RESUMO
BACKGROUND: Monoamine oxidase A (MAOA) is best known for its role in neuro-transmitter regulation. Monoamine oxidase inhibitors are used to treat atypical depression. MAOA is highly expressed in high grade prostate cancer and modulates tumorigenesis and progression in prostate cancer. Here, we investigated the potential role of MAOA inhibitors (MAOAIs) in relation to the androgen receptor (AR) pathway and resistance to antiandrogen treatment in prostate cancer. METHODS: We examined MAOA expression and the effect of MAOI treatment in relation to AR-targeted treatments using the LNCaP, C4-2B, and 22Rv1 human prostate cancer cell lines. MAOA, AR-full length (AR-FL), AR splice variant 7 (AR-V7), and PSA expression was evaluated in the presence of MAOAIs (clorgyline, phenelzine), androgenic ligand (R1881), and antiandrogen (enzalutamide) treatments. An enzalutamide resistance cell line was generated to test the effect of MAOAI treatment in this model. RESULTS: We observed that MAOAIs, particularly clorgyline and phenelzine, were effective at decreasing MAOA activity in human prostate cancer cells. MAOAIs significantly decreased growth of LNCaP, C4-2B, and 22Rv1 cells and produced additive growth inhibitory effects when combined with enzalutamide. Clorgyline decreased expression of AR-FL and AR-V7 in 22Rv1 cells and was effective at decreasing growth of an enzalutamide-resistant C4-2B cell line with increased AR-V7 expression. CONCLUSIONS: MAOAIs decrease growth and proliferation of androgen-sensitive and castration-resistant prostate cancer cells. Clorgyline, in particular, decreases expression of AR-FL and AR-V7 expression and decreases growth of an enzalutamide-resistant cell line. These findings provide preclinical validation of MAOA inhibitors either alone or in combination with antiandrogens for therapeutic intent in patients with advanced forms of prostate cancer.
Assuntos
Clorgilina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fenelzina/farmacologia , Feniltioidantoína/análogos & derivados , Neoplasias de Próstata Resistentes à Castração , Receptores Androgênicos/metabolismo , Antagonistas de Androgênios/farmacologia , Benzamidas , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Gradação de Tumores , Nitrilas , Feniltioidantoína/farmacologia , Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologiaRESUMO
Reactive oxygen species (ROS) play a critical role in cell signaling and proliferation. NADPH oxidase 1 (NOX1), a membrane-bound flavin dehydrogenase that generates O2ÌÌ, is highly expressed in colon cancer. To investigate the role that NOX1 plays in colon cancer growth, we used shRNA to decrease NOX1 expression stably in HT-29 human colon cancer cells. The 80-90% decrease in NOX1 expression achieved by RNAi produced a significant decline in ROS production and a G1/S block that translated into a 2-3-fold increase in tumor cell doubling time without increased apoptosis. The block at the G1/S checkpoint was associated with a significant decrease in cyclin D1 expression and profound inhibition of mitogen-activated protein kinase (MAPK) signaling. Decreased steady-state MAPK phosphorylation occurred concomitant with a significant increase in protein phosphatase activity for two colon cancer cell lines in which NOX1 expression was knocked down by RNAi. Diminished NOX1 expression also contributed to decreased growth, blood vessel density, and VEGF and hypoxia-inducible factor 1α (HIF-1α) expression in HT-29 xenografts initiated from NOX1 knockdown cells. Microarray analysis, supplemented by real-time PCR and Western blotting, revealed that the expression of critical regulators of cell proliferation and angiogenesis, including c-MYC, c-MYB, and VEGF, were down-regulated in association with a decline in hypoxic HIF-1α protein expression downstream of silenced NOX1 in both colon cancer cell lines and xenografts. These studies suggest a role for NOX1 in maintaining the proliferative phenotype of some colon cancers and the potential of NOX1 as a therapeutic target in this disease.
