RESUMO
BACKGROUND: We previously reported that 3'-ethynylcytidine (ECyd, TAS-106), an RNA polymerases inhibitor, enhances the anti-tumor efficacy of platinum in several tumor types in both in vitro and in vivo tumor models. However, the molecular mechanisms underlying the ECyd-induced enhancement remain elusive. METHODS: Cisplatin (CDDP)-resistant head and neck cancer KB cells were established by stepwise dose escalation with CDDP. The combination effect of ECyd and CDDP were assessed using isobologram analysis. The transcriptional and post-translational statuses of several molecules were detected using real-time PCR, immunoblot analysis and immunocytochemistry. Xenograft assays were used to confirm the mechanisms underlying the ECyd induced enhancement of CDDP anti-tumor efficacy in vivo. RESULTS: ECyd sensitized KB to CDDP by inhibiting the drug transporter Vault complex (Vaults). First, we showed that Vaults were overexpressed in CDDP-resistant KB cells. The suppression of major vault protein (MVP) by RNA interference restored the sensitivity to CDDP. Next, we showed that ECyd significantly sensitized the resistant cells to CDDP, compared with the parental paired cell line. A molecular analysis revealed that ECyd inhibited the synthesis of vRNAs as well as the induction of MVP, both of which are critical components of Vaults as a drug transporter. Furthermore, we found that the synergistic effect of ECyd and CDDP was correlated with the MVP expression level when the effect was analyzed in additional cancer cell lines. Finally, we demonstrated that ECyd decreased the vRNAs expression level in xenograft tumor. CONCLUSIONS: Our data indicated the ability of ECyd to cancel the resistance of cancer cells to CDDP by inhibiting the Vaults function and the decrease of Vaults expression itself, and the ability of the combination therapy with CDDP and ECyd to offer a new strategy for overcoming platinum resistance. Moreover, the study results suggest that Vaults could be a biomarker for stratifying patients who may benefit from the combination therapy with ECyd and platinum.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Citidina/análogos & derivados , Neoplasias Nasofaríngeas/tratamento farmacológico , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Animais , Carcinoma , Linhagem Celular Tumoral , Citidina/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patologia , Transplante de Neoplasias , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although 5-fluorouracil (5-FU) plus leucovorin (LV) is a standard chemotherapy regimen for colorectal cancer, the factors that determine the LV-mediated enhancement of the antitumor activity of 5-FU have remained unknown. We investigated the roles of folylpolyglutamate synthase (FPGS) and gamma-glutamyl hydrolase (GGH), which are the main enzymes involved in folate metabolism, in the effect of LV. LV enhanced the anticancer activity of 5-FU and the level of reduced folate in human colon cancer cells. Small-interfering RNA (siRNA) transfected into DLD-1 cells to downregulate FPGS reduced the basal level of reduced folate, the folate level after LV treatment, and the enhancement of 5-fluoro-2'-deoxyuridine (FdUrd)-induced cytotoxicity elicited by LV. By contrast, the downregulation of GGH by siRNA increased cellular sensitivity to FdUrd combined with LV. These results suggest that FPGS and GGH expression levels in tumors are determinants of the efficacy of LV in enhancing the antitumor activity of 5-FU.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Fluoruracila/administração & dosagem , Leucovorina/administração & dosagem , Peptídeo Sintases/metabolismo , gama-Glutamil Hidrolase/metabolismo , Animais , Antineoplásicos/administração & dosagem , Neoplasias do Colo/patologia , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Resultado do TratamentoRESUMO
Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP(-/-) UP(-/-)) mice. TP activities were inhibited in TP(-/-) UP(-/-) mice, and the level of thymidine in the plasma of TP(-/-) UP(-/-) mice was higher than for TP(-/-) mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP(-/-) UP(-/-) mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T(2) maps in the brain and axonal edema by electron microscopic study of the brain in TP(-/-) UP(-/-) mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.
