RESUMO
Temozolomide (TMZ) generates DNA adducts that are repaired by direct DNA and base excision repair mechanisms. Methoxyamine (MX, TRC-102) potentiates TMZ activity by binding to apurinic and apyrimidinic (AP) sites after removal of N3-methyladenine and N7-methylguanine, inhibiting site recognition of AP endonuclease. We conducted a phase I trial to determine the maximum tolerated dose and dose-limiting toxicities (DLTs) of intravenous MX when given with oral TMZ. Patients with advanced solid tumors and progression on standard treatment were enrolled to a standard 3 + 3 dose escalation trial assessing escalating doses of TMZ and MX. Tumor response was assessed per RECIST and adverse events (AEs) by CTCAEv3. Pharmacokinetics (PK) of MX and COMET assays on peripheral blood mononuclear cells were performed. 38 patients were enrolled-median age 59.5 years (38-76), mean number of cycles 2.9 [1-13]. No DLTs were observed. Cycle 1 grade 3 AEs included fatigue, lymphopenia, anemia, INR, leukopenia, neutropenia, allergic reaction, constipation, psychosis and paranoia. Cycle 2-13 grade 4 AEs included thrombocytopenia and confusion. A partial response was seen in 1 patient with a pancreatic neuroendocrine tumor (PNET) and six additional patients, each with different tumor types, demonstrated prolonged stable disease. MX PK was linear with dose and was not affected by concomitant TMZ. TMZ 200 mg/m2 daily × 5 may be safely administered with MX 150 mg/m2 intravenously once on day 1 with minimal toxicity. Further studies assessing this drug combination in select tumor types where temozolomide has activity may be warranted.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Hidroxilaminas/uso terapêutico , Neoplasias/tratamento farmacológico , Temozolomida/uso terapêutico , Adulto , Idoso , Antineoplásicos Alquilantes/administração & dosagem , Antineoplásicos Alquilantes/efeitos adversos , Antineoplásicos Alquilantes/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Área Sob a Curva , Reparo do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Meia-Vida , Humanos , Hidroxilaminas/administração & dosagem , Hidroxilaminas/efeitos adversos , Hidroxilaminas/farmacocinética , Masculino , Dose Máxima Tolerável , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Temozolomida/efeitos adversos , Temozolomida/farmacocinéticaRESUMO
BACKGROUND: Renal cell carcinoma (RCC) patients treated with tyrosine kinase inhibitors (TKI) typically respond initially, but usually develop resistance to therapy. We utilised transcriptome analysis to identify gene expression changes during development of sunitinib resistance in a RCC patient-derived xenograft (PDX) model. METHODS: RCC tumours were harvested during pre-treatment, response and escape phases. Direct anti-proliferative effects of sunitinib plus MEK inhibitor were assessed. Activation status (phosphorylation) of MEK1/2 and ERK1/2 was determined, myeloid-derived suppressor cells (MDSC) sub-fractions were quantitated and G-CSF was measured by ELISA. RESULTS: During the response phase, tumours exhibited 91% reduction in volume, characterised by decreased expression of cell survival genes. After 4-week treatment, tumours developed resistance to sunitinib, associated with increased expression of pro-angiogenic and cell survival genes. During tumour escape, cellular movement, inflammatory response and immune cell trafficking genes were induced, along with intra-tumoural accumulation of MDSC. In this PDX model, either continuous treatment with sunitinib plus MEK inhibitor PD-0325901, or switching from sunitinib to PD-0325901 was effective. The combination of PD-0325901 with TKI suppressed intra-tumoural phospho-MEK1/2, phospho-ERK1/2 and MDSC. CONCLUSIONS: Continuous treatment with sunitinib alone did not maintain anti-tumour response; addition of MEK inhibitor abrogated resistance, leading to improved anti-tumour efficacy.