Assuntos
Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Colo/metabolismo , Ciclina D1/metabolismo , Células HT29 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , NADPH Oxidase 1 , Transplante de Neoplasias , Fenótipo , Fosforilação , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Mutation of the tumor suppressor TP53 gene occurs in greater than half of all human cancers. In addition to loss of tumor suppressor function of wild-type TP53, gain-of-function mutations endow cancer cells with more malignant properties. R273 is a mutation hotspot with the p.R273H, p.R273C, and p.R273G variants occurring most commonly in patient samples. To better understand the consequences of these R273 mutations, we constructed cancer cell lines expressing TP53 p.R273H, p.R273C, or p.R273G and explored their characteristics. We found that p.R273H and p.R273C, but not p.R273G, enhanced proliferation, invasion, and drug resistance in vitro. Furthermore, breast cancer susceptibility protein 1 was upregulated by mutant TP53 p.R273H and p.R273C in response to DNA damage and repair. Transcriptional analysis of the TP53-R273 mutants by RNA-seq confirmed that the apoptosis pathway was less active in p.R273H and p.R273C, compared with R273G. Molecular dynamics simulation further revealed that TP53-R273G binds more tightly to DNA than TP53-R273H or TP53-R273C. These findings indicate that mutation of TP53 at a single codon has different effects, and likely clinical implications. p.R273H and p.R273C lead to a more aggressive phenotype than p.R273G. These findings may contribute to future diagnosis and therapy in TP53 mutant cancers.
Assuntos
Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Invasividade Neoplásica/genética , Proteína Supressora de Tumor p53/genética , Apoptose/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/administração & dosagem , Feminino , Humanos , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Proteína Supressora de Tumor p53/químicaRESUMO
BACKGROUND: Cancer is the result of a multistep process of genomic alterations, including mutations in key regulatory proteins that result in loss of balanced gene expression and subsequent malignant transformation. Throughout the various stages of colorectal carcinoma (CRC), complex genetic alterations occur, of which over-expression of growth factors, such as vascular endothelial growth factor, fibroblast growth factor and platelet-derive growth factor and their corresponding receptor tyrosine kinases, have been shown to correlate with invasiveness, tumor angiogenesis, metastasis, recurrence, and poor prognosis of colorectal cancer. To evaluate the therapeutic effect, we combined Dovitinib, an orally bioavailable, potent inhibitor of class III-V receptor tyrosine kinases with chemotherapeutic drug, oxaliplatin in preclinical models of colon cancer. METHODS: Human colon cancer cells with different RAS-RAF mutation status (HCT-116, HT-29, SW-480, CaCO2 and LS174T) were treated with a combination of Dovitinib and Oxaliplatin at low dosage followed by assays to investigate the effect of the combination on cell proliferation, cell migration, cell apoptosis and signaling pathways involved in molecular mechanism of drug(s). The antitumor effects of either of the drugs were compared to the combination using human colon carcinoma cell line HT-29 xenograft model. Treated vs untreated tumor sections were also compared for proliferation and angiogenesis markers by immunohistochemistry. RESULTS: The combination of dovitinib and oxaliplatin showed higher in vitro cytotoxicity in colon cell lines irrespective of their RAS-RAF status as compared to either of the drugs alone. Simultaneous inhibition of MAP kinase and AKT pathways and induction of apoptosis via activation of caspases 9/caspases 3 contributed to the synergistic effect of this combination therapy. In the xenograft model, the combination showed a significantly higher antitumor activity. Immunohistochemistry of post treatment tumors showed a significant decrease in proliferation and angiogenesis as compared to either of the treatments alone. CONCLUSIONS: This study demonstrates the synergistic antitumor activity of combination of dovitinib and oxaliplatin against colon cancer with different RAS-RAF status. The combination also showed its antitumor efficacy in a multidrug resistant phenotype xenograft model. This provides a basis for further investigation for its potential in clinical setting for colorectal cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Mutação , Animais , Benzimidazóis/administração & dosagem , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Sinergismo Farmacológico , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Proteína Oncogênica p21(ras)/genética , Compostos Organoplatínicos/administração & dosagem , Oxaliplatina , Quinolonas/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/genéticaRESUMO
CRLX101 is a nanopharmaceutical consisting of cyclodextrin-based polymer molecule and camptothecin. The CRLX101 nanoparticle is designed to concentrate and slowly release camptothecin in tumors over an extended period of time. Tumor biopsy and blood samples collected from patients with advanced solid malignancies before and after CRLX101 treatment are subjected to immunohistochemistry and pharmacogenomics. The expression of Topoisomerase-1, Ki-67, CaIX, CD31 and VEGF decreased after CRLX101 treatment. The expressions of these proteins are inversely proportional with survival duration of the patients. The Drug Metabolism Enzymes and Transporters (DMET) array shows an allele frequency in patients similar to global populations with none of the SNPs associated with toxicity. The results suggest that the observed lower toxicity is not likely to be due to different genotypes in SNPs. CRLX101 demonstrates a promising anti-tumor activity in heavily pre-treated or treatment-refractory solid tumor malignancies presumably by inhibition of proliferation and angiogenesis correlating with tumor growth inhibition. From the clinical editor: In this cancer treatment study clinical samples collected from patients were subjected to immunohistochemistry and pharmacogenomics. The expressions of key proteins that are inversely proportional with survival duration of the patients decreased after treatment with CRLX101, a camptothecin slow-release nanoparticle conjugate. This anti-tumor activity in heavily pre-treated and treatment resistant solid tumors, promises a novel therapeutic approach.
