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1.
Mol Cancer ; 13: 259, 2014 Dec 02.
Article in English | MEDLINE | ID: mdl-25466244

ABSTRACT

BACKGROUND: Lung cancer is the major cause of cancer-related deaths and many cases of Non Small Cell Lung Cancer (NSCLC), a common type of lung cancer, have frequent genetic/oncogenic activation of EGFR, KRAS, PIK3CA, BRAF, and others that drive tumor growth. Some patients though initially respond, but later develop resistance to erlotinib/gefitinib with no option except for cytotoxic therapy. Therefore, development of novel targeted therapeutics is imperative to provide improved survival benefit for NSCLC patients. The mTOR cell survival pathway is activated in naïve, or in response to targeted therapies in NSCLC. METHODS: We have discovered P7170, a small molecule inhibitor of mTORC1/mTORC2/ALK1 and investigated its antitumor efficacy using various in vitro and in vivo models of human NSCLC. RESULTS: P7170 inhibited the phosphorylation of AKT, S6 and 4EBP1 (substrates for mTORC2 and mTORC1) levels by 80-100% and growth of NSCLC cells. P7170 inhibited anchorage-independent colony formation of NSCLC patient tumor-derived cells subsistent of disease sub-types. The compound also induced apoptosis in NSCLC cell lines. P7170 at a well-tolerated daily dose of 20 mg/kg significantly inhibited the growth of NSCLC xenografts independent of different mutations (EGFR, KRAS, or PIK3CA) or sensitivity to erlotinib. Pharmacokinetic-pharmacodynamic (PK-PD) analysis showed sub-micro molar tumor concentrations along with mTORC1/C2 inhibition. CONCLUSIONS: Our results provide evidence of antitumor activity of P7170 in the erlotinib -sensitive and -insensitive models of NSCLC.


Subject(s)
Activin Receptors, Type II/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Proliferation/drug effects , Imidazoles/pharmacology , Lung Neoplasms/drug therapy , Multiprotein Complexes/antagonists & inhibitors , Quinolines/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Animals , Apoptosis/drug effects , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/pharmacology , Erlotinib Hydrochloride , HeLa Cells , Humans , Lung Neoplasms/metabolism , Male , Mechanistic Target of Rapamycin Complex 1 , Mechanistic Target of Rapamycin Complex 2 , Mice , Mice, Nude , Phosphatidylinositol 3-Kinases/pharmacology , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/pharmacology , Proto-Oncogene Proteins p21(ras) , Quinazolines/pharmacology , ras Proteins/pharmacology
2.
Neoplasia ; 14(6): 519-25, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22787433

ABSTRACT

Checkpoint kinase 1 (Chk1) inhibition sensitizes pancreatic cancer cells and tumors to gemcitabine. We hypothesized that Chk1 inhibition would sensitize pancreatic cancer stem cells to gemcitabine. We tested this hypothesis by using two patient-derived xenograft models (designated J and F) and the pancreatic cancer stem cell markers CD24, CD44, and ESA. We determined the percentage of marker-positive cells and their tumor-initiating capacity (by limiting dilution assays) after treatment with gemcitabine and the Chk1 inhibitor, AZD7762. We found that marker-positive cells were significantly reduced by the combination of gemcitabine and AZD7762. In addition, secondary tumor initiation was significantly delayed in response to primary tumor treatment with gemcitabine + AZD7762 compared with control, gemcitabine, or AZD7762 alone. Furthermore, for the same number of stem cells implanted from gemcitabine- versus gemcitabine + AZD7762-treated primary tumors, secondary tumor initiation at 10 weeks was 83% versus 43%, respectively. We also found that pS345 Chk1, which is a measure of DNA damage, was induced in marker-positive cells but not in the marker-negative cells. These data demonstrate that Chk1 inhibition in combination with gemcitabine reduces both the percentage and the tumor-initiating capacity of pancreatic cancer stem cells. Furthermore, the finding that the Chk1-mediated DNA damage response was greater in stem cells than in non-stem cells suggests that Chk1 inhibition may selectively sensitize pancreatic cancer stem cells to gemcitabine, thus making Chk1 a potential therapeutic target for improving pancreatic cancer therapy.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Deoxycytidine/analogs & derivatives , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Pancreatic Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Animals , Antimetabolites, Antineoplastic/administration & dosage , Checkpoint Kinase 1 , Deoxycytidine/administration & dosage , Deoxycytidine/pharmacology , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Pancreatic Neoplasms/drug therapy , Protein Kinase Inhibitors/administration & dosage , Tumor Burden/drug effects , Xenograft Model Antitumor Assays , Gemcitabine
3.
Toxicol Lett ; 196(2): 110-6, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20394812

ABSTRACT

2,2',4,4',5,5'-Hexachlorobiphenyl (PCB-153) is a non-metabolizable environmental chemical contaminant commonly found in breast milk of PCB exposed individuals, suggesting that chronic exposure to PCB-153 could have adverse health effects. We have shown previously that PCB-153 increased reactive oxygen species levels in non-tumorigenic MCF-10A human mammary epithelial cells, which were associated with DNA damage, growth inhibition, and cytotoxicity. This study investigates the hypothesis that PCB-153 exposure coordinates cell cycle progression and cellular metabolism by inhibiting cyclin D1 accumulation. PCB-153 treated MCF-10A cells exhibited a dose and time dependent decrease in cyclin D1 protein levels. The decrease in cyclin D1 protein levels was associated with an inhibition in AKT and GSK-3beta phosphorylation, which correlated with an increase in cyclin D1-T286 phosphorylation. Fibroblasts carrying a mutant form of cyclin D1 (T286A) were resistant to PCB-153 induced degradation of cyclin D1. Pre-treatment of cells with a proteasome inhibitor (MG132) suppressed PCB-153 induced decrease in cyclin D1 protein levels. Interestingly, suppression in cyclin D1 accumulation was associated with an increase in cellular glucose consumption, and hexokinase II and pyruvate kinase protein levels. These results suggest that cyclin D1 coordinates cell cycle progression and cellular metabolism in PCB-153 treated non-tumorigenic human mammary epithelial cells.


