leupeptins and Carcinoma--Non-Small-Cell-Lung

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and accounts for 85% of all lung carcinomas. The hepatocyte growth factor receptor (c-Met) has been considered as a potential therapeutic target for NSCLC. Proteasome inhibition induces cell apoptosis and has been used as a novel therapeutic approach for treating diseases including NSCLC; however, the effects of different proteasome inhibitors on NSCLC have not been fully investigated. The aim of this study is to determine a precise strategy for treating NSCLC by targeting c-Met using different proteasome inhibitors. Three proteasome inhibitors, bortezomib, MG132, and ONX 0914, were used in this study. Bortezomib (50 nM) significantly reduced c-Met levels and cell viability in H1299 and H441 cells, while similar effects were observed in H460 and A549 cells when a higher concentration (100 nM) was used. Bortezomib decreased c-Met gene expression in H1299 and H441 cells, but it had no effect in A549 and H460 cells. MG-132 at a low concentration (0.5 M) diminished c-Met levels in H441 cells, while neither a low nor a high concentration (20 M) altered c-Met levels in A549 and H460 cells. A higher concentration of MG-132 (5 M) was required for decreasing c-Met levels in H1299 cells. Furthermore, MG-132 induced cell death in all four cell types. Among all the four cell lines, H441 cells expressed higher levels of c-Met and appeared to be the most susceptible to MG-132. MG-132 decreased c-Met mRNA levels in both H1299 and H441 cells. ONX 0914 reduced c-Met levels in H460, H1299, and H441 cells but not in A549 cells. c-Met levels were decreased the most in H441 cells treated with ONX 0914. ONX 0914 did not alter cell viability in H441; however, it did induce cell death among H460, A549, and H1299 cells. This study reveals that different proteasome inhibitors produce varied inhibitory effects in NSCLS cell lines.

Topics: Antineoplastic Agents; Apoptosis; Bortezomib; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; Leupeptins; Lung Neoplasms; Oligopeptides; Proteasome Inhibitors; Proto-Oncogene Proteins c-met

Alterations in KEAP1/ NF-E2 p45-related factor 2 (NFE2L2/Nrf2) signaling pathway have been reported in 23% lung adenocarcinoma patients, suggesting that deregulation of the pathway is a major cancer driver. Here we report that mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) drives tumor growth and drug resistance by up regulating transcription factor Nrf2. In non-small cell lung cancer (NSCLC) cells and xenografts, MKP-1 knockdown triggered the down-regulation of the metabolic enzymes and cytoprotective proteins, which are the target genes of Nrf2. Consequently, the cell growth was markedly inhibited with decrease of tumor metabolisms and GSH contents. Moreover, MKP-1 silencing sensitized NSCLC cells to cisplatin treatment. Mechanistically, MKP-1 inhibited the ubiquitylation of Nrf2 via a direct interaction with the transcription factor. Nrf2 was hence stabilized and its transcriptional program was activated. Notably, Nrf2 elevated MKP-1 expression at transcriptional level. In human lung adenoma tumor samples, high levels of expression of MKP-1, Nrf2, and its target gene heme oxygenase 1 were closely correlated. Thus, MKP-1 and Nrf2 form a forward feedback loop in lung cancer cells, which stabilizing and activating Nrf2 to promote anabolic metabolism and GSH biosynthesis. This study uncovers a novel role of MKP-1 in the malignant evolution of cancers.

Topics: Adenoma; Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Dual Specificity Phosphatase 1; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Leupeptins; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasms, Experimental; NF-E2-Related Factor 2