Assuntos
Antineoplásicos/farmacologia , Camptotecina/farmacologia , Ciclodextrinas/farmacologia , Nanopartículas , Neoplasias/tratamento farmacológico , Farmacogenética , Antineoplásicos/uso terapêutico , Sequência de Bases , Camptotecina/química , Camptotecina/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Ciclodextrinas/química , Ciclodextrinas/uso terapêutico , Citocinas/sangue , Primers do DNA , Humanos , Neovascularização Patológica/prevenção & controle , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.
Assuntos
Processamento Alternativo/fisiologia , Antígenos CD/biossíntese , Moléculas de Adesão Celular/biossíntese , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Antígenos CD/genética , Moléculas de Adesão Celular/genética , Éxons/fisiologia , Células HEK293 , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/genética , Humanos , Íntrons/fisiologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Camptothecin showed remarkable anticancer activity in animal models of cancer but was restricted in clinical use for its adverse toxicity in patients. The preclinical efficacy of CRLX101, a nanoparticle (NP) assembly containing cyclodextrin-based polymer and camptothecin was evaluated by in vitro cytotoxicity in gastric cancer cell lines and in vivo antitumor effects in human gastric cancer cell line BGC823 xenografts. Treated tumor sections were analyzed for presence of NPs and compared with vehicle control tumors for hypoxia and angiogenesis. Gastric cancer cell lines showed high in vitro cytotoxicity for CRLX101 and also showed strong antitumor activity in vivo. Electron micrographs revealed the intracellular presence of NPs in close proximity to vesicles. A significant decrease in expressions of carbonic anhydrase, VEGF, and CD31 proteins in treated tumors indicated an inhibition of hypoxia and angiogenesis. The results provide preclinical data for gastric adenocarcinoma. FROM THE CLINICAL EDITOR: This study describes a nanoparticle assembly containing cyclodextrin-based polymer and camptothecin, resulting in increased bioavailability of camptothecin, an effective but toxic anti-cancer agent. The antitumor effects and safety profile were demonstrated in a gastric carcinoma cell line.
Assuntos
Antineoplásicos/administração & dosagem , Camptotecina/análogos & derivados , Nanopartículas/química , Neoplasias Gástricas/tratamento farmacológico , Animais , Antineoplásicos/química , Camptotecina/administração & dosagem , Camptotecina/química , Linhagem Celular Tumoral , Ciclodextrinas/química , Portadores de Fármacos/química , Humanos , Irinotecano , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To examine whether combining arsenic trioxide (ARS) and pamidronate (PAM), anticancer drugs that generate reactive oxygen species (ROS), enhanced targeting of redox sensitive growth signals, we studied cloning efficiency, protein tyrosine phosphatase (PTPase) activity, and epidermal growth factor receptor (EGFR) phosphorylation in DU-145 and PC-3 human prostate cancer cells in response to treatment with ARS and/or PAM for 24 h. IC50 concentrations in a clonogenic assay for ARS and PAM were 9 and 20 µM, respectively, in DU-145 cells; and 2 and 12 µM, in PC-3 cells. When combined, ARS and PAM demonstrated additive cytotoxicity in the DU-145 line (combination index [CI] of 1.10) and synergy for PC-3 cells (CI of 0.86). ARS (20 µM for 24 h) inhibited PTPase activity by 36 ± 7 %, p < 0.05 vs. untreated controls, in DU-145 cells; and by 58 ± 8%, p < 0.05, in the PC-3 line. PAM (20 µM for 24 h) decreased PTPase activity by 24 ± 9%, p = 0.06, and 8 ± 1%, p < 0.01, in DU-145 and PC-3 cells, respectively. Combining ARS and PAM significantly inhibited PTPase activity in both cell lines at lower concentrations of each drug. Pretreatment with N-acetyl-L-cysteine reversed ARS- and PAM-induced inhibition of PTPase activity. PTPase inhibition by ARS and/or PAM treatment in both DU-145 and PC-3 cells was associated with prolonged EGFR activation. These experiments demonstrate additive or synergistic cell killing by the ARS/PAM combination in DU-145 or PC-3 cells and suggest that enhanced antitumor activity may be related to alterations in receptor tyrosine kinase signaling that occur, in part, due to ROS-mediated PTPase inhibition.
