cytokeratin 18 (P < 0.01; 24 hours). Cleaved cytokeratin 18 levels decreased below baseline between 72 and 360 hours for ABT-737-treated and control mice whereas intact cytokeratin 18 decreased below the level of detection at 8 and 15 days in ABT-737-treated mice only. Apoptosis in tumors reflected changes in circulating cytokeratin 18 (cleaved caspase-3, P < 0.05 at 2 hours and P < 0.001 at 6, 12, and 24 hours; caspase-cleaved cytokeratin 18, P < 0.05 at 15 days, for drug treated versus controls).. ABT-737 caused tumor regression by apoptosis in H146 xenografts that mapped to a drug-specific, early increase in circulating cleaved cytokeratin 18 that subsequently declined. Circulating, intact cytokeratin 18 levels correlated with tumor burden. Cleaved caspase-3 and caspase-cleaved cytokeratin 18 in tumor correlated with treatment (P < 0.05, 2 hours; P < 0.001, 6, 12, and 24 hours; cleaved caspase-3, P < 0.05, 15 days; caspase-cleaved cytokeratin 18), indicating that events in plasma were tumor derived. These circulating biomarker data will be translated to clinical trials wherein serial tumor biopsies are rarely obtained.

Topics: Animals; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Biomimetic Materials; Biphenyl Compounds; Butylated Hydroxytoluene; Caspase 3; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; Keratin-18; Lung Neoplasms; Mice; Nitrophenols; Piperazines; Small Cell Lung Carcinoma; Sulfonamides; Xenograft Model Antitumor Assays

Bcl-2 is a central regulator of cell survival that is overexpressed in the majority of small cell lung cancers (SCLC) and contributes to both malignant transformation and therapeutic resistance. We compared primary SCLC xenografts prepared from de novo human tumors with standard cell line-based xenografts in the evaluation of a novel and highly potent small molecule inhibitor of Bcl-2, ABT-737. ABT-737 induced dramatic regressions in tumors derived from some SCLC cell lines. In contrast, only one of three primary xenograft SCLC tumors showed significant growth inhibition with ABT-737. Explanations for this apparent dichotomy may include relatively low expression of Bcl-2 in the primary xenografts or inherent differences in the model systems. The addition of etoposide to ABT-737 in the primary xenografts resulted in significant decreases in tumor growth, underscoring the clinical potential of ABT-737 in combination therapy. To identify factors that may contribute to resistance to ABT-737 and related inhibitors, we isolated resistant derivatives of an initially sensitive cell line-based xenograft. Acquired resistance in this model was associated with decreases in the expression of the primary target Bcl-2, of proapoptotic partners of Bcl-2 (Bax and Bim), and of Bcl-2:Bim heterodimers. Expression profiling reveals 85 candidate genes demonstrating consistent changes in gene expression with acquired resistance. Taken together, these data have specific implications for the clinical development of Bcl-2 inhibitors for SCLC and broader implications for the testing of novel anticancer strategies in relevant preclinical models.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biphenyl Compounds; Carcinoma, Small Cell; Caspase 3; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Activation; Etoposide; Humans; Lung Neoplasms; Mice; Mice, Nude; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Xenograft Model Antitumor Assays

ABT-737 is a subnanomolar inhibitor of the antiapoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w. Although ABT-737 triggers extensive cell death in many small-cell lung carcinoma (SCLC) cell lines, some of the SCLC cell lines and the majority of the cancer cell lines derived from other solid tumors were found to be resistant to ABT-737. To better understand the mechanism of resistance to ABT-737, we screened a short interfering RNA library consisting of short interfering RNA against 4000 'druggable' targets in an SCLC-derived cell line, NCI-H196. By comparing the knockdowns with phenotypes, all of the three top 'hits' from the screen were found to result from off-target gene silencing. Interestingly, the three off-target siRNAs were found to knock down an antiapoptotic Bcl-2 family protein Mcl-1 owing to the complementation between their seed regions with the 3' untranslated region (3' UTR) of Mcl-1. Furthermore, reducing the level of Mcl-1 using siRNAs or the small-molecule compounds Bay43-9006 and Seliciclib was sufficient to overcome the resistance to ABT-737 in the resistant SCLC cell line and cancer cell lines derived from other solid tumors. These results provide further evidence that Mcl-1 is the major factor that causes resistance to ABT-737 in cancer cells derived from diverse solid tumors, and the combination of Mcl-1 downregulating agents with ABT-737 could be potent therapeutic regimens for patient with ABT-737-resistant SCLC and many other types of solid tumors.