Assuntos
Benzamidas/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Difenilamina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indóis/farmacologia , Neoplasias Renais/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Adulto , Animais , Benzamidas/farmacologia , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Linhagem Celular Tumoral , Difenilamina/farmacologia , Difenilamina/uso terapêutico , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/uso terapêutico , Neoplasias Renais/enzimologia , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células Supressoras Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Pirróis/uso terapêutico , Receptores de Interleucina-2/deficiência , Sunitinibe , Carga Tumoral/efeitos dos fármacos , Evasão Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The elevated level of replication stress is an intrinsic characteristic of cancer cells. Targeting the mechanisms that maintain genome stability to further increase replication stress and thus induce severe genome instability has become a promising approach for cancer treatment. Here, we identify histone deacetylase 8 (HDAC8) as a drug target whose inactivation synergizes with the inhibition of checkpoint kinases to elicit substantial replication stress and compromise genome integrity selectively in cancer cells. We showed that simultaneous inhibition of HDAC8 and checkpoint kinases led to extensive replication fork collapse, irreversible cell-cycle arrest, and synergistic vulnerability in various cancer cells. The efficacy of the combination treatment was further validated in patient tumor-derived organoid (PDO) and xenograft mouse (PDX) models, providing important insights into patient-specific drug responses. Our data revealed that HDAC8 activity was essential for reducing the acetylation level of structural maintenance of chromosomes protein 3 (SMC3) ahead of replication forks and preventing R loop formation. HDAC8 inactivation resulted in slowed fork progression and checkpoint kinase activation. Our findings indicate that HDAC8 guards the integrity of the replicating genome, and the cancer-specific synthetic lethality between HDAC8 and checkpoint kinases provides a promising replication stress-targeting strategy for treating a broad range of cancers.
RESUMO
PURPOSE: TRC102, a small-molecule base-excision repair inhibitor, potentiates the cytotoxicity of pemetrexed and reverses resistance by binding to chemotherapy-induced abasic sites in DNA. We conducted a phase I clinical trial combining pemetrexed and TRC102 with cisplatin-radiation in stage III nonsquamous non-small cell lung cancer (NS-NSCLC). PATIENTS AND METHODS: Fifteen patients were enrolled from 2015 to 2019. The primary objective was to determine the dose-limiting toxicity and maximum tolerated dose of TRC102 in combination with pemetrexed, cisplatin, and radiotherapy. Secondary objectives were to assess toxicity, tumor response, and progression-free survival at 6 months. Based on our preclinical experiments, pemetrexed-TRC102 was given on day 1, and cisplatin/radiotherapy was initiated on day 3. This schedule was duplicated in the second cycle. After completion, two additional cycles of pemetrexed-cisplatin were given. Toxicities were assessed using NCI CTACAE versions 4/5. RESULTS: The median age was 69 years (45-79) with the median follow-up of 25.7 months (range, 7.9-47.4). No dose-limiting toxicities and no grade 5 toxicity were seen. Hematologic and gastrointestinal toxicities were the most common side effects. No clinical radiation pneumonitis was seen. Of 15 evaluable patients, three had complete response (20%), and 12 had partial response (80%). The 6-month progression-free survival was 80%, and the 2-year overall survival was 83%. CONCLUSIONS: Pemetrexed-TRC102 combined with cisplatin/radiotherapy in NS-NSCLC is safe and well tolerated. The recommended phase II dose is 200 mg TRC102 along with cisplatin-pemetrexed. No additional safety signal was seen beyond the expected CRT risks. A phase II trial, integrating post-CRT immunotherapy with this aggressive DNA-damaging regimen, is warranted.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Cisplatino , Reparo do DNA , Glutamatos/efeitos adversos , Guanina/efeitos adversos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Pemetrexede/efeitos adversos , Platina/uso terapêuticoRESUMO
Despite advances in melanoma treatment, more than 70% of patients with distant metastasis die within 5 years. Proactive treatment of early melanoma to prevent metastasis could save lives and reduce overall healthcare costs. Currently, there are no treatments specifically designed to prevent early melanoma from progressing to metastasis. We used the Connectivity Map to conduct an in silico drug screen and identified 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) as a drug class that might prevent melanoma metastasis. To confirm the in vitro effect of statins, RNA sequencing was completed on A375 cells after treatment with fluvastatin to describe changes in the melanoma transcriptome. Statins induced differential expression in genes associated with metastasis and are used in commercially available prognostic tests for melanoma metastasis. Finally, we completed a chart review of 475 patients with melanoma. Patients taking statins were less likely to have metastasis at the time of melanoma diagnosis in both univariate and multivariate analyses (24.7% taking statins vs. 37.6% not taking statins, absolute risk reduction = 12.9%, P = 0.038). These findings suggest that statins might be useful as a treatment to prevent melanoma metastasis. Prospective trials are required to verify our findings and to determine the mechanism of metastasis prevention.