Assuntos
Timidina Fosforilase/deficiência , Uridina Fosforilase/deficiência , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Marcação de Genes , Humanos , Intestino Delgado/enzimologia , Fígado/enzimologia , Camundongos , Camundongos Knockout , Encefalomiopatias Mitocondriais/enzimologia , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/patologia , Fenótipo , Timidina Fosforilase/genética , Timidina Fosforilase/fisiologia , Uridina Fosforilase/genética , Uridina Fosforilase/fisiologiaRESUMO
1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS-106), is a new antitumor cytidine analogue, inhibiting RNA synthesis. In this study we investigated the cellular growth inhibition, intracellular metabolism, cell cycle phase specificity, and RNA synthesis of TAS-106 compared with those of 5-fluorouracil (5-FU), known to possess both DNA- (inhibition of thymidylate synthase activity) and RNA-synthesis-inhibiting activity (inhibition of RNA function). The IC50 values of TAS-106 and 5-FU ranged from 0.0173 to 3.11 microM, and from 6.80 to >1,000 microM, respectively, in a panel of 10 human tumor cells, indicating that TAS-106 possesses greater cytotoxicity than 5-FU. Using excess thymidine-synchronized cells, TAS-106 and 5-FU appeared to exert their cytotoxic effects independently of the cell cycle. The intracellular metabolism and the effect on pre-rRNA processing of TAS-106 differed from those of 5-FU. More than 50% of 5-FU incorporated into the cells was in the unchanged form, while 5-FU incorporated into RNA was approximately 20%. On the other hand, TAS-106 was incorporated in a time-dependent manner into the cells and rapidly converted to its mono-, di- and tri-phosphate form, however, the amount incorporated into RNA fraction was very small. 5-FU incorporated into RNA was confirmed to impair the normal processing of ribosomal RNA (formation of 34/32S RNA from 45S RNA), however, TAS-106 did not affect pre-rRNA processing and may be involved in the inhibition of the synthesis of ribosomal RNA. We concluded that intracellular accumulation and retention of the active metabolite of TAS-106, 3'-ethynylcytidine 5'-triphosphate (ECTP), may contribute to its potent cytotoxicity. The unique mechanism of antitumor activity and intensive cellular metabolism of TAS-106 could contribute to cancer chemotherapy through the pathways different from those of 5-FU or other antitumor nucleosides.
Assuntos
Antineoplásicos/farmacologia , Citidina/análogos & derivados , RNA Ribossômico/antagonistas & inibidores , Ribonucleosídeos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citidina/análise , Citidina/metabolismo , Citidina/farmacologia , Fluoruracila/análise , Fluoruracila/metabolismo , Fluoruracila/farmacologia , Humanos , Concentração Inibidora 50 , Precursores de RNA/antagonistas & inibidores , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA Ribossômico/química , RNA Ribossômico/metabolismoRESUMO
Dihydropyrimidine dehydrogenase is the most extensively investigated predictive marker for individual response to 5-fluorouracil. Clinical responses to the anticancer agent, along with various reports, have clearly shown that dihydropyrimidine dehydrogenase activity is closely correlated to its mRNA levels, but the regulatory mechanisms of its expression have remained unclear. We attempted to clarify the mechanisms and found that activator protein (AP-1) is probably one of the key factors in the transcriptional regulation of DPYD in cancer cells, and that phorbol 12-myristate 13-acetate (PMA) plus ionomycin treatment enhances transcription of DPYD via AP-1 activation. In this study, we characterized our previously subcloned 5' region of human DPYD, an approximately 3.0-kb fragment (accession no. AB162145). Luciferase reporter assay showed that the clone showed strong promoter activities in 293T and HSC42 cells, and comparative analysis using 5' deletion mutants suggested the existence of several positive and negative regulatory regions, including putative binding sites for AP-1, SP-1, and nuclear factor-kappaB. PMA/ionomycin treatment increased the mRNA level of DPYD in HSC42 cells, and electrophoretic gel mobility shift assay showed that the complex on the putative AP-1 binding site was drastically induced by PMA/ionomycin treatment. The complexes formed were competed out by preincubation with the cold-consensus AP-1 binding site, and the DNA binding complex formed on the site contained c-Jun and c-Fos, which are components of AP-1 transcription factor. We further identified the functional AP-1 binding site (nucleotide positions from -290 to -280), whose nucleotide mutations abolished PMA/ionomycin-induced DPYD promoter activation.
