RESUMO
DNA topoisomerase I inhibitors, both synthetic and of natural origin, are receiving increasing consideration primarily as drugs against refractory tumors. Alkannin and shikonin, two enantiomeric dyes from Alkanna tinctoria and Lithospermum erythrorhizon, have been known over many centuries as dyestuff, wound healing, anti-inflammatory, antibacterial and antitumor substances. Although multiple mechanisms appear to be implicated, their potency is associated with the inhibition of topoisomerase I and with the redox properties of the naphthazarin scaffold. Here, the synthesis of new naphthalene and naphthoquinone derivatives inspired by alkannin and shikonin is described and their structural and biological properties were examined. Different oxidation states of the naphthalene nucleus were examined to observe the effect of this parameter on cytotoxicity. Antiproliferative activities against a panel of human cancer cell lines were evaluated and the implication of topoisomerase I was assessed.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Naftalenos/síntese química , Naftalenos/farmacologia , Naftoquinonas/síntese química , Naftoquinonas/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/farmacologiaRESUMO
Amyloid-beta peptides (Abeta) generated by proteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases play an important role in the pathogenesis of Alzheimer's disease (AD). There is mounting evidence that the lipid matrix of neuronal cell membranes plays an important role in the accumulation of Abeta peptides into senile plaques, one of the hallmarks of AD. With the aim to clarify the molecular basis of the interaction between Abeta and cellular membranes, we investigated the effects of various phospholipids (PLs) and a PL-rich diet on Abeta production. Here we show that modulation of Abeta production and Abeta42:40 ratio is not limited to individual fatty acids, rather it is the composition of the PLs of the membrane bilayer, that influences the specificity and level of the regulated intramembranous proteolysis of APP by the gamma-secretase complex. We show that Abeta levels in the conditioned media, in response to some of the PL supplements, is increased in the center and decreased on either side of a graph that resembles bell-shaped distribution. This means that the PLs have less of a tendency to produce unusually extreme effects on Abeta production in SP-C99 transfected Cos-7 cultured cells. We proposed a mechanism-based hypothesis to rationalize PLs' effects on Abeta production.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfolipídeos/farmacologia , Esfingomielinas/farmacologia , Animais , Células COS , Cardiolipinas/farmacologia , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática/métodos , Fosfatidilinositóis/farmacologiaRESUMO
BACKGROUND: Aberrant expression of microRNAs, small non-coding RNA molecules that post-transcriptionally repress gene expression, seems to be causatively linked to the pathogenesis of cancer. In this context, miR-21 was found to be overexpressed in different human cancers (e.g. glioblastoma, breast cancer). In addition, it is thought to be endowed with oncogenic properties due to its ability to negatively modulate the expression of tumor-suppressor genes (e.g. PTEN) and to cause the reversion of malignant phenotype when knocked- down in several tumor models. On the basis of these findings, miR-21 has been proposed as a widely exploitable cancer-related target. However, scanty information is available concerning the relevance of miR-21 for prostate cancer. In the present study, we investigated the role of miR-21 and its potential as a therapeutic target in two prostate cancer cell lines, characterized by different miR-21 expression levels and PTEN gene status. RESULTS: We provide evidence that miR-21 knockdown in prostate cancer cells is not sufficient per se i) to affect the proliferative and invasive potential or the chemo- and radiosensitivity profiles or ii) to modulate the expression of the tumor-suppressors PTEN and Pdcd4, which in other tumor types were found to be regulated by miR-21. We also show that miR-21 is not differently expressed in carcinomas and matched normal tissues obtained from 36 untreated prostate cancer patients subjected to radical prostatectomy. CONCLUSIONS: Overall, our data suggest that miR-21 is not a central player in the onset of prostate cancer and that its single hitting is not a valuable therapeutic strategy in the disease. This supports the notion that the oncogenic properties of miR-21 could be cell and tissue dependent and that the potential role of a given miRNA as a therapeutic target should be contextualized with respect to the disease.