Assuntos
Reposicionamento de Medicamentos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Transcriptoma/efeitos dos fármacos , Idoso , Simulação por Computador , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Melanoma/genética , Melanoma/mortalidade , Melanoma/secundário , Pessoa de Meia-Idade , Prognóstico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Análise de SobrevidaRESUMO
Thymidylate synthase (TS) inhibitors including fluoropyrimidines [e.g., 5-Fluorouracil (5-FU) and 5-Fluorodeoxyuridine (5-FdU, floxuridine)] and antifolates (e.g., pemetrexed) are widely used against solid tumors. Previously, we reported that shRNA-mediated knockdown (KD) of uracil DNA glycosylase (UDG) sensitized cancer cells to 5-FdU. Because p53 has also been shown as a critical determinant of the sensitivity to TS inhibitors, we further interrogated 5-FdU cytotoxicity after UDG depletion with regard to p53 status. By analyzing a panel of human cancer cells with known p53 status, it was determined that p53-mutated or -deficient cells are highly resistant to 5-FdU. UDG depletion resensitizes 5-FdU in p53-mutant and -deficient cells, whereas p53 wild-type (WT) cells are not affected under similar conditions. Utilizing paired HCT116 p53 WT and p53 knockout (KO) cells, it was shown that loss of p53 improves cell survival after 5-FdU, and UDG depletion only significantly sensitizes p53 KO cells. This sensitization can also be recapitulated by UDG depletion in cells with p53 KD by shRNAs. In addition, sensitization is also observed with pemetrexed in p53 KO cells, but not with 5-FU, most likely due to RNA incorporation. Importantly, in p53 WT cells, the apoptosis pathway induced by 5-FdU is activated independent of UDG status. However, in p53 KO cells, apoptosis is compromised in UDG-expressing cells, but dramatically elevated in UDG-depleted cells. Collectively, these results provide evidence that loss of UDG catalyzes significant cell death signals only in cancer cells mutant or deficient in p53.Implications: This study reveals that UDG depletion restores sensitivity to TS inhibitors and has chemotherapeutic potential in the context of mutant or deficient p53. Mol Cancer Res; 16(2); 212-21. ©2017 AACR.
Assuntos
Desoxiuridina/análogos & derivados , Mutação , Neoplasias/genética , RNA Interferente Pequeno/farmacologia , Proteína Supressora de Tumor p53/genética , Uracila-DNA Glicosidase/deficiência , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desoxiuridina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes/métodos , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Neoplasias/tratamento farmacológico , Pemetrexede/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
beta-Lapachone, an o-naphthoquinone, induces a novel caspase- and p53-independent apoptotic pathway dependent on NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 reduces beta-lapachone to an unstable hydroquinone that rapidly undergoes a two-step oxidation back to the parent compound, perpetuating a futile redox cycle. A deficiency or inhibition of NQO1 rendered cells resistant to beta-lapachone. Thus, beta-lapachone has great potential for the treatment of specific cancers with elevated NQO1 levels (e.g., breast, non-small cell lung, pancreatic, colon, and prostate cancers). We report the development of mono(arylimino) derivatives of beta-lapachone as potential prodrugs. These derivatives are relatively nontoxic and not substrates for NQO1 when initially diluted in water. In solution, however, they undergo hydrolytic conversion to beta-lapachone at rates dependent on the electron-withdrawing strength of their substituent groups and pH of the diluent. NQO1 enzyme assays, UV-visible spectrophotometry, high-performance liquid chromatography-electrospray ionization-mass spectrometry, and nuclear magnetic resonance analyses confirmed and monitored conversion of each derivative to beta-lapachone. Once converted, beta-lapachone derivatives caused NQO1-dependent, mu-calpain-mediated cell death in human cancer cells identical to that caused by beta-lapachone. Interestingly, coadministration of N-acetyl-l-cysteine, prevented derivative-induced cytotoxicity but did not affect beta-lapachone lethality. Nuclear magnetic resonance analyses indicated that prevention of beta-lapachone derivative cytotoxicity was the result of direct modification of these derivatives by N-acetyl-l-cysteine, preventing their conversion to beta-lapachone. The use of beta-lapachone mono(arylimino) prodrug derivatives, or more specifically a derivative converted in a tumor-specific manner (i.e., in the acidic local environment of the tumor tissue), should reduce normal tissue toxicity while eliciting tumor-selective cell killing by NQO1 bioactivation.
