RESUMO
Since it is important to lower the death threshold in tumor cells and to enhance response to genotoxic damage, the mechanisms of bromodeoxyuridine (BrdU)-mediated radiosensitization were now examined in human C8161 melanoma. This revealed that anti-apoptotic Bcl-2, and cell cycle controlling hyperphosphorylated Rb and cyclin A were downregulated by BrdU, even without irradiation. BrdU pretreatment and subsequent UV irradiation (10 J/m(2)) accelerated an early increase in the ratio of pro-apoptotic Bax to that of Bcl-2, increased apoptosis-associated PARP cleavage and potentiated DNA damage compared to irradiated unsensitized cells. BrdU also synergized with radiation to increase autophagic features, such as perinuclear vacuole formation. More specifically, conversion of LC3B-I into LC3B-II by immune blotting and an increased pattern of cytoplasmic and nuclear LC3B by fluorescent immunostaining, supported induction of autophagy in BrdU-pretreated cells irradiated with 25 J/m(2). This report shows for the first time that radiation sensitizers like BrdU enhance and modify radiation-induced cell death by accelerating an increased bax/bcl-2 ratio in unirradiated cells, and subsequently increasing radiation-induced apoptosis and/or autophagy depending on radiation dosage.
Assuntos
Apoptose , Autofagia , Dano ao DNA , Melanoma/radioterapia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias Cutâneas/radioterapia , Bromodesoxiuridina/farmacologia , Linhagem Celular Tumoral , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Melanoma/tratamento farmacológico , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína do Retinoblastoma/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Raios Ultravioleta , Vacúolos/metabolismoRESUMO
2-acetyl furanonaphthoquinone (FNQ) is a naturally occurring drug with enhanced toxicity versus glucose-starved tumor cells, which frequently show topoisomerase II drug resistance. Since loss of p53 tumor suppressor function or overexpression of the anti-apoptotic bcl-2 gene can decrease susceptibility to some cancer therapies, we now investigated the effect of FNQ against genetically matched C8161 melanoma cell lines transduced to express unequal levels of Bcl-2, or engineered to harbour a functional wt p53 for comparison with dominant-negative mutant p53 R175H. Cells with differing p53 genotype showed susceptibility to FNQ. However, this response was attenuated in those overexpressing mutant p53, although a brief p53 induction was early seen in FNQ-treated wt p53 cells. Cells susceptible to FNQ showed cleavage of anti-apoptotic Mcl-1, sustained activation of the c-Jun N-terminal Kinase (p-JNK), and apoptosis-associated PARP fragmentation, all of which were counteracted in bcl-2 overexpressing cells. Suppression of JNK activation with the specific inhibitor, SP600125 also prevented FNQ-mediated cell death. Our data suggests that Bcl-2, persistent JNK phosphorylation and cleavage of anti-apoptotic Mcl-1 are key events controlling susceptibility to FNQ.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Furanos/farmacologia , Melanoma/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Naftoquinonas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Caspases/genética , Caspases/metabolismo , Caspases/farmacologia , Linhagem Celular Tumoral , Humanos , Melanoma/genética , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/farmacologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/farmacologiaRESUMO
Copper and two molecules of diethyl dithiocarbamate [DEDTC] form the Cu[DEDTC](2) complex, which shows cytotoxicity against melanoma and carcinoma cells, making it a potentially useful anti-cancer agent. The differential response to Cu[DEDTC](2) in susceptible human SKBR3 carcinoma and C8161 melanoma cell variants of moderate and high resistance to this organometallic complex was evaluated in this study. Both cell lines underwent apoptosis-associated PARP cleavage, changes in expression of nuclear NFkB p65, p21WAF1 and cyclin A, with loss of clonogenicity in response to this agent. However, a threefold greater concentration [IC(50) 0.6 microM DEDTC: 0.3 microM Cu] was required to kill moderately resistant C8161 melanoma compared to highly susceptible SKBR3 cells. Decreased susceptibility to Cu[DEDTC](2) in C8161 melanoma correlated with greater levels of glutathione peroxidase and catalase, and a fourfold lower requirement for N-acetyl cysteine (1mM) to overcome toxicity. Whereas melanoma cells selected for resistance to [0.8 microM DEDTC: 0.4 microM Cu] showed persistent catalase and GPx activity, melanoma cells with moderate susceptibility showed decreased catalase and Gpx when responding to treatment. Cytotoxic response in moderately susceptible C8161 melanoma cells involved an early accumulation of pro-apoptotic Bax in the G2 cell cycle phase, followed by an increased ratio of pro-apoptotic Bak to anti-apoptotic Mcl-1 in mitochondria. Our data suggests that Cu[DEDTC](2) toxicity is mediated through an increase in pro-apoptotic Bak/Bax via disruption of the peroxide and thiol metabolism.
