RESUMO
Platinum-based drugs remain as the cornerstone of cancer chemotherapy; however, development of multidrug resistance presents a therapeutic challenge. This study aims at understanding the molecular mechanisms underlying resistance to cisplatin and unraveling surrogate signaling networks that could revert sensitivity to apoptosis stimuli. We made use of three different sets of cell lines, A549 and H2030 non-small-cell lung cancer (NSCLC) and A2780 ovarian cancer cells and their cisplatin-resistant variants. Here we report that cisplatin-resistant cell lines displayed a multidrug-resistant phenotype. Changes in mitochondrial metabolism and defective mitochondrial signaling were unraveled in the resistant cells. More interestingly, a marked increase in sensitivity of the resistant cells to death receptor-induced apoptosis, in particular TRAIL (TNF-related apoptosis-inducing ligand)-mediated execution, was observed. Although this was not associated with an increase in gene transcription, a significant increase in the localization of TRAIL death receptor, DR4, to the lipid raft subdomains of plasma membrane was detected in the resistant variants. Furthermore, exposure of cisplatin-resistant cells to TRAIL resulted in upregulation of inducible nitric oxide synthase (iNOS) and increase in nitric oxide (NO) production that triggered the generation of peroxynitrite (ONOO(-)). Scavenging ONOO(-) rescued cells from TRAIL-induced apoptosis, thereby suggesting a critical role of ONOO(-) in TRAIL-induced execution of cisplatin-resistant cells. Notably, preincubation of cells with TRAIL restored sensitivity of resistant cells to cisplatin. These data provide compelling evidence for employing strategies to trigger death receptor signaling as a second-line treatment for cisplatin-resistant cancers.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Ácido Peroxinitroso/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Microdomínios da Membrana/metabolismo , Transporte Proteico , Espécies Reativas de Nitrogênio/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
BACKGROUND: The aim of this study was to determine the maximum-tolerated dose (MTD), safety, pharmacokinetics, and pharmacodynamics of OPB-51602, an oral, direct signal transduction activator of transcription 3 (STAT3) inhibitor, in patients with refractory solid tumors. PATIENTS AND METHODS: Three cohorts were studied: cohort A, a sequential dose escalation of OPB-51602 administered intermittently (days 1-14 every 21 days); cohort B, an expansion cohort evaluating the dose lower than the MTD; cohort C, evaluating continuous daily dosing. RESULTS: Fifty-one patients were studied at 2, 4, and 5 mg per day dosing. The MTD was 5 mg; first-cycle dose-limiting toxicities (DLTs) were grade 3 hyponatremia in one patient, and grade 3 dehydration in another. Intermittent dosing of both 2 and 4 mg doses were tolerable, and the recommended phase II dose was 4 mg. Cohort B investigated 4 mg intermittently, whereas cohort C investigated 4 mg continuously. Common toxicities included fatigue, nausea/vomiting, diarrhea, anorexia, and early-onset peripheral neuropathy. Drug-induced pneumonitis occurred in two patients in cohort C. Continuous dosing was associated with a higher incidence of peripheral neuropathy and a lower mean relative dose intensity, compared with intermittent dosing. Steady-state pharmacokinetics was characterized by high oral clearance, mean elimination half-life ranging from 44 to 61 h, and a large terminal-phase volume of distribution. An active metabolite, OPB-51822, accumulated to a greater extent than OPB-51602. Flow cytometry of peripheral blood mononuclear cells demonstrated pSTAT3 (Tyr(705)) inhibition following exposure. Two patients achieved partial responses at 5 mg intermittently and 4 mg continuously; both had epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC) with prior EGFR tyrosine kinase inhibitor exposure. CONCLUSION: OPB-51602 demonstrates promising antitumor activity, particularly in NSCLC. Its long half-life and poorer tolerability of continuous dosing, compared with intermittent dosing, suggest that less frequent dosing should be explored. CLINICALTRIALSGOV IDENTIFIER: NCT01184807.