Assuntos
MicroRNAs/genética , Neoplasias da Próstata/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Raios gama , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Técnicas de Silenciamento de Genes , Humanos , Masculino , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Paclitaxel/farmacologia , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Próstata/efeitos da radiação , Neoplasias da Próstata/patologia , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/efeitos da radiaçãoRESUMO
Tumor cell growth, even in advanced stages of ovarian cancer, is nearly always restricted to the peritoneal cavity; therefore, repeated intraperitoneal injections of oligodeoxynucleotides containing dinucleotides with unmethylated CpG motifs (CpG-ODN) recruiting and activating innate effector cells throughout the abdominal cavity to the tumor site might control tumor cell growth and ascites formation. After a single CpG-ODN treatment, in IGROV-1 ovarian tumor ascites-bearing athymic mice, the number of tumor cells declined rapidly and markedly, and ascites volumes declined shortly after treatment (5 h), increasing thereafter at a slower rate than in controls. When administered every 7 days for 4 weeks, CpG-ODN had only a marginal effect on survival time, whereas administration 5 days/wk for 3 or 4 weeks led to a significantly increased survival time as compared with controls (P<0.005) and completely controlled ascites growth without apparent toxicity, although a disorganization of lymphoid organs was observed. Bio-plex assay of cytokine levels in peritoneal fluid of ascites-bearing mice after CpG-ODN treatment revealed an increase in interleukin (IL)-6, IL-10, IL-12, and interferon-gamma at 24 hours, which returned to control mice levels at 48 to 96 hours, whereas the high levels of angiogenic factors remained unchanged. Depletion of natural killer or monocytes/macrophages only slightly influenced the CpG-ODN-induced reduction of ascites tumor cells, indicating that the antitumor activity might not be related to a specific cell/cytokine but rather to the repertoire of cells and cytokines accumulated in the peritoneal cavity. Thus, our data suggest a relevant role for repeated activation of cells and cytokines of innate immunity in the therapy of ovarian cancer patients with malignant ascites.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Ascite/tratamento farmacológico , Oligodesoxirribonucleotídeos/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , Animais , Ascite/etiologia , Esquema de Medicação , Feminino , Humanos , Injeções Intraperitoneais , Camundongos , Camundongos Nus , Oligodesoxirribonucleotídeos/imunologia , Neoplasias Ovarianas/complicações , Neoplasias Ovarianas/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ST1968 (namitecan), a novel 7-modified hydrophilic camptothecin, was found to be effective against tumor models relatively resistant to topotecan and irinotecan. Based on this observation, this study was designed to investigate the cellular and antitumor effects of ST1968 in a subline of A431, squamous cell carcinoma, selected for resistance to topotecan (A431/TPT). This model was characterized by a slow growth rate, associated with downregulation of EGFR and topoisomerase I. In contrast to other camptothecins (SN38 and gimatecan), ST1968 was able to overcome almost completely the resistance at cellular level. The cellular pharmacokinetics indicated a comparable accumulation and retention of ST1968 in sensitive and resistant cells, in spite of expression of the efflux transporter, P-glycoprotein, in resistant cells. The uptake and retention of topotecan were dramatically reduced in both tumor cell lines, but more evident in the resistant one. In contrast to topotecan, ST1968 retained an outstanding efficacy in vivo against the resistant tumor (A431/TPT). The results are consistent with the interpretation that ST1968 was able to overcome the most relevant mechanisms associated with the development of topotecan resistance (i.e., slow proliferation and target downregulation) owing to its peculiar pharmacokinetic behaviour.
Assuntos
Camptotecina/análogos & derivados , Carcinoma de Células Escamosas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Topotecan/uso terapêutico , Animais , Camptotecina/farmacologia , Camptotecina/uso terapêutico , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Humanos , Camundongos , Camundongos Nus , Topotecan/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Vacuolar-H(+)-ATPase plays a critical role in the cellular balance of protons, thus regulating intracellular pH and contributing to apoptosis resistance, drug resistance, and invasive and metastatic behavior of cells. NiK-12192, a vacuolar-H(+)-ATPase inhibitor, caused a reduction in the volume and/or acidity of lysosomes, a polarization of alphavbeta5 integrin distribution, and a number of floating live cells, whereas signs of apoptosis appeared only after 72 h of treatment. In conclusion, NiK-12192, by affecting vacuolar- H(+)-ATPase activity (and intracellular pH), causes a modification of structures crucial for cell adhesion and induces cell death, likely by a modality involving an anoikis-mediated apoptosis.