Assuntos
Antineoplásicos/farmacologia , Ditiocarb/farmacologia , Resistencia a Medicamentos Antineoplásicos , Peroxidases/análise , Compostos de Sulfidrila/análise , Proteína Killer-Antagonista Homóloga a bcl-2/análise , Proteína X Associada a bcl-2/análise , Carcinoma/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cobre , Humanos , Melanoma/patologiaRESUMO
Since diethyl dithiocarbamate (DEDTC) forms complexes with either zinc or copper, and 8-hydroxyquinoline (8-OHQ) also complexes with copper, we now compared the cytotoxic activity of Cu[DEDTC]2, Zn[DEDTC]2 and Cu[8-OHQ]2. This report shows that at nanomolar levels, only copper-[DEDTC]2, suppresses proliferation and clonogenicity of SKBR3 human breast carcinoma, concurrently with induction of apoptosis-associated PARP fragmentation. Susceptibility to these agents was paralleled by reactive oxygen generation (ROS) and greater expression of anti-oxidant enzymes like MnSOD and catalase, with no comparable effect on Cu/Zn superoxide dismutase. The lethal effects of Cu[DEDTC]2 manifested when adding the two separate aqueous components or the preformed synthetic complexes in DMSO, was prevented by N-acetyl cysteine or glutathione, with no comparable protection afforded by non-thiol anti-oxidants like mannitol or DMSO. Exogenously added catalase also protected cells from Cu[DEDTC]2, suggesting that this complex may kill after the levels of superoxide anion [O2*-] dismutated by MnSOD increase hydrogen peroxide-related stress. Cu[DEDTC]2 also induced p21WAF1, a cdk inhibitor usually not inducible in mutant p53 tumors like SKBR3 carcinoma, correlating with dephosphorylation of the Sp1 transcription factor. Concentrations of Cu[DEDTC]2 cytotoxic for SKBR3 carcinoma did not induce comparable damage versus normal diploid human WI-38 fibroblasts. In contrast to the cytotoxic effect of nM levels of Cu[DEDTC]2 against SKBRR3 cells, no response was seen in the same cells exposed to 20 microM cis-platin. Since neither DEDTC bound to zinc, nor copper bound to 8-OHQ showed comparable cytotoxicity, our results suggest that the greater activity of copper-DEDTC reflects a specific structure-activity relationship for the active complex. Since Cu[DEDTC]2 shows more effectiveness than other metal-chelator complexes, it may be worth further investigation as an alternative to cancer therapies.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Quelantes/farmacologia , Cobre/farmacologia , Ditiocarb/análogos & derivados , Compostos Organometálicos/farmacologia , Antineoplásicos/química , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quelantes/química , Cobre/química , Ditiocarb/química , Ditiocarb/farmacologia , Fluoresceínas/metabolismo , Humanos , Hidroxiquinolinas/química , Hidroxiquinolinas/farmacologia , Compostos Organometálicos/química , Oxiquinolina , Zinco/química , Zinco/farmacologiaRESUMO
MEK1/2 inhibitors like U0126 can potentiate or antagonize the antitumor activity of cytotoxic agents such as cisplatin, paclitaxel or vinblastine, depending on the drug or the target cells. We now investigated whether U0126, differentially regulates melanoma signaling in response to UV radiation or betulinic acid, a drug lethal against melanoma. This report shows that U0126 inhibits early response (ERK) kinase activation and cyclin A expression in wt p53 C8161 melanoma exposed to either UV radiation or betulinic acid. However, U0126 does not protect from UV damage, but counteracts betulinic acid-mediated apoptosis in the same cells. Protection from the latter drug by joint treatment with U0126 was also evident in wt p53 MelJuso melanoma and mutant p53 WM164 melanoma. The latter cells were the most responsive to betulinic acid, showing a selective decline in the cdk4 protein, without a comparable change in other key cell cycle proteins like cdc2, cdk2, cdk7 or cyclin A, prior to apoptosis-associated PARP fragmentation. Laser scanning cytometry also showed that betulinic acid induced a significant increase in chromatin condensation in WM164 melanoma irrespective of whether they were in adherent form or as multicellular spheroids. All these betulinic acid-induced changes were counteracted by U0126. Our data show for the first time that (a) cdk4 protein is an early target of betulinic acid-induced apoptosis and (b) unrestricted ERK signaling favours betulinic acid-induced apoptosis, but this is counteracted by U0126, partly through counteracting chromatin condensation and restoring Akt activation decreased by betulinic acid treatment.