Assuntos
Antineoplásicos/farmacocinética , Biomarcadores Tumorais/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Fator de Transcrição STAT3/antagonistas & inibidores , Administração Oral , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/sangue , Ásia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biotransformação , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Relação Dose-Resposta a Droga , Esquema de Medicação , Receptores ErbB/genética , Feminino , Meia-Vida , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Masculino , Dose Máxima Tolerável , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Mutação , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
Certain classes of tumor cells respond favorably to TRAIL due to the presence of cell surface death receptors DR4 and DR5. Despite this preferential sensitivity, resistance to TRAIL remains a clinical problem and therefore the heightened interest in identifying compounds to revert tumor sensitivity to TRAIL. We recently demonstrated that the phosphatidylinositide-3-kinase (PI3K) inhibitor, LY294002, and its inactive analog LY303511, sensitized tumor cells to vincristine-induced apoptosis, independent of PI3K/Akt pathway. Intrigued by these findings, we investigated the effect of LY303511 on TRAIL-induced apoptosis in HeLa cells. Preincubation of cells with LY30 significantly amplified TRAIL signaling as evidenced by enhanced DNA fragmentation, caspases 2, 3, 8, and 9 activation, and reduction in the tumor colony formation. This increase in TRAIL sensitivity involved mitochondrial membrane permeabilization resulting in the egress of cytochrome c and second mitochondrial activator of caspase/direct IAP-binding protein with low PI, cleavage of X-linked inhibitor of apoptosis protein, and activation of caspase 9. We link this execution signal to the ability of LY30 to downregulate cFLIP(S) and oligomerize DR5, thus facilitating the signaling of the death initiating signaling complex. The subsequent exposure to TRAIL resulted in processing/activation of caspase 8 and cleavage of its substrate, the BH3 protein Bid. These data provide a novel mechanism of action of this small molecule with the potential for use in TRAIL-resistant tumors.
Assuntos
Apoptose/efeitos dos fármacos , Cromonas/farmacologia , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Mitocôndrias/fisiologia , Piperazinas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Caspase 8/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Células HT29 , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/fisiologia , Morfolinas/farmacologiaRESUMO
At least two mechanisms of early cytosolic acidification during apoptotic signaling have been described, one that involves caspase 8 activation downstream of receptor ligation and another dependent on mitochondria-derived hydrogen peroxide during merocil-induced apoptosis. Here, we show that Bcl-2 inhibits both mechanisms of acidification. Moreover, Bcl-2 overexpression resulted in a slightly elevated constitutive level of superoxide anion and pH in CEM leukemia cells. Interestingly, decreasing intracellular superoxide concentration with an inhibitor of the beta-nicotinamide adenine dinucleotide phosphate oxidase or by transient transfection with a dominant-negative form of the guanosine triphosphate-binding protein Rac1 resulted in a significant increase in the sensitivity of CEM/Bcl-2 cells to CD95- or merocil-induced apoptosis. This increase in sensitivity was a direct result of a significant increase in caspase 8 activation and caspase 8-dependent acidification in the absence of caspase 9 activity or cytochrome c release. These findings suggest a mechanism of switching from mitochondria-dependent to mitochondria-independent death signaling in the same cell, provided the intracellular milieu is permissive for upstream caspase 8 activation, and could have implications for favorably tailoring tumor cells for drug treatment even when the mitochondrial pathway is compromised by Bcl-2.
Assuntos
Apoptose/fisiologia , Líquido Intracelular/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Superóxidos/metabolismo , Receptor fas/metabolismo , Apoptose/efeitos dos fármacos , Caspase 8 , Caspase 9 , Caspases/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/efeitos dos fármacos , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tiobarbitúricos/farmacologia , Receptor fas/farmacologia , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
We recently showed that two photoproducts of merocyanine 540, C2 and C5, triggered cytochrome C release; however, C5 was inefficient in inducing caspase activity and apoptosis in leukemia cells, unlike C2. Here we show that HL60 cells acidified upon exposure to C2 but not C5. The intracellular drop in pH and caspase activation were dependent upon hydrogen peroxide production, and were inhibited by scavengers of hydrogen peroxide. On the contrary, caspase inhibitors did not block hydrogen peroxide production. In turn, increased intracellular hydrogen peroxide concentration was downstream of superoxide anion produced within 2 h of exposure to C2. Inhibitor of NADPH oxidase diphenyleneiodonium neither inhibited superoxide production nor caspase activation triggered by C2. However, exposure of purified mitochondria to C2 resulted in significantly increased superoxide production. Furthermore, cytochrome C release from isolated mitochondria induced by C2 was completely inhibited in the presence of scavengers of hydrogen peroxide. Contrarily, scavenging hydrogen peroxide had no effect on the cyclosporin A-sensitive mitochondrial permeability transition induced by C5. Our data suggest a scenario where drug-induced hydrogen peroxide production induces intracellular acidification and release of cytochrome C, independent of the inner membrane pore, thereby creating an intracellular environment permissive for caspase activation.