RESUMO
The majority of eukaryotic genes undergo alternative splicing, an evolutionarily conserved phenomenon, to generate functionally diverse protein isoforms from a single transcript. The fact that defective pre-mRNA splicing can generate non-functional and often toxic proteins with catastrophic effects, accurate removal of introns and joining of exons is vital for cell homeostasis. Thus, molecular tools that could either silence a disease-causing gene or regulate its expression in trans will find many therapeutic applications. Here we present two RNA-based approaches, namely RNAi and theophylline-responsive riboswitch that can regulate gene expression by loss-of-function and modulation of splicing, respectively. These strategies are likely to continue to play an integral role in studying gene function and drug discovery.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Aptâmeros de Nucleotídeos/farmacologia , Interferência de RNA , Teofilina/farmacologia , Aptâmeros de Nucleotídeos/metabolismo , Linhagem Celular Tumoral , Humanos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Teofilina/metabolismo , TransfecçãoRESUMO
BACKGROUND: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a cell adhesion molecule expressed in a variety of cell types is a putative tumor suppressor gene. Alternative splicing of CEACAM1 generates 11 different splice variants, which include 1-4 ectodomains with either short or long cytoplasmic domain generated by the exclusion (CEACAM1-S) or inclusion (CEACAM1-L) of exon 7. Studies in rodents indicate that optimal ratios of CEACAM1 splice variants are required to inhibit colonic tumor cell growth. RESULTS: We show that CEACAM1 is expressed in a tissue specific manner with significant differences in the ratios of its short (CEACAM1-S) and long (CEACAM1-L) cytoplasmic domain splice variants. Importantly, we find dramatic differences between the ratios of S:L isoforms in normal breast tissues versus breast cancer specimens, suggesting that altered splicing of CEACAM1 may play an important role in tumorogenesis. Furthermore, we have identified two regulatory cis-acting elements required for the alternative splicing of CEACAM1. Replacement of these regulatory elements by human beta-globin exon sequences resulted in exon 7-skipped mRNA as the predominant product. Interestingly, while insertion of exon 7 in a beta-globin reporter gene resulted in its skipping, exon 7 along with the flanking intron sequences recapitulated the alternative splicing of CEACAM1. CONCLUSION: Our results indicate that a network of regulatory elements control the alternative splicing of CEACAM1. These findings may have important implications in therapeutic modalities of CEACAM1 linked human diseases.