Assuntos
Benzamidas/farmacologia , Indóis/farmacologia , Lisossomos/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/ultraestrutura , Relação Dose-Resposta a Droga , Citometria de Fluxo , Células HCT116 , Células HL-60 , Células HT29 , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Concentração Inibidora 50 , Lisossomos/química , Lisossomos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Confocal , Microscopia Eletrônica , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Receptores de Vitronectina/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The cell ability of tumor cells to tolerate stress conditions is a typical feature of solid tumors. In particular, the resistance to oxidative stress of melanoma cells likely contributes to their intrinsic drug resistance. In an attempt to develop novel strategies for overcoming the mechanisms of cellular protection against oxidative stress, in this study we have explored the efficacy of the combination of two prooxidant agents in two human melanoma cell clones. The selected clones are characterized by a marked difference in expression of gamma-glutamyltransferase, which is known to produce a persistent low level of oxidative stress resulting in the stimulation of protective systems. The gamma-glutamyltransferase-overexpressing clone exhibited a low susceptibility to arsenic trioxide-induced apoptosis, associated with low reactive oxygen species induction and increased catalase activity. The combination of arsenic trioxide with subtoxic concentrations of ascorbic acid resulted in a sensitization to apoptotic cell death. The expression of protective mechanisms, in particular catalase activity, accounted for the behavior of the resistant clone. The sensitization achieved by the combination was associated with a cellular response involving the ASK1/p38 axis, which is implicated in the regulation of catalase expression and the activation of apoptotic signals. In conclusion, the results of our study provide evidence that a rational combination of prooxidant agents may be effective in overcoming cellular tolerance to oxidative stress.
Assuntos
Arsenicais/uso terapêutico , Ácido Ascórbico/metabolismo , Inibidores do Crescimento/uso terapêutico , Melanoma/tratamento farmacológico , Óxidos/uso terapêutico , gama-Glutamiltransferase/metabolismo , Apoptose , Trióxido de Arsênio , Catalase/metabolismo , Linhagem Celular Tumoral , Citoproteção , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Humanos , MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 5/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Estresse Oxidativo , gama-Glutamiltransferase/genéticaRESUMO
Limited information is available concerning the expression and role of microRNAs in prostate cancer. In this study, we investigated the involvement of miR-205 in prostate carcinogenesis. Significantly lower miR-205 expression levels were found in cancer than in normal prostate cell lines as well as in tumor compared with matched normal prostate tissues, with a particularly pronounced reduction in carcinomas from patients with local-regionally disseminated disease. Restoring the expression of miR-205 in prostate cancer cells resulted in cell rearrangements consistent with a mesenchymal-to-epithelial transition, such as up-regulation of E-cadherin and reduction of cell locomotion and invasion, and in the down-regulation of several oncogenes known to be involved in disease progression (i.e., interleukin 6, caveolin-1, EZH2). Our evidence suggests that these events are driven by the concurrent repression of specific predicted miR-205 targets, namely N-chimaerin, ErbB3, E2F1, E2F5, ZEB2, and protein kinase Cepsilon. Strikingly, the latter seemed to play a direct role in regulating epithelial-to-mesenchymal transition. In fact, its down-regulation led to a cell phenotype largely reminiscent of that of cells ectopically expressing miR-205. Overall, we showed for the first time that miR-205 exerts a tumor-suppressive effect in human prostate by counteracting epithelial-to-mesenchymal transition and reducing cell migration/invasion, at least in part through the down-regulation of protein kinase Cepsilon.