Topics: 3' Untranslated Regions; Antineoplastic Agents; Base Sequence; bcl-X Protein; Benzenesulfonates; Biphenyl Compounds; Blotting, Western; Carcinoma, Small Cell; Cell Line, Tumor; Cell Survival; DNA-Binding Proteins; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Niacinamide; Nitrophenols; Phenylurea Compounds; Piperazines; Proto-Oncogene Proteins c-bcl-2; Purines; Pyridines; Receptor, Fibroblast Growth Factor, Type 2; RNA Interference; RNA, Small Interfering; Roscovitine; Sorafenib; Sulfonamides; Transmembrane Activator and CAML Interactor Protein; Zinc Fingers

ABT-737 is a novel and potent Bcl-2 antagonist with single-agent activity against small-cell lung cancer (SCLC) cell lines. Here, we evaluated the contribution of Bcl-2 family members to the in vitro cellular response of several SCLC cell lines to ABT-737. Relatively higher levels of Bcl-2, Bcl-X(L), Bim and Noxa, and lower levels of Mcl-1 characterized naïve SCLC cell lines that were sensitive to ABT-737. Conversely, a progressive decrease in the relative levels of Bcl-2 and Noxa and a progressive increase in Mcl-1 levels characterized the increased resistance of H146 cells following chronic exposure to ABT-737. Knockdown of Mcl-1 with small interfering RNA sensitized two resistant SCLC cell lines H196 and DMS114 to ABT-737 by enhancing the induction of apoptosis. Likewise, up-regulation of Noxa sensitized H196 cells to ABT-737. Combination treatment with DNA-damaging agents was extremely synergistic with ABT-737 and was associated with the down-regulation of Mcl-1 and the up-regulation of Noxa, Puma, and Bim in H196 cells. Thus, SCLC cells sensitive to ABT-737 expressed the target proteins Bcl-2 and Bcl-X(L), whereas Mcl-1 and factors regulating Mcl-1 function seem to contribute to the overall resistance of SCLC cells to ABT-737. Overall, these observations provide further insight as to the mechanistic bases for ABT-737 efficacy in SCLC and will be helpful for profiling patients and aiding in the rational design of combination therapies.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Biphenyl Compounds; Carboplatin; Carcinoma, Small Cell; Cell Growth Processes; Cell Line, Tumor; Down-Regulation; Drug Synergism; Etoposide; Humans; Lung Neoplasms; Membrane Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Nitrophenols; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Sulfonamides; Transfection; Up-Regulation

Cytotoxic chemotherapies are standard of care for patients suffering from advanced non-small cell lung cancer (NSCLC). However, objective responses are only achieved in 20% of cases and long-term survival is rarely observed. Clinically applied anticancer drugs exert at least some of their activities by inducing apoptosis. A critical step in apoptotic signal transduction is the permeabilization of the mitochondrial outer membrane (MOM), which is regulated by the BCL-2 family of proteins. Hence, therapeutic targeting of BCL-2 proteins is a promising approach to increase the drug-sensitivity of cancers. To this end we have assessed the impact of conditional expression of the proapoptotic multidomain (BH1-2-3) protein BAK, which directly permeabilizes the MOM, and the BH3-mimetic ABT-737, which acts indirectly by derepressing BH1-2-3 proteins, on apoptosis and drug sensitivity of NSCLC cells. Conditionally expressed BAK sensitized resistant NSCLC cells to drug-induced apoptosis. In contrast, ABT-737 was ineffective in those NSCLC cells expressing high levels of the anti-apoptotic MCL-1 protein. Tissue microarray analysis of tumor samples from 84 chemotherapy-naïve NSCLC patients revealed MCL-1 expression in 56% of cases, thus supporting the relevance of this resistance factor in a clinical setting. Enforced expression of the BH3-only protein NOXA, which targets MCL-1, overcame resistance to ABT-737. Moreover, combining conditionally expressed BAK with ABT-737 enhanced apoptosis in NSCLC cells independently of their MCL-1 status. In conclusion, the heterogeneity of apoptosis defects observed in drug-resistant NSCLC demands individually tailored molecular therapies. Targeting the MOM permeabilizer BAK appears to have a broader apoptogenic activity than the BH3-only mimetic ABT-737.