Lung cancer remains the top cancer killer worldwide, with squamous cell carcinoma (SCC) as the second commonest histologic subtype. Arsenic trioxide (ATO) was previously shown to suppress growth of lung cancer. Fibroblast growth factor receptor (FGFR) amplification was recently demonstrated in lung SCC, with specific FGFR inhibitor (e.g. PD173074) developed as a potential targeted therapy. Therefore the combination effects of ATO and PD173074 in SCC was studied.. The combination of ATO/PD173074 was studied in a proof-of-principle model using a lung SCC cell line with FGFR1 overexpression: SK-MES-1. The effects of ATO and/or PD173074 on cell viability and protein expression were studied by MTT assay and Western blot respectively. Cell cycle analysis, phosphatidylserine externalization and mitochondrial membrane depolarization were monitored by flow cytometry. FGFR1 knockdown was performed with siRNAs. Proteasome inhibitor (MG-132) was used to study the degradation mechanism. In vivo effect of ATO and/or PD173074 was investigated using a nude mice xenograft model.. Combined ATO/PD173074 reduced cell viability along with increased sub-G1 population, phosphatidylserine externalization and mitochondrial membrane depolarization more significantly than single treatments. Downregulation of FGFR1, p-Akt, Akt, p-Src, Src, p-c-Raf, c-Raf, Erk and survivin as well as upregulation of p-Erk and cleaved PARP were observed upon ATO and/or PD treatment. MG-132 partially reversed the degradation of Akt, Src, c-Raf and Erk induced by ATO/PD, suggestive of ubiquitin-independent proteasome-dependent degradation. However, the mechanism of FGFR1 downregulation remained unknown. Downregulation of FGFR1, Akt, Src, c-Raf and Erk as well as cleaved PARP elevation induced by ATO and/or PD were confirmed in vivo.. Massive protein degradation (FGFR1, Akt, Src, c-Raf and Erk) was induced by ATO and/or PD173074 treatment mainly mediated by activation of proteasomal degradation in SCC cell line SK-MES-1 in vitro and in vivo.

Topics: Animals; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Drug Therapy, Combination; Female; Leupeptins; Lung Neoplasms; Mice; Mice, Nude; Oxides; Pyrimidines; Receptor, Fibroblast Growth Factor, Type 1; Xenograft Model Antitumor Assays

The current treatment for advanced nonsmall cell lung cancer (NSCLC) remains unsatisfactory due to resistance to chemotherapy and ionizing radiation. The ubiquitin-proteasome system (UPS) regulates multiple cellular processes that are crucial for the proliferation and survival of all kinds of cells. Carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132), a specific and selective reversible inhibitor of the 26S proteasome, represents a novel approach for cancer therapy. However, whether MG132 can potentiate the effect of radiation against the growth and metastasis of NSCLC is not clear. We found that MG132 inhibited the proliferation of human NSCLC cell lines (A549 and H1299) in a dose- and time-dependent manner by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Then MG132 at a nontoxic dose (100 nM) was selected for following studies. Pretreatment of A549 and H1299 cells with 100 nM MG132 before ionizing radiation (IR) potentiated the anticancer effect of IR. Moreover, pretreatment with 100 nM MG132 before IR-enhanced radiation induced cell cycle arrest by decreased CyclinD1 but increased Wee1 expression in A549 and H1299 cells. In addition, pretreatment of MG132 combined with IR significantly suppressed cell migration and invasion abilities in NSCLC cell lines, which was accompanied by decreased expression of matrix metalloproteinase (MMP)-2 and MMP-9 in NSCLC cell lines. Taken together, our results demonstrate that MG132 enhances the antigrowth and antimetastatic effects of irradiation in NSCLC cells by modulating expression of cell cycle and invasion- related genes.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Humans; Leupeptins; Lung Neoplasms; Neoplasm Metastasis; NF-kappa B; Nuclear Proteins; Proteasome Inhibitors; Protein-Tyrosine Kinases; Radiation Tolerance

The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis.. API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction.. API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.