Assuntos
Processamento Alternativo/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Citoplasma/metabolismo , Linhagem Celular Tumoral , Éxons/genética , Regulação Neoplásica da Expressão Gênica , Saúde , Humanos , Íntrons/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sequências Reguladoras de Ácido NucleicoRESUMO
The cellular metabolism of doxorubicin generates reactive oxygen species with significant potential to damage DNA. Such DNA damage can result in mutations if not adequately repaired by cellular DNA repair pathways. Secondary malignancies have been reported in patients who have received doxorubicin-containing chemotherapeutic regimens; however, the underlying molecular mechanism(s) to explain the development of these tumors remains under active investigation. We have previously demonstrated the presence of DNA bases modified by oxidation in the peripheral blood mononuclear cells of patients with breast cancer following treatment with doxorubicin. In those studies, doxorubicin was administered by continuous infusion over 96 h to minimize the risk of cardiac toxicity. To evaluate potential mechanisms underlying doxorubicin-induced DNA base oxidation in non-malignant tissues, MCF-10A breast epithelial cells were cultured for 96 h with the same doxorubicin concentration achieved in vivo (0.1 microM). During doxorubicin exposure, MCF-10A cells underwent growth arrest and apoptosis, developed elevated levels of reactive oxygen species, and demonstrated a time-dependent and significant increase in the levels of 11 oxidized DNA bases, as determined by gas chromatography/mass spectroscopy. Diminished expression of DNA repair enzymes was also observed over the same time course. Thus, clinically achievable concentrations of doxorubicin induce a level of oxidative stress in MCF-10A cells that is capable of oxidizing DNA bases and significantly altering cellular proliferation.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Mama/citologia , Dano ao DNA , DNA/química , Doxorrubicina/administração & dosagem , Células Epiteliais/efeitos dos fármacos , Estresse Oxidativo , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sequência de Bases/efeitos dos fármacos , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Reparo do DNA , Doxorrubicina/farmacologia , Células Epiteliais/citologia , Feminino , Citometria de Fluxo , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Oxirredução , Perfusão , Fatores de TempoRESUMO
The pan-PI3K inhibitors are one treatment option for triple-negative breast cancer (TNBC). However, this treatment is ineffective for unknown reasons. Here, we report that aberrant expression of wingless-type MMTV integration site family (WNT) and activated WNT signals, which crosstalk with the PI3K-AKT-mTOR signaling pathway through GSK3ß, plays the most critical role in resistance to pan-PI3K inhibitors in TNBC cells. GDC-0941 is a pan-PI3K inhibitor that activates the WNT/beta-catenin pathway in TNBC cells through stimulation of WNT secretion. GDC-0941-triggered WNT/beta-catenin pathway activation was observed in MDA-MB-231 and HCC1937 cells, which are TNBC cell lines showing aberrant WNT/beta-catenin activation, and not in SKBR3 and MCF7 cells. This observation is further investigated in vivo. GDC-0941 exhibited minimal tumor inhibition in MDA-MB-231 cells, but it significantly suppressed tumor growth in HER-positive SK-BR3 cells. In vivo mechanism study revealed the activation of WNT/beta-catenin pathway by GDC-0941. A synergistic effect was observed when combined treatment with GDC-0941 and the WNT inhibitor LGK974 at low concentrations in MDA-MB-231 cells. These findings indicated that WNT pathway activation conferred resistance in TNBC cells treated with GDC-0941. This resistance may be further circumvented through combined treatment with pan-PI3K and WNT inhibitors. Future clinical trials of these two inhibitors are warranted.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indazóis/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Sulfonamidas/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Feminino , Imunofluorescência , Humanos , Camundongos , Camundongos Nus , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazinas/farmacologia , Piridinas/farmacologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Células Tumorais Cultivadas , Proteínas Wnt/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Autophagy is the principal catabolic response to nutrient starvation and is necessary to clear dysfunctional or damaged organelles, but excessive autophagy can be cytotoxic or cytostatic and contributes to cell death. Depending on the abundance of enzymes involved in molecule biosynthesis, cells can be dependent on uptake of exogenous nutrients to provide these molecules. Argininosuccinate synthetase 1 (ASS1) is a key enzyme in arginine biosynthesis, and its abundance is reduced in many solid tumors, making them sensitive to external arginine depletion. We demonstrated that prolonged arginine starvation by exposure to ADI-PEG20 (pegylated arginine deiminase) induced autophagy-dependent death of ASS1-deficient breast cancer cells, because these cells are arginine auxotrophs (dependent on uptake of extracellular arginine). Indeed, these breast cancer cells died in culture when exposed to ADI-PEG20 or cultured in the absence of arginine. Arginine starvation induced mitochondrial oxidative stress, which impaired mitochondrial bioenergetics and integrity. Furthermore, arginine starvation killed breast cancer cells in vivo and in vitro only if they were autophagy-competent. Thus, a key mechanism underlying the lethality induced by prolonged arginine starvation was the cytotoxic autophagy that occurred in response to mitochondrial damage. Last, ASS1 was either low in abundance or absent in more than 60% of 149 random breast cancer biosamples, suggesting that patients with such tumors could be candidates for arginine starvation therapy.