Assuntos
Apoptose/efeitos dos fármacos , Butadienos/farmacologia , Melanoma/metabolismo , Melanoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Triterpenos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Ciclina A/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Dano ao DNA/efeitos dos fármacos , Regulação para Baixo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Triterpenos Pentacíclicos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Ácido BetulínicoRESUMO
Overexpression of EGF receptors and constitutive cyclin D1 expression are frequently associated with human squamous carcinomas. We have now investigated whether these parameters influence susceptibility to okadaic acid induced cell death in EGF-receptor overexpressing mutant p53 A431 human carcinoma. Exposure of these cells to 20 nM okadaic acid induced apoptosis-associated caspase 3 activation, DNA fragmentation, cleavage of Poly ADP-Ribose Polymerase (PARP), p53-independent expression of pro-apoptotic bax, and loss of proliferation-promoting cyclin D1. All these alterations were antagonized by concurrent addition of exogenous EGF. Ectopic overexpression of the cyclin D1 gene in A431 carcinoma conferred resistance to 20 nM okadaic acid irrespective of exogenous EGF, associated with a parallel induction of anti-apoptotic bcl-2. Treatment with a subtoxic concentration of a bispecific bcl-2/bcl xL antisense oligonucleotide cooperated with okadaic acid to down-regulate bcl-2 and sensitize cyclin D1-overexpressing cells to okadaic acid. Although EGF protects EGF-R proficient epithelial cells from diverse apoptotic stimuli through Mcl-1, this is the first report demonstrating that cyclin D1 overexpression provides an EGF independent protection from okadaic acid-induced cell death through induction of bcl-2. We also show that this anti-apoptotic effect of cyclin D1 overexpression, can be partly antagonized with antisense strategies that down-regulate anti-apoptotic bcl-2 family members.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclinas/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Apoptose/fisiologia , Sequência de Bases , Caspase 3/metabolismo , Linhagem Celular Tumoral , Ciclina D , Fator de Crescimento Epidérmico/farmacologia , Expressão Gênica/efeitos dos fármacos , Genes bcl-1 , Genes bcl-2 , Humanos , Ácido Okadáico/farmacologia , Oligodesoxirribonucleotídeos Antissenso/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genéticaRESUMO
Solid tumors are often placed under stress conditions, such as glucose starvation which may result in topoisomerase II drug resistance. In this study, we investigated whether glucose deprivation or substitution by fructose regulates tumor cell apoptosis induced by 2-acetyl furanonaphthoquinone (FNQ). We now show that FNQ exerts much greater antitumor activity than either 7-methoxy 2-ethyl FNQ or 2-ethyl FNQ. Whereas 0.8 microM FNQ induces apoptosis after 16 hours in glucose-supplemented conditions irrespective of bcl-2 overexpression in K1735 melanoma, 0.5 microM FNQ is also effective within 12 hours in low glucose or in fructose-supplemented medium. Under the latter conditions, apoptosis-associated PARP cleavage and cytosolic cytochrome C are increased, together with induction and partial translocation to mitochondria of phosphorylated Jun-N-terminal kinase and massive upregulation of mitochondrial Mn superoxide dismutase. We propose that mitochondrial colocalization of these activities is important in this synergistic anti-tumor effect of FNQ and glucose depletion. Since glucose limitation slows proliferation and decreases efficacy of some genotoxic drugs that trigger apoptosis in rapidly dividing cells, we propose evaluating FNQ as a novel therapeutic anti-cancer adjuvant against slowly proliferating tumors.