Assuntos
Apoptose/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Canais Iônicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Pirimidinonas/metabolismo , Pirimidinonas/farmacologia , Inibidores de Caspase , Caspases/metabolismo , Ciclosporina/farmacologia , Grupo dos Citocromos c/metabolismo , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Sequestradores de Radicais Livres/farmacologia , Células HL-60 , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Luz , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismoRESUMO
Induction of mitochondrial permeability transition (MPT) and cytosolic translocation of cytochrome C are considered essential components of the apoptotic pathway. Hence, there is the realization that mitochondrial-specific drugs could have potential for use as chemotherapeutic agents to trigger apoptosis in tumor cells. Recently, we showed that photoproducts of merocyanine 540 (pMC540) induced tumor cell apoptosis. In this study, we focused on identifying mitochondrial-specific compounds from pMC540 and studied their apoptotic potential. One purified fraction, C5, induced a drop in mitochondrial transmembrane potential and cytosolic translocation of cytochrome C in HL60 human leukemia cells. Moreover, the addition of C5 to purified rat liver mitochondria induced MPT as indicated by mitochondrial matrix swelling, which was completely inhibited by cyclosporin A, an inhibitor of the inner-membrane pore. Supernatant of C5-treated mitochondria showed a dose-dependent increase in cytochrome C, which was also inhibited in the presence of cyclosporin A, strongly indicating a direct effect on the inner-membrane pore. Despite the strong mitochondrial reactivity, C5 elicited minimal cytotoxicity (less than 25%) against HL60 leukemia and M14 melanoma cells because of inefficient caspase activation. However, prior exposure to C5 significantly enhanced the apoptotic response to etoposide or the CD95 receptor. Thus, we demonstrate that MPT induction and cytochrome C release by the novel compound C5, in the absence of effective caspase activation, is insufficient for triggering efficient apoptosis in tumor cells. However, when used in combination with known apoptosis inducers, such compounds could enhance the sensitivity of tumor cells to apoptosis. (Blood. 2000;95:1773-1780)
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Grupo dos Citocromos c/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Melanoma/patologia , Mitocôndrias/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Pirimidinonas/farmacologia , Radiossensibilizantes/farmacologia , Neoplasias Cutâneas/patologia , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/efeitos da radiação , Transporte Biológico/efeitos dos fármacos , Inibidores de Caspase , Ciclosporina/farmacologia , Citosol/enzimologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Fluorescência , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/ultraestrutura , Proteínas de Neoplasias/antagonistas & inibidores , Oligopeptídeos/farmacologia , Permeabilidade/efeitos dos fármacos , Fotoquímica , Pirimidinonas/química , Pirimidinonas/efeitos da radiação , Radiossensibilizantes/química , Radiossensibilizantes/efeitos da radiação , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
Intracellular superoxide (O(2)*- was manipulated in M14 melanoma cells by overexpression or repression of Cu/Zn SOD using a tetracycline-inducible expression system. Scavenging intracellular O(2)*- increased tumor cell sensitivity to daunorubicin, etoposide, and pMC540, whereas expression of the antisense SOD mRNA significantly decreased cell sensitivity to drug treatment. Whereas Cu/Zn SOD overexpressing cells exhibited higher activation of the executioner caspase 3 upon drug exposure, caspase 3 activation was significantly lower when Cu/Zn SOD was repressed by antisense expression. These data show that intracellular O(2)*- regulates tumor cell response to drug-induced cell death via a direct or indirect effect on the caspase activation pathway.
Assuntos
Apoptose , Superóxidos/metabolismo , Caspase 3 , Caspases/metabolismo , Daunorrubicina/farmacologia , Interações Medicamentosas , Ativação Enzimática , Etoposídeo/farmacologia , Humanos , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Pirimidinonas/farmacologia , RNA Mensageiro/metabolismo , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Células Tumorais Cultivadas , Receptor fas/metabolismoRESUMO
If the interplay between caspase proteases and mitochondria decide the fate of the cell during apoptosis, they may constitute useful molecular targets for novel drug design. We have shown that photoactivated merocyanine 540 (pMC540) triggers caspase-mediated apoptosis in HL60 leukemia and M14 melanoma cells. Because pMC540 is a mixture of photoproducts, we set out to purify the biologically active component(s) from this mixture and to investigate their ability to directly activate intracellular caspases and/or trigger mitochondrial events associated with apoptosis. Two photoproducts, namely C1 and C2, purified and characterized by mass spectroscopy and nuclear magnetic resonance (NMR) analysis, effectively induced apoptosis in HL60 and M14 cells. Interestingly, both C1 and C2 induced non-receptor-dependent activation of caspase 8, which was responsible for the downstream activation of caspase 3 and cell death. Both compounds induced the release of cytochrome C from mitochondria of tumor cells and from purified rat liver mitochondria; however, different mechanisms were operative in cytochrome C translocation in response to C1 or C2. C1-induced cytochrome C release was mediated by the mitochondrial permeability transition (MPT) pore and accompanied by a decrease in mitochondrial transmembrane potential (triangle uppsim), whereas cytochrome C release in response to C2 was independent of MPT pore opening. These findings do not exclude the possibility that changes in mitochondrial triangle uppsim are critical for apoptosis in some instances, but support the notion that this may not be a universal step in the apoptotic process. Thus, identification of two novel anticancer agents that directly activate effector components of the apoptotic pathway could have potential implications for the development of newer chemotherapeutic drugs.