Assuntos
Genes Supressores de Tumor , MicroRNAs/genética , Neoplasias da Próstata/genética , Proteína Quinase C-épsilon/genética , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Regulação para Baixo , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Masculino , Mesoderma/patologia , MicroRNAs/biossíntese , Invasividade Neoplásica , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteína Quinase C-épsilon/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Homeobox 2 de Ligação a E-box com Dedos de ZincoRESUMO
The extracellular gamma-glutamyltransferase-mediated metabolism of glutathione has been implicated in prooxidant events which may have impact on cellular functions including drug resistance. This study was performed in two GGT-transfected melanoma clones to explore the hypothesis that GGT expression in tumour cells is implicated in modulation of cell behaviour under stress conditions. Our results show that GGT-overexpression in melanoma cells was associated with resistance to oxidative stress produced by prooxidant agents such as hydrogen peroxide and ascorbic acid. In GGT-overexpressing cells, ability to tolerate oxidative stress was evidenced by the presence of a moderate level of ROS and lack of DNA damage response following treatment with H(2)O(2). Cellular response to oxidative stress induced by ascorbic acid was detectable only in the clone with low GGT activity which also exhibited an increased susceptibility to apoptosis. The increased resistance of the GGT-overexpressing clone was not related to intracellular GSH content but rather to the increased expression of catalase and to a reduced efficiency of iron-mediated formation of toxic free radicals. Taken together, these findings are consistent with a contribution of GGT in the mechanisms of drug resistance, because induction of oxidative stress is a relevant event in the apoptotic response to cytotoxic agents.
Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Melanoma/enzimologia , Estresse Oxidativo/fisiologia , gama-Glutamiltransferase/metabolismo , Apoptose/fisiologia , Western Blotting , Catalase/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromos c/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
On the basis of the evidence that vacuolar H(+)-ATPase is implicated in the development of the metastatic phenotype, we have explored the possibility to target the enzyme function in an attempt to control the metastatic behavior of tumor cells. In this study, we used an indole derivative, NiK-12192 [4-(5,6-dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6,6-tetramethyl-piperidin-4-yl)-benzamide], recently identified as an effective inhibitor of vacuolar H(+)-ATPase, as a potential antimetastatic agent in the treatment of NSCLC H460 xenograft, which is able to induce lung metastases in mice. Oral administration of NiK-12192 caused a significant inhibition of formation of spontaneous metastases. In contrast, the drug exhibited a negligible effect on the development of artificial metastases (i.e., after i.v. injection of tumor cells), thus supporting that the drug affects the early events of the metastatic process (e.g., migration and invasion). Cellular effects are consistent with this interpretation. In conclusion, the available results show for the first time that a vacuolar H(+)-ATPase inhibitor is effective in modulation of the metastatic behavior of a lung carcinoma, supporting its potential therapeutic interest as a novel treatment approach.
Assuntos
Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Indóis/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Indóis/farmacologia , Integrina alfaVbeta3/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Topotecan/uso terapêutico , ATPases Vacuolares Próton-Translocadoras/metabolismo , Cicatrização/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The proto-oncogene c-myc is involved in multiple cell pathways with opposite effects on cell outcome of death or proliferation. It has been proposed that these different roles depend on the sequestration of c-Myc protein in cellular compartments and/or its phosphorylation. We speculated that subcellular localization of c-Myc protein and of its phosphorylated form (P-c-Myc) could have a role in the different response to paclitaxel (PTX) in two prostate carcinoma cell lines, PC3 and DU145, which undergo either multinucleation or c-myc-dependent apoptosis, respectively. c-myc is amplified only in PC3, but a similar extent of c-Myc phosphorylation was observed in both cell lines after PTX treatment. We found that PTX-induced upregulation of c-myc in DU145 cells, not occurring in PC3 cells, cannot be ascribed to a different protein localization, and that a comparable c-Myc and P-c-Myc nuclear translocation occurs in both cell lines after drug treatment. Thus, subcellular localization of c-Myc and P-c-Myc is not crucial in determining the mode of cell death in these prostate carcinoma cell lines.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma/tratamento farmacológico , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Paclitaxel/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Carcinoma/metabolismo , Linhagem Celular Tumoral , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Proto-Oncogene MasRESUMO