Assuntos
Di-Hidrouracila Desidrogenase (NADP)/genética , Neoplasias Gástricas/enzimologia , Fator de Transcrição AP-1/fisiologia , Transcrição Gênica/fisiologia , Sítios de Ligação , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , Di-Hidrouracila Desidrogenase (NADP)/biossíntese , Humanos , Ionomicina/farmacologia , Rim/enzimologia , Rim/fisiologia , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Neoplasias Gástricas/genética , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , TransfecçãoRESUMO
We evaluated the therapeutic effect of TS-1, a novel oral fluoropyrimidine, in combination with leucovorin with in vivo experiments using a murine tumor xenograft model fed a low folate diet. The reduced folate levels in the tumors of mice fed a low folate diet were significantly lower than those in the tumors of mice fed a normal diet. In addition, the basal reduced folate levels in the tumors of mice fed a low folate diet were comparable to those in human colorectal cancer tissues. Furthermore, when leucovorin was orally administered, the reduced folate levels in the tumors of mice fed a low folate diet were more than two-fold higher than those of mice fed the normal diet. The leucovorin-induced potentiation of TS-1 antitumor activity was obviously higher in COL-1 tumor-bearing mice fed a low folate diet than in mice fed a normal diet. The remarkable increase in reduced folate levels following the administration of leucovorin contributed to the leucovorin-induced potentiation of TS-1 antitumor activity in this low-folate-diet animal model. These results suggest that rodent models fed a low folate diet are suitable for in vivo evaluation of reduced folate drugs like leucovorin. In this model, the combination of leucovorin with TS-1 resulted in a higher antitumor efficacy than TS-1 alone or the combination of UFT and leucovorin, suggesting that TS-1 and leucovorin combination therapy may be an effective regimen for patients with colorectal cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/patologia , Leucovorina/administração & dosagem , Ácido Oxônico/administração & dosagem , Tegafur/administração & dosagem , Animais , Dieta , Modelos Animais de Doenças , Combinação de Medicamentos , Avaliação de Medicamentos , Ácido Fólico/administração & dosagem , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de NeoplasiasRESUMO
PURPOSE: Dihydropyrimidine dehydrogenase (DPD), the initial rate-limiting enzyme in the degradation of 5-fluorouracil (5-FU), is known to be a principal factor in clinical responses to the anticancer agent 5-FU, and various reports have clearly demonstrated that DPD activity is closely correlated to mRNA levels. However, the regulatory mechanisms of DPD gene (DPYD) expression remain unclear. In this study, the regulatory mechanisms have been intensively studied. EXPERIMENTAL DESIGN AND RESULTS: A subcloned 3.0-kb fragment of the 5' region of DPYD contains a total of 60 CpG sites, suggesting that methylation status may affect the repression of DPYD. The clone showed various promoter activities that were largely correlated with mRNA levels in most cell lines, except HSC3 and HepG2. Bisulfite sequencing analysis revealed that various CpG sites around the transcription start site were abnormally methylated in cells with low DPYD expression: Reversal of hypermethylation by 5-azacytidine treatment significantly increased DPYD expression in HSC3 and HepG2 cells that showed strong promoter activity. In HepG2, in vitro methylation of the DPYD promoter directly decreased promoter activity, and 5-azacytidine treatment restored higher DPYD expression in a dose- and time-dependent manner, along with decreased sensitivity to 5-FU. CONCLUSIONS: We found that DPD activity was controlled, at least in part, at the transcription level of DPYD and that aberrant methylation of the DPYD promoter region acted as one of the repressors of DPYD expression and affected sensitivity to 5-FU in cancer cells. Our new results could lead to a more precise understanding of the molecular basis of 5-FU response.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Metilação de DNA , Di-Hidrouracila Desidrogenase (NADP)/genética , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Adenocarcinoma/patologia , Carcinoma/patologia , Carcinoma Hepatocelular/patologia , Carcinoma de Células Escamosas/patologia , Neoplasias do Colo/patologia , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Bucais/patologia , Polimorfismo Genético , Regiões Promotoras Genéticas , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Yeast cells (Sacchromyces cerevisiae) in 0.9% NaCl solution containing phloxine B (dye) were treated by an application of a rectangular electric pulse. We input microscopic images of the yeast suspensions after the application into a computer, and measured whether each cell dyes or not, the phase in the cell cycle, and each cell size, using the software we had developed. After those measurements, we discussed the relationship between the yield of electropermeabilization (the ratio of dyed cells to the total cell number) and the phase in the cell cycle, and cell size. From the results, it was found that the yeast cells from S-phase to M-phase (S-M phase) in the cell cycle tend to be more permeated than G1-phase yeast cells, and in both phases the yield decreases with the increase in cell size.