Topics: Apoptosis; beta-Transducin Repeat-Containing Proteins; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Line, Tumor; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glycogen Synthase Kinase 3; Heterocyclic Compounds, 3-Ring; Humans; Leupeptins; Myeloid Cell Leukemia Sequence 1 Protein; Nucleosides; Phosphorylation; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-akt; Ubiquitin-Protein Ligases

In this study, the effect of MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal-H) at a low dose on radiotherapeutic efficacy and its accurate mechanism of radiosensitization were investigated in human non-small cell lung cancer. The effect of MG132 on ionizing radiation (IR)-induced cytotoxicity, cell proliferation and survival of A549 cells was evaluated. The protein expression modulated by MG132 and IR were inspected by Western blot analysis. To determine in vivo radiotherapeutic efficacy, tumor growth delay was analyzed in a A549 tumor-bearing xenograft mouse model after single or repeated treatment of MG132 and/or IR. Induction of apoptosis and change of c-Myc expression in the tumor tissue was explored by histological analysis. MG132 at a non-toxic dose enhanced the radiation-induced cytotoxicity of A549 cells, accompanying a significant decrease of c-Myc expression. Suppression of c-Myc expression by small interfering RNA (siRNA) displayed enhancement of radiosensitivity similarly to MG132 treatment. Tumor growth in the xenograft mice was markedly delayed by systemic administration of MG132 combined with IR. In vivo down-regulation of c-Myc and increased induction of apoptosis were simultaneously observed in the tumor tissues followed by combinational treatment of MG132 and IR. The results reveal a novel mechanism for proteasome inhibitor-mediated radiosensitization in which c-Myc down-regulation is involved.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Survival; Down-Regulation; Humans; Leupeptins; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Proteasome Inhibitors; Proto-Oncogene Proteins c-myc; Radiation-Sensitizing Agents; Xenograft Model Antitumor Assays

Proteasome inhibitors (PIs) are promising new therapeutic agents for treating non-small cell lung carcinoma (NSCLC). To investigate the mechanisms of action of PIs, we analyzed the proapoptotic activities of PIs (MG132 or Bortezomib) in NSCLC cells. We found that both MG132 (>1 microM) and Bortezomib (>0.025 microM) induced a significant apoptosis in NCI-H1703, a PI-sensitive NSCLC cell line, through initially activating the intrinsic apoptosis pathway, leading to the activation of a positive feedback mechanism (PFM), which then conveyed apoptosis signaling from the intrinsic pathway to the extrinsic pathway with formation of a signaling loop for maximal caspase activation. Mcl-1 and Noxa were identified to be the major anti-apoptotic and proapoptotic proteins, respectively, in PI-induced apoptosis and mutually exclusive in protein stability. Although the Mcl-1 protein was upregulated by proteasome inhibition, it was also subjected to caspase 3-dependent cleavage governed by the PFM. Moreover, it was revealed that Mcl-1 protein cleavage contributed to PFM-governed apoptosis in following inter-related ways: reducing the anti-apoptotic Mcl-1; generating the truncated proapoptotic Mcl-1(S); and inducing a shift of balance between Mcl-1 and Noxa. It was further manifested that tumor necrosis factor-related apoptosis-inducing ligand boosted MG132's proapoptotic activity through strengthening the PFM in both NCI-H1703 and NCI-H358, a PI-resistant NSCLC cell line. Therefore, this study provides a basis for enhancing the efficacy of PIs in treating NSCLC.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Feedback, Physiological; Humans; Leupeptins; Lung Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; TNF-Related Apoptosis-Inducing Ligand

The PML tumor suppressor controls key pathways for growth suppression, induction of apoptosis, and cellular senescence. PML loss occurs frequently in human tumors through unknown posttranslational mechanisms. Casein kinase 2 (CK2) is oncogenic and frequently upregulated in human tumors. Here we show that CK2 regulates PML protein levels by promoting its ubiquitin-mediated degradation dependent on direct phosphorylation at Ser517. Consequently, PML mutants that are resistant to CK2 phosphorylation display increased tumor-suppressive functions. In a faithful mouse model of lung cancer, we demonstrate that Pml inactivation leads to increased tumorigenesis. Furthermore, CK2 pharmacological inhibition enhances the PML tumor-suppressive property in vivo. Importantly, we found an inverse correlation between CK2 kinase activity and PML protein levels in human lung cancer-derived cell lines and primary specimens. These data identify a key posttranslational mechanism that controls PML protein levels and provide therapeutic means toward PML restoration through CK2 inhibition.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Apoptosis; Carcinoma, Non-Small-Cell Lung; Casein Kinase II; Cell Line; Cell Line, Transformed; Cell Line, Tumor; Enzyme Activation; Enzyme Inhibitors; Genes, Tumor Suppressor; Green Fluorescent Proteins; Hemagglutinins; Humans; Leupeptins; Lung Neoplasms; Mice; Mice, Transgenic; Molecular Sequence Data; Neoplasm Proteins; NIH 3T3 Cells; Nuclear Proteins; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Promyelocytic Leukemia Protein; Proteasome Endopeptidase Complex; Protein Structure, Tertiary; Protein Subunits; RNA, Small Interfering; Sequence Deletion; Serine; Sorbitol; Transcription Factors; Transcriptional Activation; Triazoles; Tumor Suppressor Proteins; Ubiquitin