Assuntos
Antineoplásicos/uso terapêutico , Apoptose , Furanos/uso terapêutico , Glucose/deficiência , Melanoma/tratamento farmacológico , Naftoquinonas/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Citocromos c/análise , Citocromos c/metabolismo , Glicólise , Humanos , MAP Quinase Quinase 4/análise , MAP Quinase Quinase 4/metabolismo , Melanoma/metabolismo , Mitocôndrias/enzimologia , Fosforilação , Poli(ADP-Ribose) Polimerases/análise , Poli(ADP-Ribose) Polimerases/metabolismo , Transporte Proteico , Neoplasias Cutâneas/metabolismo , Superóxido Dismutase/análise , Superóxido Dismutase/metabolismo , Ativação Transcricional , Células Tumorais Cultivadas , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Differentiated melanocytic cells produce melanin, through several redox reactions including tyrosinase-catalyzed DOPA oxidation to DOPA quinone. We now developed a method based on DOPA oxidase in-gel detection and Sypro Ruby fluorometric normalization to investigate induction of specific DOPA oxidase isoforms in response to hydrogen peroxide-mediated stress, and to ask whether this is associated with p53-dependent adaptive responses. This report shows that hydrogen peroxide leads to comparable induction of 60 and 55 kDa DOPA oxidases in poorly pigmented B16 melanoma, in contrast to sole induction of a major 55 kDa DOPA oxidase in their highly pigmented counterparts. In the latter cells, this response also increases p53 concomitant with joint induction of p53-activated proteins like the cell-cycle inhibitor p21WAF1 and pro-apoptotic bax, with no comparable effect on expression of anti-apoptotic bcl-2. Together, these data suggest that response to hydrogen peroxide involves p53-mediated growth-restrictive signaling and unequal induction of specific DOPA oxidases in melanocytic cells with unequal basal pigmentation.
Assuntos
Ciclinas/metabolismo , Peróxido de Hidrogênio/farmacologia , Melanoma Experimental/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Diferenciação Celular , Inibidor de Quinase Dependente de Ciclina p21 , Relação Dose-Resposta a Droga , Ativação Enzimática , Isoenzimas/metabolismo , Melanoma Experimental/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Pigmentação da Pele , Proteína X Associada a bcl-2RESUMO
Redox imbalance due to oxidative stress or excessive antioxidant levels can alter apoptotic responses. Recently, antioxidants like N-acetylcysteine (NAC) were reported to inhibit H(2)O(2)-mediated necrotic cell death, although they were inactive against apoptosis induced by other agents like etoposide. NAC was also found to kill preferentially tumor cells compared to normal fibroblasts at 20-50mM, but these concentrations are lethal to normal splenocytes. We now demonstrate that 10mM NAC, a non-toxic concentration, can enhance the UV radiation-mediated apoptosis of human C8161 melanoma cells. Compared to treatment with UV radiation alone, combination treatment with NAC doubled the ratio of activated caspase-3 to pro-caspase-3 and produced greater fragmentation of the retinoblastoma protein and the E2F-4 transcription factor without affecting the E2F-1 protein. These effects of joint NAC-UV radiation treatment were counteracted by the overexpression of the bcl-2 gene. To our knowledge, this report is the first to: (i) demonstrate a synergy between DNA-damaging agents, like UV radiation, and antioxidants, like NAC, and (ii) show that a Bcl-2-inhibitable E2F-4 fragmentation occurs concurrently with caspase-3 activation and apoptosis.