Assuntos
Arginina/metabolismo , Argininossuccinato Sintase/deficiência , Neoplasias da Mama/metabolismo , Mitocôndrias/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Arginina/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias da Mama/patologia , Neoplasias da Mama/prevenção & controle , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Humanos , Hidrolases/farmacologia , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Polietilenoglicóis/farmacologia , Prognóstico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Iodonium-class flavoprotein dehydrogenase inhibitors have been demonstrated to possess antiproliferative potential and to inhibit reactive oxygen production in human tumor cells, although the mechanism(s) that explains the relationship between altered cell growth and the generation of reactive oxygen species (ROS) remains an area of active investigation. Because of the ability of these compounds to inhibit the activity of flavoprotein-containing epithelial NADPH oxidases, we chose to examine the effects of several iodonium-class flavoprotein inhibitors on human colon cancer cell lines that express high, functional levels of a single such oxidase (NADPH oxidase 1, or Nox1). We found that diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodonium diphenyl inhibited the growth of Caco2, HT-29, and LS-174T colon cancer cells at concentrations (10-250nM for DPI, 0.5-2.5µM for DTI, and 155nM to 10µM for iodonium diphenyl) substantially lower than needed for DU145 human prostate cancer cells, which do not possess functional NADPH oxidase activity. Drug treatment was associated with decreased H2O2 production and diminished intracellular ROS levels, lasting up to 24h, after short-term (1-h) exposure to the iodonium analogs. Decreased tumor cell proliferation was caused, in part, by a profound block in cell cycle progression at the G1/S interface in both LS-174T and HT-29 cells exposed to either DPI or DTI; and the G1 block was produced, for LS-174T cells, by upregulation of p27 and a drug concentration-related decrease in the expression of cyclins D1, A, and E that was partially prevented by exogenous H2O2. Not only did DPI and DTI decrease intracellular ROS, they both also significantly decreased the mRNA expression levels of Nox1, potentially contributing to the prolonged reduction in tumor cell reactive oxygen levels. We also found that DPI and DTI significantly decreased the growth of both HT-29 and LS-174T human tumor xenografts, at dose levels that produced peak plasma concentrations similar to those utilized for our in vitro experiments. These findings suggest that iodonium analogs have therapeutic potential for NADPH oxidase-containing human colon cancers in vivo and that at least part of their antineoplastic mechanism of action may be related to targeting Nox1.
Assuntos
Ciclo Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Flavoproteínas/antagonistas & inibidores , NADPH Oxidases/metabolismo , Oniocompostos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Tiofenos/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclinas/biossíntese , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Expressão Gênica , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Camundongos , NADPH Oxidase 1 , NADPH Oxidases/genética , Transplante de Neoplasias , Neoplasias da Próstata/metabolismo , RNA Mensageiro/biossíntese , Transplante HeterólogoRESUMO
Ribonucleotide reductase (RNR) is an attractive target for anticancer agents given its central function in DNA synthesis, growth, metastasis, and drug resistance of cancer cells. The current clinically established RNR inhibitors have the shortcomings of short half-life, drug resistance, and iron chelation. Here, we report the development of a novel class of effective RNR inhibitors addressing these issues. A novel ligand-binding pocket on the RNR small subunit (RRM2) near the C-terminal tail was proposed by computer modeling and verified by site-directed mutagenesis and nuclear magnetic resonance (NMR) techniques. A compound targeting this pocket was identified by virtual screening of the National Cancer Institute (NCI) diverse small-molecule database. By lead optimization, we developed the novel RNR inhibitor COH29 that acted as a potent inhibitor of both recombinant and cellular human RNR enzymes. COH29 overcame hydroxyurea and gemcitabine resistance in cancer cells. It effectively inhibited proliferation of most cell lines in the NCI 60 human cancer panel, most notably ovarian cancer and leukemia, but exerted little effect on normal fibroblasts or endothelial cells. In mouse xenograft models of human cancer, COH29 treatment reduced tumor growth compared with vehicle. Site-directed mutagenesis, NMR, and surface plasmon resonance biosensor studies confirmed COH29 binding to the proposed ligand-binding pocket and offered evidence for assembly blockade of the RRM1-RRM2 quaternary structure. Our findings offer preclinical validation of COH29 as a promising new class of RNR inhibitors with a new mechanism of inhibition, with broad potential for improved treatment of human cancer.