The cyclooxygenase-2 (COX-2) inhibitor celecoxib is an approved drug in the clinic for colon cancer chemoprevention and has been tested for its chemopreventive and therapeutic efficacy in various clinical trials. Celecoxib induces apoptosis in a variety of human cancer cells including lung cancer cells. Our previous work has shown that celecoxib induces death receptor 5 expression, resulting in induction of apoptosis and enhancement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human lung cancer cells. In the current study, we further show that celecoxib down-regulated the expression of cellular FLICE-inhibitory protein (c-FLIP), a major negative regulator of the death receptor-mediated extrinsic apoptotic pathway, through a ubiquitin/proteasome-dependent mechanism independent of COX-2 in human lung cancer cells. Overexpression of c-FLIP, particularly FLIP(L), inhibited not only celecoxib-induced apoptosis but also apoptosis induced by the combination of celecoxib and TRAIL. These results thus indicate that c-FLIP down-regulation also contributes to celecoxib-induced apoptosis and enhancement of TRAIL-induced apoptosis, which complements our previous finding that the extrinsic apoptotic pathway plays a critical role in celecoxib-induced apoptosis in human lung cancer cells. Collectively, we conclude that celecoxib induces apoptosis in human lung cancer cells through activation of the extrinsic apoptotic pathway, primarily by induction of death receptor 5 and down-regulation of c-FLIP.

Topics: Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; CASP8 and FADD-Like Apoptosis Regulating Protein; Celecoxib; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Down-Regulation; Drug Synergism; Flow Cytometry; Humans; Leupeptins; Lung Neoplasms; Pyrazoles; RNA, Small Interfering; Sulfonamides; TNF-Related Apoptosis-Inducing Ligand; Transfection; Ubiquitin

Non-small cell lung cancer frequently presents as a locally advanced disease. In this setting, radiation has a prominent role in cancer therapy. However, tumor adaptation to oxidative stress may lessen the efficacy of radiation therapy. Recent studies demonstrate that proteasome inhibitors increase the efficacy of radiation against a range of tumors. Although proteasome inhibition impacts on NF-kappaB translocation, the precise mechanism through which proteasome inhibitors induce tumor cell death and promote radiation efficacy remains unclear. The purpose of this study is to evaluate the potential of the proteasome inhibitor, MG-132, to improve the efficacy of radiation therapy and to determine whether its effect is linked to the suppression of the antioxidant enzyme, manganese superoxide dismutase (MnSOD). Human NSCLC (A549) cells were utilized both in vivo and in vitro to evaluate proteasome inhibition on radiation response. In vivo, mice that received combined treatments of 2.5 microg/g body weight MG-132 and 30 Gy demonstrated a delay in tumor regrowth in comparison to the 30 Gy control group. In vitro, clonegenic survival assays confirmed a dose-dependent enhancement of radiation sensitivity in combination with MG-132 and a significant interaction between the two. The levels of IkappaB-alpha, a NF-kappaB target gene and also an inhibitor of NF-kappaB nuclear translocation, decreased in a time-dependent manner following administration of MG-132 confirming the inhibition of the 26S proteasome. The MnSOD protein level was increased consistent with lower levels of IkappaB-alpha, confirming a NF-kappaB-mediated effect. Cells treated with radiation demonstrated an induction of MnSOD; however, the administration of MG-132 suppressed this induction These results support the hypothesis that proteasome inhibitors such as MG-132 can increase the efficacy of radiation therapy, in part, by suppression of cytoprotective NF-kappaB-mediated MnSOD expression.