Hepatocyte growth factor (HGF) and its tyrosine kinase receptor Met play a pivotal role in the tumor metastatic phenotype and represent attractive therapeutic targets. We investigated the biochemical and biological effects of the tyrosine kinase inhibitor RPI-1 on the human lung cancer cell lines H460 and N592, which express constitutively active Met. RPI-1-treated cells showed down-regulation of Met activation and expression, inhibition of HGF/Met-dependent downstream signaling involving AKT, signal transducers and activators of transcription 3 and paxillin, as well as a reduced expression of the proangiogenic factors vascular endothelial growth factor and basic fibroblast growth factor. Cell growth in soft agar of H460 cells was strongly reduced in the presence of the drug. Furthermore, RPI-1 inhibited both spontaneous and HGF-induced motility/invasiveness of both H460 and human endothelial cells. Targeting of Met signaling by alternative methods (Met small interfering RNA and anti-phosphorylated Met antibody intracellular transfer) produced comparable biochemical and biological effects. Using the spontaneously metastasizing lung carcinoma xenograft H460, daily oral treatment with well-tolerated doses of RPI-1 produced a significant reduction of spontaneous lung metastases (-75%; P < 0.001, compared with control mice). In addition, a significant inhibition of angiogenesis in primary s.c. tumors of treated mice was observed, possibly contributing to limit the development of metastases. The results provide preclinical evidence in support of Met targeting pharmacologic approach as a new option for the control of tumor metastatic dissemination.
Assuntos
Indóis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Pequenas/irrigação sanguínea , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/patologia , Adesão Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Metástase Neoplásica , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The vacuolar-H(+)-ATPase, functionally expressed in cell membranes, is known to play a relevant role in intracellular pH regulatory mechanisms, because it is implicated in pumping protons into the extracellular environment or in sequestrating excess protons into acidic vacuolar compartments. Because tumor cells exist in a hypoxic microenvironment and produce acidic metabolites, this regulatory mechanism is recognized as a protective function. This study was designed to investigate the effect of NiK-12192 [4-(5,6-dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6,6-tetramethyl-piperidin-4-yl)-benzamide], an indole derivative identified as an effective inhibitor of vacuolar-H(+)-ATPase, on the cytotoxic activity of two camptothecins, i.e., topotecan and SN-38 (7-ethyl-10-hydroxycamptothecin, the active metabolite of irinotecan). The cellular studies performed in two pairs of human colon carcinoma cell lines, i.e., LoVo and LoVo/DX (overexpressing P-glycoprotein) and HT29 and HT29/Mit (overexpressing breast cancer resistant protein), indicated an enhancement of the antiproliferative effect of camptothecins by concomitant exposure to subtoxic concentrations of NiK-12192. Studies of subcellular distribution indicated that whereas topotecan alone localized mainly in mitochondria and endoplasmic compartment, the simultaneous presence of NiK-12192 caused a cytoplasmic redistribution. In HT29/Mit cells, NiK-12192 reverted the pattern of acidification induced by topotecan. The potentiation of topotecan efficacy by NiK-12192 was documented by an increased efficacy of the combination in both the HT29 tumor xenografts, being more evident in the topotecan-resistant HT29/Mit tumor. In conclusion, the vacuolar-H(+)-ATPase inhibitor NiK-12192 was able to potentiate the cytotoxic/antitumor effects of camptothecins, either in in vitro or in in vivo systems. Such findings support a potential interest for the use of vacuolar-H(+)-ATPase inhibitors in combination therapy to improve camptothecin efficacy.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Camptotecina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Topotecan/farmacologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/análise , Animais , Camptotecina/farmacologia , Feminino , Células HT29 , Humanos , Irinotecano , Camundongos , Proteínas de Neoplasias/análise , Neoplasias Experimentais/tratamento farmacológico , Coloração e Rotulagem , Topotecan/farmacocinéticaRESUMO
Atypical retinoids are potent inducers of apoptosis, but activation of the apoptotic pathway seems to be independent of retinoid receptors. Previous studies with a novel adamantyl retinoid, ST1926, have shown that apoptosis induction is associated with an early genotoxic stress. To better understand the relevance of these events, we have selected a subline of the H460 lung carcinoma cell line resistant to ST1926. Resistant cells exhibited cross-resistance to a related molecule, CD437, but not cross-resistance to agents with different mechanisms of action. In spite of a lack of defects in intracellular drug accumulation, induction of DNA strand breaks in resistant cells required exposure to a substantially higher concentration, which was consistent with the degree of resistance. At drug concentrations causing a similar antiproliferative effect (IC80) and a comparable extent of DNA lesions in sensitive and resistant cells, the apoptotic response was a delayed and less marked event in resistant cells, thus indicating a reduced susceptibility to apoptosis. In spite of recognition of DNA lesions in resistant cells, as supported by phosphorylation of p53 and histone H2AX, resistant cells exhibited no activation of the mitochondrial pathways of apoptosis. Following exposure to equitoxic drug concentrations, only sensitive cells exhibited a typical stress/DNA damage response, with activation of the S-phase checkpoint. The cellular resistance to ST1926 reflects alterations responsible for a reduced generation of DNA lesions and for an enhanced tolerance of the genotoxic stress, resulting in lack of activation of the intrinsic pathway of apoptosis. The defective DNA damage response, accompanied by a reduced susceptibility to apoptosis in resistant cells, provides further support to the involvement of genotoxic stress as a critical event in mediating apoptosis induction by ST1926.