Assuntos
Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Conformação Proteica/efeitos dos fármacos , Ribonucleotídeo Redutases/antagonistas & inibidores , Animais , Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Western Blotting , Ciclo Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Simulação por Computador , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Feminino , Citometria de Fluxo , Meia-Vida , Humanos , Hidroxiureia/farmacologia , Subunidade alfa de Receptor de Interleucina-2/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutagênese Sítio-Dirigida , Mutação/genética , Neoplasias/metabolismo , Neoplasias/patologia , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/genética , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em Tandem , Tiazóis/farmacologia , GencitabinaRESUMO
The present study was performed to investigate the possible role of mTOR inhibitors in restoring chemosensitivity to adriamycin/cisplatin and elucidate the underlying mechanism. Combining adriamycin/cisplatin with torisel synergistically inhibited the cell proliferation in human oropharyngeal carcinoma cell line KB and its multidrug-resistant subclone KB/7D. Combining adriamycin and torisel inhibited the phosphorylation of 4EBP-1 and p70S6K, the proteins involved in mTOR pathway, increased expression of γH2AX indicative of DNA damage, triggered cell cycle arrest at G2/M and apoptosis. We conclude that chromatin decondensation by DNA damage provided an easy access for torisel to block the translation of proteins essential for DNA repair thereby restoring the chemosensitivity.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , DNA Topoisomerases Tipo II/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Inibidores da Topoisomerase II/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Dano ao DNA , Reparo do DNA , Doxorrubicina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Fatores de Iniciação em Eucariotos/metabolismo , Everolimo , Feminino , Neoplasias de Cabeça e Pescoço/enzimologia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Sirolimo/administração & dosagem , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/metabolismo , Inibidores da Topoisomerase II/administração & dosagemRESUMO
Cyclin D1 (CCND1) is a known cell cycle regulator whose overexpression is a hallmark of mantle cell lymphoma (MCL). Although molecular techniques have unified the diagnostic approach to MCL, no therapeutic advances have been made to target this particular pathway. The significance of CCND1 in the pathogenesis and treatment of MCL has yet to be defined. We have taken advantage of RNA interference (RNAi) to down-regulate CCND1 expression in two MCL cell lines (Granta-519 and Jeko-1) to investigate the cytotoxic effect of combining RNAi with conventional chemotherapeutic agents. We designed four small interfering RNAs (siRNAs) specific to CCND1, one specific to CCND2, and one dual-targeting siRNA that simultaneously down-regulates CCND1 and CCND2. Etoposide and doxorubicin were used as chemotherapeutics in combination with the siRNAs. The transfected siRNAs in MCL cell lines triggered 40-60% reduction in target mRNA and protein levels. Importantly, the siRNA-mediated reduction in cyclins resulted in decreased IC(50) (50% inhibitory concentration) values for both doxorubicin and etoposide. The combination of siRNA-mediated inhibition of the cyclins along with chemotherapeutic agents could potentially be used to lower the effective doses of the chemotherapeutic agents and reduce drug-related toxicities.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Ciclina D2/genética , Interferência de RNA , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Ciclina D2/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Humanos , Immunoblotting , Concentração Inibidora 50 , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/metabolismo , Linfoma de Célula do Manto/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Bortezomib demonstrates synergism with gemcitabine via a fixed-dose rate (FDR). The aim of this phase I trial in solid tumors was to establish the maximum tolerated dose (MTD) and safety data for this combination. PATIENTS AND METHODS: Twenty-nine patients with a median age of 63 (range 36-84) years and median Karnofsky Performance Status of 90 (range 60-100) were enrolled and treated with bortezomib (1.0 or 1.3 mg/m(2)) on days 1, 4, 8 and 11 and FDR gemcitabine (750, 1,000, or 1,250 mg/m(2)) on days 1 and 8 of each 21-day cycle. Response was evaluated every two cycles. RESULTS: Dose-limiting toxicities were grade 4 thrombocytopenia and neutropenia and grade 3 liver function test abnormalities. The MTD was bortezomib 1 mg/m2 and FDR gemcitabine 1,250 mg/m(2). The median number of cycles delivered was 3 (range 1-28). There was one partial response and six cases of stable disease. The median duration of response was 8.5 (range 3-20) months. CONCLUSION: FDR gemcitabine and bortezomib combination can be delivered effectively with acceptable toxicity.