Assuntos
NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/farmacologia , Pró-Fármacos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Humanos , Espectroscopia de Ressonância Magnética , Naftoquinonas/química , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Pró-Fármacos/síntese química , Pró-Fármacos/química , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta , Relação Estrutura-AtividadeRESUMO
Layer-by-layer self-assembled polyelectrolyte shells are a new class of micro/nanocapsules with unique physicochemical properties for potential applications in drug/gene delivery. The objective of this study was to investigate the interactions of polyelectrolyte shells ( approximately 1 mum in diameter) with MCF-7 breast cancer cells and identify key parameters that affect such interactions. Tailoring of surface properties of polyelectrolyte shells was achieved by choosing different outermost layer materials, including cationic polymers, anionic polymers, and lipid bilayers. Different surface compositions led to a wide range of electrostatic potentials from -46 to +47 mV in phophate-buffered saline buffer. Confocal microscopy studies showed that the polyelectrolyte shells were internalized into the cell cytoplasm, but not into the nuclei. Correlation of cell uptake with shell surface compositions was complicated by the adsorption of serum proteins on the surface of polyelectrolyte shells, particularly polycation-coated shells. To prevent protein adsorption, poly(ethylene glycol) (PEG) grafted poly(ethyleneimine) (PEI) copolymers (1:1, 1:5, 1:10 graft ratios) were synthesized and introduced on the shell surface. Shells coated with PEI-PEG copolymers effectively reduced protein adsorption whereas PEI-PEG copolymers with lower graft ratios achieved higher cell uptake efficiency after 24 h of incubation with MCF-7 cells.
Assuntos
Cápsulas , Linhagem Celular Tumoral , Polímeros , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Cápsulas/química , Cápsulas/metabolismo , Sistemas de Liberação de Medicamentos , Endocitose , Humanos , Teste de Materiais , Microscopia Eletrônica , Polímeros/química , Polímeros/metabolismo , Propriedades de SuperfícieRESUMO
Beta-lapachone (beta-Lap) triggers apoptosis in a number of human breast and prostate cancer cell lines through a unique apoptotic pathway that is dependent upon NQO1, a two-electron reductase. Recently, our laboratory showed that beta-lap-exposed MCF-7 cells exhibited an early increase in intracellular cytosolic Ca(2+) from endoplasmic reticulum stores, and that BAPTA-AM (an intracellular Ca(2+) chelator) blocked these early increases and partially inhibited all aspects of beta-lap-induced apoptosis. We now show that exposure of NQO1-expressing breast cancer cells to beta-lap stimulates a unique proteolytic apoptotic pathway involving mu-calpain activation. No apparent activation of m-calpain was noted. Upon activation, mu-calpain translocated to the nucleus concomitant with specific nuclear proteolytic events. Apoptotic responses in beta-lap-exposed NQO1-expressing cells were significantly delayed and survival enhanced by exogenous over-expression of calpastatin, a natural inhibitor of mu- and m-calpains. Furthermore, purified mu-calpain cleaved PARP to a unique fragment (approximately 60 kDa), not previously reported for calpains. We provide evidence that beta-lap-induced, mu-calpain-stimulated apoptosis does not involve any known apoptotic caspases; the activated fragments of caspases were not observed after beta-lap exposures, nor were there any changes in the pro-enzyme forms as measured by Western blot analyses. The ability of beta-lap to trigger an apparently novel, p53-independent, calpain-mediated apoptotic cell death further support the development of this drug for improved breast cancer therapy.