Assuntos
Apoptose , Caspases/metabolismo , Grupo dos Citocromos c/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Melanoma/metabolismo , Pirimidinonas/farmacologia , Animais , Antineoplásicos/uso terapêutico , Caspase 3 , Caspase 8 , Caspase 9 , Ativação Enzimática/efeitos dos fármacos , Humanos , Leucemia Promielocítica Aguda/patologia , Melanoma/patologia , Potenciais da Membrana , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Fotoquímica , Pirimidinonas/química , Pirimidinonas/uso terapêutico , Ratos , Células Tumorais CultivadasRESUMO
Resveratrol, a constituent of grapes and other food products, has been shown to prevent carcinogenesis in murine models. We report here that resveratrol induces apoptotic cell death in HL60 human leukemia cell line. Resveratrol-treated tumor cells exhibit a dose-dependent increase in externalization of inner membrane phosphatidylserine and in cellular content of subdiploid DNA, indicating loss of membrane phospholipid asymmetry and DNA fragmentation. Resveratrol-induced cell death is mediated by intracellular caspases as observed by the dose-dependent increase in proteolytic cleavage of caspase substrate poly (ADP-ribose) polymerase (PARP) and the ability of caspase inhibitors to block resveratrol cytotoxicity. We also show that resveratrol treatment enhances CD95L expression on HL60 cells, as well as T47D breast carcinoma cells, and that resveratrol-mediated cell death is specifically CD95-signaling dependent. On the contrary, resveratrol treatment of normal human peripheral blood lymphocytes (PBLs) does not affect cell survival for up to 72 hours, which correlates with the absence of a significant change in either CD95 or CD95L expression on treated PBLs. These data show specific involvement of the CD95-CD95L system in the anti-cancer activity of resveratrol and highlight the chemotherapeutic potential of this natural product, in addition to its recently reported chemopreventive activity.
Assuntos
Anticarcinógenos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Rosales/química , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologia , Receptor fas/fisiologia , Anticorpos Monoclonais/farmacologia , Anticarcinógenos/isolamento & purificação , Neoplasias da Mama/patologia , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Fragmentação do DNA , DNA de Neoplasias/metabolismo , Proteína Ligante Fas , Feminino , Células HL-60/efeitos dos fármacos , Células HL-60/patologia , Humanos , Imunoglobulina G/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Lipídeos de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfatidilserinas/metabolismo , Resveratrol , Estilbenos/isolamento & purificação , Células Tumorais Cultivadas , Receptor fas/imunologiaRESUMO
The molecular events involved in tumor cell death induced by novel photoproducts of merocyanine 540 (pMC540) are poorly understood. Using HL60 leukemia and M14 melanoma cell lines we investigated the role of the apoptotic pathway in pMC540-mediated cell death. Tumor cells exposed to pMC540 showed cell size shrinkage and an increase in the sub-diploid DNA content. A loss of membrane phospholipid asymmetry associated with apoptosis was induced by pMC540 in both tumor cell lines as evidenced by the externalization of phosphatidylserine. A dose-dependent increase in caspase-3 protease activity suppressed by the tetrapeptide inhibitor DEVD-CHO was observed in both cell lines. Western blot analysis of poly (ADP-ribose) polymerase, a caspase substrate, showed the classical cleavage pattern (116 to 89 kDa) associated with apoptosis in pMC540-treated cell lysates. Furthermore, caspase inhibition blocked the externalization of membrane PS, indicating that the loss of membrane phospholipid asymmetry is a downstream event of caspase activation. These findings demonstrate that tumor cell death induced by pMC540 is mediated by caspase proteases.