Subject(s)
Cell Cycle/drug effects , Energy Metabolism/drug effects , Environmental Pollutants/toxicity , Epithelial Cells/drug effects , Mammary Glands, Human/drug effects , Polychlorinated Biphenyls/toxicity , Animals , Cyclin D1/genetics , Cyclin D1/metabolism , Dose-Response Relationship, Drug , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Glucose/metabolism , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Hexokinase/metabolism , Humans , Leupeptins/pharmacology , Mammary Glands, Human/metabolism , Mammary Glands, Human/pathology , Mice , Mutation , NIH 3T3 Cells , Phosphorylation , Protease Inhibitors/pharmacology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Pyruvate Kinase/metabolism , Time Factors
4.
Cancer Biol Ther ; 8(20): 1962-71, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19738419

ABSTRACT

Manganese superoxide dismutase (SOD2) is a nuclear encoded and mitochondria localized antioxidant enzyme that converts mitochondria derived superoxide to hydrogen peroxide. This study investigates the hypothesis that mitochondria derived reactive oxygen species (ROS) regulate ionizing radiation (IR) induced transformation in normal cells. Mouse embryonic fibroblasts (MEFs) with wild type SOD2 (+/+), heterozygous SOD2 (+/-), and homozygous SOD2 (-/-) genotypes were irradiated with equitoxic doses of IR, and assayed for transformation frequency, cellular redox environment, DNA damage, and cell cycle checkpoint activation. Transformation frequency increased ( approximately 5-fold) in SOD2 (-/-) compared to SOD2 (+/+) MEFs. Cellular redox environment (GSH, GSSG, DHE and DCFH-oxidation) did not show any significant change within 24 h post-IR. However, a significant increase in cellular ROS levels was observed at 72 h post-IR in SOD2 (-/-) compared to SOD2 (+/+) MEFs, which was consistent with an increase in GSSG in SOD2 (-/-) MEFs. Late ROS accumulation was associated with an increase in micronuclei frequency in SOD2 (-/-) MEFs. Exit from G(2) was accelerated in irradiated SOD2 (+/-) and SOD2 (-/-) compared to SOD2 (+/+) MEFs. These results support the hypothesis that SOD2 activity and mitochondria generated ROS regulate IR induced transformation in mouse embryonic fibroblasts.


Subject(s)
Cell Transformation, Neoplastic/radiation effects , Fibroblasts/radiation effects , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Animals , Cell Survival/radiation effects , Cells, Cultured , DNA Damage , Embryo, Mammalian/cytology , Fibroblasts/cytology , Fibroblasts/metabolism , Flow Cytometry , Genotype , Glutathione/metabolism , Immunoblotting , Mice , Mice, Knockout , Micronuclei, Chromosome-Defective/radiation effects , Radiation, Ionizing , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism
5.
Free Radic Biol Med ; 45(8): 1094-102, 2008 Oct 15.
Article in English | MEDLINE | ID: mdl-18691649

ABSTRACT

Polychlorinated biphenyls (PCBs) are environmental chemical contaminants believed to adversely affect cellular processes. We investigated the hypothesis that PCB-induced changes in the levels of cellular reactive oxygen species (ROS) induce DNA damage resulting in cytotoxicity. Exponentially growing cultures of human nonmalignant breast epithelial cells (MCF10A) were incubated with PCBs for 3 days and assayed for cell number, ROS levels, DNA damage, and cytotoxicity. Exposure to 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) or 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), a metabolite of 4-chlorobiphenyl (PCB3), significantly decreased cell number and MTS reduction and increased the percentage of cells with sub-G1 DNA content. Results from electron paramagnetic resonance (EPR) spectroscopy showed a 4-fold increase in the steady-state levels of ROS, which was suppressed in cells pretreated with catalase. EPR measurements in cells treated with 4-Cl-BQ detected the presence of a semiquinone radical, suggesting that the increased levels of ROS could be due to the redox cycling of 4-Cl-BQ. A dose-dependent increase in micronuclei frequency was observed in PCB-treated cells, consistent with an increase in histone 2AX phosphorylation. Treatment of cells with catalase blunted the PCB-induced increase in micronuclei frequency and H2AX phosphorylation that was consistent with an increase in cell survival. Our results demonstrate a PCB-induced increase in cellular levels of ROS causing DNA damage, resulting in cell killing.


Subject(s)
Breast/drug effects , Breast/enzymology , Catalase/metabolism , Epithelial Cells/drug effects , Polychlorinated Biphenyls/toxicity , Breast/pathology , Cells, Cultured , Electron Spin Resonance Spectroscopy , Epithelial Cells/enzymology , Epithelial Cells/pathology , Female , Histones/drug effects , Histones/metabolism , Humans , Immunoblotting , Micronuclei, Chromosome-Defective/drug effects , Reactive Oxygen Species/metabolism
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