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Calpaína/metabolismo , NAD(P)H Desidrogenase (Quinona)/farmacologia , Naftoquinonas/farmacologia , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/farmacologia , Calpaína/antagonistas & inibidores , Caspases/metabolismo , Núcleo Celular/metabolismo , Ensaio de Unidades Formadoras de Colônias , Inibidores de Cisteína Proteinase/farmacologia , Citosol/metabolismo , Ativação Enzimática , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Microscopia Confocal , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Transporte Proteico , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Heat shock protein 27 (Hsp27) is a chaperone protein, and its expression is increased in response to various stress stimuli including anticancer chemotherapy, which allows the cells to survive and causes drug resistance. We previously identified lead compounds that bound to Hsp27 and tubulin via proteomic approaches. Systematic ligand based optimization in the current study significantly increased the cell growth inhibition and apoptosis inducing activities of the compounds. Compared to the lead compounds, one of the new derivatives exhibited much better potency to inhibit tubulin polymerization but a decreased activity to inhibit Hsp27 chaperone function, suggesting that the structural modification dissected the dual targeting effects of the compound. The most potent compounds 20 and 22 exhibited strong cell proliferation inhibitory activities at subnanomolar concentration against 60 human cancer cell lines conducted by Developmental Therapeutic Program at the National Cancer Institute and represented promising candidates for anticancer drug development.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células HCT116 , Proteínas de Choque Térmico HSP27/metabolismo , Células HT29 , Humanos , Células K562 , Células MCF-7 , Modelos Químicos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/metabolismo , Estrutura Molecular , Polimerização/efeitos dos fármacos , Relação Estrutura-Atividade , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/químicaRESUMO
Improving patient outcome by personalized therapy involves a thorough understanding of an agent's mechanism of action. ß-Lapachone (clinical forms, Arq501/Arq761) has been developed to exploit dramatic cancer-specific elevations in the phase II detoxifying enzyme NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is dramatically elevated in solid cancers, including primary and metastatic [e.g., triple-negative (ER-, PR-, Her2/Neu-)] breast cancers. To define cellular factors that influence the efficacy of ß-lapachone using knowledge of its mechanism of action, we confirmed that NQO1 was required for lethality and mediated a futile redox cycle where â¼120 moles of superoxide were formed per mole of ß-lapachone in 2 minutes. ß-Lapachone induced reactive oxygen species (ROS), stimulated DNA single-strand break-dependent poly(ADP-ribose) polymerase-1 (PARP1) hyperactivation, caused dramatic loss of essential nucleotides (NAD(+)/ATP), and elicited programmed necrosis in breast cancer cells. Although PARP1 hyperactivation and NQO1 expression were major determinants of ß-lapachone-induced lethality, alterations in catalase expression, including treatment with exogenous enzyme, caused marked cytoprotection. Thus, catalase is an important resistance factor and highlights H2O2 as an obligate ROS for cell death from this agent. Exogenous superoxide dismutase enhanced catalase-induced cytoprotection. ß-Lapachone-induced cell death included apoptosis-inducing factor (AIF) translocation from mitochondria to nuclei, TUNEL+ staining, atypical PARP1 cleavage, and glyceraldehyde 3-phosphate dehydrogenase S-nitrosylation, which were abrogated by catalase. We predict that the ratio of NQO1:catalase activities in breast cancer versus associated normal tissue are likely to be the major determinants affecting the therapeutic window of ß-lapachone and other NQO1 bioactivatable drugs.