Assuntos
Dano ao DNA , Resistencia a Medicamentos Antineoplásicos , Adamantano/análogos & derivados , Adamantano/farmacologia , Antineoplásicos/farmacologia , Apoptose , Western Blotting , Caspases/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Cinamatos/farmacologia , Citocromos c/metabolismo , DNA/química , DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Histonas/química , Humanos , Marcação In Situ das Extremidades Cortadas , Potenciais da Membrana , Mitocôndrias/metabolismo , Fosforilação , Retinoides/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
To investigate the cellular/molecular basis of the activity of a novel lipophilic camptothecin, gimatecan (ST1481), against slowly proliferating cells, we performed a comparative study of topotecan and gimatecan in human bladder cancer models (HT1376 and MCR). Gimatecan was significantly more effective than topotecan in inhibiting the growth of HT1376 tumor, thus reflecting antiproliferative potency. In both HT1376 and MCR cells, gimatecan caused a persistent S-phase arrest, indicating an efficient DNA damage checkpoint. This response was consistent with a cytostatic effect, because no evidence of apoptosis was detected. In contrast to gimatecan, topotecan at equitoxic concentrations caused an early and persistent downregulation of topoisomerase I. Modulation of protein level could not be solely ascribed to the proteasome-mediated degradation of the enzyme because the proteasome inhibitor PS341 sensitized MCR but not HT1376 cells to camptothecins, suggesting alternative mechanisms of drug-induced topoisomerase I downregulation. Indeed, the two camptothecins caused a differential inhibition of topoisomerase I transcription, which is more marked in topotecan-treated cells. The HT1376 model was more sensitive to this immediate decrease of mRNA level. Our data document a marked antitumor activity of gimatecan against a bladder carcinoma model. A limited downregulation of topoisomerase I by gimatecan provides additional insights into the cellular basis of drug potency.