Topics: Active Transport, Cell Nucleus; Animals; Antioxidants; Blotting, Western; Body Weight; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Combined Modality Therapy; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Female; Humans; I-kappa B Proteins; Leupeptins; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; NF-kappa B; NF-KappaB Inhibitor alpha; Oxidative Stress; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Transport; Radiation Tolerance; Reactive Oxygen Species; Superoxide Dismutase; Time Factors

The role of the ubiquitin-proteasome pathway during roscovitine induced apoptosis was evaluated in the non-small cell lung carcinoma cell line MR65. To this end specific inhibitors of proteasome activity, MG132 and lactacystin were used. Addition of MG132 or lactacystin, 1 h prior to the addition of the CDK-inhibitor roscovitine to the cell cultures inhibited apoptosis significantly, as measured by PS exposure, cytokeratin 18 cleavage and caspase-3 activation. Furthermore, we show that inhibition of proteasome activation prior to induction of apoptosis by roscovitine prevents loss of mitochondrial inner transmembrane potential (DeltaPsim). In addition we found that MG132 and lactacystin prevent release of cytochrome c from the mitochondrion. In contrast to the above findings we see no effect of proteasome inhibition in Fas-mediated apoptosis. Taken together our data suggest a specific role for proteasomes very early in roscovitine-induced apoptosis, upstream from the caspase cascade and mitochondrion.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Lung Neoplasms; Membrane Potentials; Mitochondria; Proteasome Endopeptidase Complex; Purines; Roscovitine; Signal Transduction; Tumor Cells, Cultured

Activation of signaling pathways after DNA damage induced by topoisomerase (topo) poisons can lead to cell death by apoptosis. Treatment of human nonsmall cell lung carcinoma (NSCLC-3 or NSCLC-5) cells with the topo I poison SN-38 or the topo II poison etoposide (VP-16) leads to activation of NF-kappaB before induction of apoptosis. Inhibiting the degradation of IkappaBalpha by pretreatment with the proteasome inhibitor MG-132 significantly inhibited NF-kappaB activation and apoptosis but not DNA damage induced by SN-38 or VP-16. Transfection of NSCLC-3 or NSCLC-5 cells with dominant negative mutant IkappaBalpha (mIkappaBalpha) inhibited SN-38 or VP-16 induced transcription and DNA binding activity of NF-kappaB without altering drug-induced apoptosis. Regulation of apoptosis by mitochondrial release of cytochrome c and activation of pro-caspase 9 followed by cleavage of poly(ADP-ribose) polymerase by effector caspases 3 and 7 was similar in neo and mIkappaBalpha cells treated with SN-38 or VP-16. In contrast to pretreatment with MG-132, exposure to MG-132 after SN-38 or VP-16 treatment of neo or mIkappaBalpha cells decreased cell cycle arrest in the S/G2 + M fraction and enhanced apoptosis compared with drug alone. In summary, apoptosis induced by topoisomerase poisons in NSCLC cells is not mediated by NF-kappaB but can be manipulated by proteasome inhibitors.

Topics: Apoptosis; Camptothecin; Carcinoma, Non-Small-Cell Lung; Cisplatin; DNA; DNA Damage; DNA-Binding Proteins; Enzyme Inhibitors; Etoposide; Humans; I-kappa B Proteins; Irinotecan; Leupeptins; Lung Neoplasms; NF-kappa B; NF-KappaB Inhibitor alpha; Paclitaxel; Peptide Hydrolases; Proteasome Endopeptidase Complex; Topoisomerase I Inhibitors; Topoisomerase II Inhibitors

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Camptothecin; Carcinoma, Non-Small-Cell Lung; DNA-Binding Proteins; Enzyme Inhibitors; Humans; I-kappa B Proteins; Irinotecan; Leupeptins; Lung Neoplasms; NF-kappa B; NF-KappaB Inhibitor alpha; Topoisomerase I Inhibitors; Transfection; Tumor Cells, Cultured