Assuntos
Neoplasias da Mama/tratamento farmacológico , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/administração & dosagem , Poli(ADP-Ribose) Polimerases/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Catalase/genética , Catalase/metabolismo , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Necrose/genética , Necrose/patologia , Poli(ADP-Ribose) Polimerase-1 , Espécies Reativas de Oxigênio/metabolismoRESUMO
Cyclooxygenase-2 (COX-2) inhibitor nimesulide inhibits the proliferation of various types of cancer cells mainly via COX-2 independent mechanisms, which makes it a good lead compound for anti-cancer drug development. In the presented study, a series of new nimesulide analogs were synthesized based on the structure-function analysis generated previously. Some of them displayed very potent anti-cancer activity with IC(50)s around 100 nM-200 nM to inhibit SKBR-3 breast cancer cell growth. CSUOH0901 (NSC751382) from the compound library also inhibits the growth of the 60 cancer cell lines used at National Cancer Institute Developmental therapeutics Program (NCIDTP) with IC(50)s around 100 nM-500 nM. Intraperitoneal injection with a dosage of 5 mg/kg/d of CSUOH0901 to nude mice suppresses HT29 colorectal xenograft growth. Pharmacokinetic studies demonstrate the good bioavailability of the compound.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Benzodioxóis/síntese química , Benzodioxóis/farmacologia , Inibidores de Ciclo-Oxigenase 2/síntese química , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Benzodioxóis/farmacocinética , Benzodioxóis/toxicidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacocinética , Inibidores de Ciclo-Oxigenase 2/toxicidade , Desenho de Fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Ratos , Sulfonamidas/farmacocinética , Sulfonamidas/toxicidade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We previously developed a series of anticancer agents based on cyclooxygenase-2 (COX-2) inhibitor nimesulide as a lead compound. However, the molecular targets of these agents still remain unclear. In this study, we synthesized a biotinylated probe based on a representative molecule of the compound library and performed protein pull-down assays to purify the anticancer targets of the compound. Via proteomic approaches, the major proteins bound to the probe were identified to be tubulin and heat shock protein 27 (Hsp27), and the compound inhibited tubulin polymerization by binding at the colchicine domain. However, the tubulin inhibitory effect of the compound activated the Hsp27 phosphorylation and possibly overrode the direct Hsp27 inhibitory effects, which made it difficult to solely validate the Hsp27 target. Taken together, the compound was a dual ligand of tubulin and Hsp27, inhibited tubulin polymerization, and had the potential to be a class of new chemotherapeutic agents.
Assuntos
Antineoplásicos/farmacologia , Benzodioxóis/farmacologia , Sulfonamidas/farmacologia , Moduladores de Tubulina/farmacologia , Antineoplásicos/química , Benzodioxóis/química , Biotinilação , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Modelos Moleculares , Sondas Moleculares/química , Fosforilação , Ligação Proteica , Proteômica , Relação Estrutura-Atividade , Sulfonamidas/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/químicaRESUMO
BACKGROUND: There has been substantial growth in the numbers of patients with conjunctival squamous cell carcinoma infected with HIV in East Africa. The natural history of the conjunctival squamous cell carcinoma appears to be unique in this region of the world, but the etiologic mechanism unclear and therapeutic options limited. This research was carried out to determine if conjunctival squamous cell carcinoma harbors human papillomavirus DNA and is associated with activation of the EGFR signaling pathway. Positive findings would identify etiologic causes and provide clinical guidance to improve treatment. METHODS/FINDINGS: Expression of p-MAPK/MAPK, p-Akt/Akt and p-EGFR/EGFR in cell nuclei and cytoplasm of 38 FFPE specimens were assessed by immunohistochemistry; HPV genotype was detected by qPCR assay; EGFR mutation was assessed by DNA sequencing analysis; and EGFR mRNA expression was measured using relative qPCR. Statistical analyses included two-sided Fisher exact test or chi-square test, Spearman correlation coefficient and ANOVA. HPV 18 was found in 61% of samples, with HPV 16 double-genotype in 6 patients (16%). Immunohistochemistry and qPCR data suggest that activation and expression of the EGFR signaling pathway is related to disease progression of conjunctival cancer. The associations between cytoplasmic p-MAPK, cytoplasmic p-Akt and tumor invasiveness were significant (p = 0.05 or 0.028). Nuclear p-EGFR appeared only in invasive tumors. A significant positive association between EGFR expression and disease invasiveness was observed (p = 0.01). A SNP in 10 patients and one missense mutation were found within EGFR tyrosine kinase domain. Statistical analysis indicates that patients with measurable EGFR expression more likely harbor EGFR mutations, compared to those with negative EGFR expression (35.3% vs. 0%). CONCLUSIONS/SIGNIFICANCE: We conclude that HPV types 16/18 infection is frequent in East African patients with AIDS-associated squamous cell carcinoma of the conjunctiva. EGFR activation/alteration may contribute to and sustain the high prevalence of this cancer. Our findings hint that adoption of HPV vaccination strategies may impact the incidence of conjunctival carcinoma. Agents that target the EGFR pathway may have potential therapeutic benefit.