Assuntos
Camptotecina/análogos & derivados , Camptotecina/uso terapêutico , DNA Topoisomerases Tipo I/metabolismo , Modelos Animais de Doenças , Neoplasias da Bexiga Urinária/prevenção & controle , Animais , Antineoplásicos/uso terapêutico , Apoptose , Ácidos Borônicos/farmacologia , Bortezomib , DNA Topoisomerases Tipo I/genética , Regulação para Baixo , Feminino , Humanos , Camundongos , Camundongos Nus , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fase S/efeitos dos fármacos , Inibidores da Topoisomerase I , Topotecan/uso terapêutico , Transplante Heterólogo , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismoRESUMO
Strategies targeting apoptotic pathways may have relevance to improve the efficacy of antitumor therapy. Because synthetic atypical retinoids are potent inducers of apoptosis, there is an increasing interest in exploiting their potential in novel therapeutic approaches. In the present study, we have investigated the cellular effects of the combination of a novel atypical retinoid, ST1926, and the epidermal growth factor receptor inhibitor ZD1839. The results indicated a synergistic interaction between the two drugs associated with a dramatic enhancement of apoptotic response, up-regulation of the cell death receptor DR5, and caspase 8 activation. Other molecular events induced by the cotreatment included (a) a stabilization of the ST1926-induced genotoxic stress detected by formation of phosphorylated gamma-H2AX foci and (b) a complete inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation associated with activation of the proapoptotic protein BAD (i.e., inhibition of phosphorylation on Ser112). In addition, ZD1839 itself inhibited survival pathways by causing a partial dephosphorylation of Akt and a marked down-regulation of survivin. The role of ERK-mediated survival pathways in the cellular response to the drug combination was further supported by the counteracting effect of stimulation of survival pathways by an alternative receptor tyrosine kinase and by the use of a specific inhibitor of the ERK pathway. In conclusion, the results support that the survival pathways activated by epidermal growth factor receptor are determinants of the cell susceptibility to ST1926-induced apoptosis and lowering survival signals may increase the cellular sensitivity to the atypical retinoid. The favorable pharmacologic profiles of both ST1926 and ZD1839 suggest that the combination of these well-tolerated agents may have therapeutic potential.
Assuntos
Adamantano/análogos & derivados , Adamantano/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cinamatos/farmacologia , Quinazolinas/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Caspase 8 , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA , Sinergismo Farmacológico , Ativação Enzimática , Receptores ErbB/antagonistas & inibidores , Feminino , Gefitinibe , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/biossíntese , Transdução de Sinais/efeitos dos fármacos , Neoplasias Vulvares/tratamento farmacológico , Neoplasias Vulvares/patologiaRESUMO
Filler materials for medical use present limits, such as the induction of chronic inflammation and fibrosis. In the search for synthetic materials with improved biocompatible properties, a new polyacrylamide hydrogel, Aquamid (Contura SA, Montreux, Switzerland), has been investigated in preclinical systems. In cell cultures (endothelial cells and fibroblast), no or only transient biological effects were associated with 10% Aquamid exposure. The Aquamid-host interactions were examined in mice (10 mice per group) implanted subcutaneously or in the mammary fat pad with a very large volume (1.5 ml) of the material. Blood analysis, performed after 15 and 94 days (five mice per time for each group) to detect acute or late manifestations of toxicity, did not reveal relevant abnormalities in either group of Aquamid-bearing mice compared with control mice, except for a transient thrombocytopenia and a mild leukocytosis. Histological analysis of the pellet showed the presence of a thin, poorly vascularized cyst wall in implants. Only mild mesenchymal reparative and inflammatory processes were observed, even at longer observation times (more than 400 days). No alterations in any organ were detected. Despite the large volume implanted (approximately 5 percent of mouse body weight), the Aquamid pellet maintained its original size and shape without spreading or sticking to surrounding tissues. In conclusion, the study indicated a good tolerability of the new biopolymer in preclinical systems. The clinical utility of this new compound, if confirmed by clinical randomized trials showing its atoxic properties, could be in the field of aesthetic plastic surgery as a filler material for body contouring and in reconstructive surgery and above all in cancer patients to restore surgical defects.