Topics: Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Biphenyl Compounds; Butylated Hydroxytoluene; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; Electrophoresis; Etoposide; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Immunohistochemistry; Lung Neoplasms; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Nitrophenols; Paclitaxel; Permeability; Piperazines; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sulfonamides

The epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms.. We performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called "mitochondrial") apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK-ERK1/2 (mitogen-activated protein kinase kinase-extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737.. Inhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR-MEK-ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy.

Topics: Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Biphenyl Compounds; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Line, Tumor; Drug Synergism; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Membrane Proteins; Mutation; Nitrophenols; Piperazines; Proto-Oncogene Proteins; Quinazolines; Sulfonamides

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-X(L) versus Bcl-2 (K(i)'s of 0.80 and 67 nmol/L for Bcl-X(L) and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC(50) of <500 nmol/L in cells dependent on Bcl-X(L) for survival. In addition, A-385358 enhances the in vitro cytotoxic activity of numerous chemotherapeutic agents (paclitaxel, etoposide, cisplatin, and doxorubicin) in several tumor cell lines. In A549 non-small-cell lung cancer cells, A-385358 potentiates the activity of paclitaxel by as much as 25-fold. Importantly, A-385358 also potentiated the activity of paclitaxel in vivo. Significant inhibition of tumor growth was observed when A-385358 was added to maximally tolerated or half maximally tolerated doses of paclitaxel in the A549 xenograft model. In tumors, the combination therapy also resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy.

Topics: Aniline Compounds; Animals; Antineoplastic Agents; bcl-X Protein; Biphenyl Compounds; Cell Line, Tumor; Drug Synergism; Humans; Kinetics; Lung Neoplasms; Male; Mice; Mice, SCID; Nitrophenols; Paclitaxel; Piperazines; Sulfonamides; Transplantation, Heterologous

Phosphodiesterase I (EC 3.1.4.1) activity was detected in normal human blood serum. The enzyme is stable at laboratory temperature for three days, but is inactivated at pH less than 7. The pH for optimum activity increases with the substrate concentration (under the conditions used, from pH 9.0 to 10.2) and, conversely, the Km increases with pH and buffer concentration. The enzyme is inhibited by ethylenediaminetetraacetate but not by phosphate (0.1 mol/liter). We developed a simple quantitative method for its determination, based on hydrolysis of the p-nitrophenyl ester of thymidine 5'-monophosphate and subsequent measurement of the liberated p-nitrophenol at 400 nm in NaOH (0.1 mol/liter). Normal values (mean +/- 2 SD) were determined to be 33 +/- 6.4 U/liter. Preliminary studies indicate that phosphodiesterase I activity is greater than normal in serum of patients with necrotic changes in the liver or kidney or in cases of breast cancer, but not in that of patients with myocardial infarction, bone cancer, lung cancer, or chronic liver cirrhosis.

Topics: Alkaline Phosphatase; Bone Neoplasms; Breast Neoplasms; Edetic Acid; Female; Humans; Kinetics; Liver Cirrhosis; Lung Neoplasms; Male; Myocardial Infarction; Nitrophenols; Phosphates; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Thymine Nucleotides