Assuntos
Neoplasias da Túnica Conjuntiva/complicações , Neoplasias da Túnica Conjuntiva/virologia , Receptores ErbB/genética , Infecções por HIV/complicações , Infecções por HIV/enzimologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/enzimologia , África Oriental/epidemiologia , Carcinoma in Situ/complicações , Carcinoma in Situ/epidemiologia , Carcinoma in Situ/virologia , Neoplasias da Túnica Conjuntiva/enzimologia , Neoplasias da Túnica Conjuntiva/epidemiologia , Progressão da Doença , Ativação Enzimática , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , HIV/fisiologia , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , Humanos , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação/genética , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Prevalência , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
Bax is a pro-apoptotic protein that mediates intrinsic cell-death signaling. Using a yeast-based functional screening approach, we identified interferon gamma receptor beta chain (IFNgammaR2) as a new Bax suppressor. IFNgammaR2 is a component of the IFNgamma receptor complex along with the IFNgammaR alpha chain (IFNgammaR1). Upon IFNgamma binding, a conformational change in the receptor complex occurs that activates the Jak2/STAT1 signaling cascade. We found that the C-terminal region (amino acids 296-337) of IFNgammaR2 (IFNgammaR2(296-337)) contains a novel Bax inhibitory domain. This portion does not contain the Jak2-binding domain; therefore, the antiapoptotic function of IFNgammaR2 is independent of JAK/STAT signaling. IFNgammaR2(296-337) rescued human cells from apoptosis induced by overexpression of Bax but not Bak. Overexpression of IFNgammaR2 (wild type and IFNgammaR2(296-337)) rescued cells from etoposide and staurosporine, which are known to induce Bax-mediated cell death. Interestingly, IFNgammaR2 inhibited apoptosis induced by the BH3-only protein Bim-EL, suggesting that IFNgammaR2 inhibits Bax activation through a BH3-only protein. Bax and IFNgammaR2 were co-immunoprecipitated from cell lysates prepared from HEK293 and DAMI cells. Furthermore, direct binding of purified recombinant proteins of Bax and IFNgammaR2 was also confirmed. Addition of recombinant Bcl-2 protein to cell lysates significantly reduced the interaction of IFNgammaR2 and Bax, suggesting that Bcl-2 and IFNgammaR2 bind a similar domain of Bax. We found that the C-terminal fragment (cytoplasmic domain) of IFNgammaR2 is expressed in human cancer cell lines of megakaryocytic cancer (DAMI), breast cancer (MDA-MD-468), and prostate cancer (PC3 cells). The presence of the C-terminal fragment of IFNgammaR2 may confer on cancer cells resistance to apoptotic stresses. Our discovery of the anti-Bax activity of the cytoplasmic domain of IFNgammaR2 may shed new light on the mechanism of how cell death is controlled by IFNgamma and Bax.
Assuntos
Apoptose , Receptores de Interferon/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Sítios de Ligação , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Citosol/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Receptores de Interferon/química , Receptores de Interferon/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Transfecção , Leveduras/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genéticaRESUMO
Work from the laboratory of Dr. Arthur B. Pardee has highlighted basic principles that govern cellular and molecular biological processes in living cells. Among the most important governing principles in cellular and molecular responses are: (i) threshold "restriction" responses, wherein a level of response is reached and a "point of no return" is achieved; (ii) feedback regulation; and (iii) redundancy. Lessons learned from the molecular biology of cellular stress responses in mammalian cancer versus normal cells after ionizing radiation (IR) or chemotherapeutic agent exposures reveal similar instances of these guiding principles in mammalian cells. Among these are the: (i) induction of cell death responses by beta-lapachone (beta-lap), a naphthoquinone anti-tumor agent that kills cancer cells via an NQO1 (i.e., X-ray-inducible protein-3, xip3)-dependent mechanism; (ii) induction of secretory clusterin (sCLU) in response to TGF-beta1 exposure, and the ability of induced sCLU protein to down-regulate TGF-beta1 signaling; and (iii) induction of DNA mismatch repair-dependent G(2) cell cycle checkpoint responses after exposure to alkylating agents. We have learned these lessons and now adopted strategies to exploit them for improved therapy. These examples will be discussed and compared to the pioneering findings of researchers in the Pardee laboratory over the years.