Assuntos
Resinas Acrílicas/efeitos adversos , Hidrogéis/efeitos adversos , Teste de Materiais , Ritidoplastia , Resinas Acrílicas/administração & dosagem , Animais , Bovinos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Reação a Corpo Estranho/etiologia , Reação a Corpo Estranho/patologia , Humanos , Hidrogéis/administração & dosagem , Técnicas In Vitro , Injeções Subcutâneas , Contagem de Leucócitos , Camundongos , Contagem de Plaquetas , Tela Subcutânea/patologiaRESUMO
Amplification of the c-MYC proto-oncogene is a frequent alteration in hormone refractory prostate carcinomas (HRPC). In an attempt to investigate the role of c-myc in the cellular response to paclitaxel (PTX), we used two HRPC cell lines, DU145 and PC3, characterised by different levels of the protein and by different behaviour in response to taxane. In both cell lines, PTX-induced cell death was a caspase-mediated apoptosis. In DU145 cells, PTX induced an early apoptotic response associated with upregulation of c-myc restricted to the G2/M cell population. This event appeared delayed in the presence of c-myc antisense (AS-c-myc), suggesting an upstream regulation of the protein expression. In addition, the antisense approach provided evidence of an involvement of c-myc in the apoptotic response to the taxane. In contrast, in PC3 cells, the overexpressed c-myc was not modulated by drug-treatment and the addition of AS-c-myc did not affect the cell growth inhibition of PTX. In both cell lines, PTX-induced c-myc phosphorylation was concomitant with the mitotic arrest and not related to the modulation of the activation state of AKT and MAPK kinases. Our data indicate that the cellular response to PTX of HRPC cells can involve c-myc and suggest that its pro-apoptotic role is affected by the genetic background, thus supporting a complex and differentiated HRPC cell response to taxanes.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose , Resistencia a Medicamentos Antineoplásicos/fisiologia , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Caspases/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Ativação Enzimática , Expressão Gênica , Hormônios/farmacologia , Humanos , Masculino , Fosforilação , Neoplasias da Próstata/patologia , Proto-Oncogene Mas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células Tumorais CultivadasRESUMO
Based on structure of the substrate of urease and for the purpose of designing pharmacophore models for urease inhibitors, which could be effective in physiological and pharmacological studies, a series of twenty-five 1,3,4-diazole-2(3H)-thiones-2(3H)-thiones, 1,3,4-diazoles-2(3H)-thiones, and 1,2,4-tri-3-thiones (OSNs) were designed, synthesized, and evaluated for various kinetic parameters of urease inhibition. OSNs inhibited the activity of urease(s) in a concentration dependent fashion. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of inhibition was of pure competitive type for all the 25 compounds. 5-[4-(hydroxy)phenyl]-1,3,4-thiadiazole-2(3H)-thione was found to be the most active one with a Ki value of 2 microM. The Ki values were increased with an increase in substrate concentrations. Apparently, OSNs employ a homologous mechanism of action by exploiting a common transition catalysis state and acting as ligand chelators to form octahedral complexes with the urease enzymes in an orientation-specific mode. The inhibition was slightly potentiated by lower pH and not abolished in the presence of NH2OH (a scavenger of histidine residue). Because of their safe profile in the genotoxic assay, they may be pursued in the near future for human testing
Assuntos
Inibidores Enzimáticos/farmacologia , Oxidiazóis/farmacologia , Triazóis/farmacologia , Urease/antagonistas & inibidores , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/química , Humanos , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Estrutura Molecular , Oxidiazóis/química , Tiadiazóis/química , Triazóis/química , Urease/metabolismoRESUMO
Lipophilicity of camptothecins derivatives has been reported to improve the stability of the lactone ring and to favor rapid uptake and intracellular accumulation. Recently, a novel series of lipophilic camptothecins substituted at position 7 was developed, and gimatecan (ST1481) was selected for clinical development on the basis of some favorable features, including potent cytotoxicity and the unique feature of the lack of recognition by breast cancer resistance-associated protein (BCRP). In this work the intrinsic fluorescence properties of this compound were exploited to investigate its intracellular disposition in comparison with the water-soluble camptothecin, topotecan (TPT), in HT-29 colon carcinoma cells and in a subline, HT-29/Mit, selected for resistance to mitoxantrone and overexpressing BCRP. The study was performed at single-cell level by means of microspectrofluorometry and fluorescence image analysis. The results indicated a quite different subcellular localization of TPT ST1481, since TPT localized mainly in mitochondria, whereas gimatecan exhibited a lysosomal localization. An increased persistence of DNA damage in gimatecan-treated cells was consistent with the interpretation that lysosomes represent a store of active drug. In contrast to gimatecan, which showed a similar localization in HT-29 cells and in the mitoxantrone-resistant subline, the cellular pharmacokinetic of TPT was markedly influenced by overexpression of BCRP protein in the resistant subline. In conclusion, the present results indicating a quite different behavior of the two camptothecins suggest that, apart from intracellular accumulation, subcellular distribution plays a role in their cytotoxic potency and contributes to their pharmacological features.