Assuntos
Ciclo Celular/efeitos da radiação , Permeabilidade da Membrana Celular/efeitos da radiação , Eletroporação/métodos , Interpretação de Imagem Assistida por Computador/métodos , Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/efeitos da radiação , Permeabilidade da Membrana Celular/fisiologia , Tamanho Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Campos Eletromagnéticos , Doses de Radiação , Saccharomyces cerevisiae/citologia , Estatística como AssuntoRESUMO
In this study, combination therapies using the oral fluoropyrimidine tegafur-gimeracil-oteracil (S-1) with several targeted agents or antibodies, were evaluated. First, the effects of tyrosine kinase inhibitors (erlotinib hydrochloride, sorafenib tosilate and sunitinib malate) against human non-small cell lung cancer (NSCLC), breast cancer and colorectal cancer were evaluated in vivo. The effects of the combination of S-1 and targeted antibodies (bevacizumab and cetuximab) against human colorectal cancers was also evaluated in vivo. S-1 and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib, showed a significant inhibition of growth in human NSCLC (Lu-99 and PC-9 cell lines). The antitumor activity of the combination of S-1 and erlotinib against Lu-99 and PC-9 cancer cell lines was significantly superior to either monotherapy (P<0.05). Combination therapy using the multi-tyrosine kinase inhibitors, sorafenib or sunitinib, with S-1 against breast cancer (MX-1 cell line) and NSCLC (NCI-H460 cell line) was significantly superior to either monotherapy (P<0.01). The combination of the anti-vascular endothelial growth factor antibody bevacizumab or the anti-EGFR antibody, cetuximab, with S-1 against human colorectal cancer [Col-1, KM20C (bevacizumab) and DLD-1 (cetuximab) cell lines] and a 5-fluorouracil (5-FU)-resistant cell line (KM12C/5-FU) was significantly superior to either monotherapy (p<0.01). In particular, the growth of the Col-1 cells was completely inhibited by the combination of S-1 and bevacizumab. No toxic mortalities and no significant difference in the body weight changes of the animals treated with S-1 combined with the targeted agents or with the mono-therapies were observed; therefore, the treatments appeared to be well-tolerated. Our preclinical findings indicate that the combination therapies of S-1 and targeted agents are promising treatment options.
RESUMO
Combined chemotherapy with 5-fluorouracil and leucovorin (LV) has been widely used for the treatment of patients with colorectal cancer. Given that LV effects are attributable to increased levels of reduced folate in cancer cells, we attempted here to show the in vivo role of folylpolyglutamate synthetase (FPGS), which stabilizes intracellular reduced folate, in the anticancer activities of oral fluoropyrimidines, UFT or S-1, combined with LV. To this end, HCT-15 human colon cancer cells were knocked down for FPGS expression by RNA interference. The cell line stably expressing FPGS shRNA (FPGS shRNA HCT-15) was cloned and transferred subcutaneously into nude mice fed a low-folate diet. FPGS shRNA HCT-15 tumors expressed a significantly lower level of FPGS at protein and mRNA levels than parental HCT-15 cells, and the levels of reduced folate in FPGS shRNA HCT-15 tumors became 57% of those in parent after a single administration of 10 mg/kg of LV. Notably, FPGS downregulation did not affect the tumor growth or sensitivity to fluoropyrimidine. Importantly, we observed that LV given for 14 days failed to enhance the anticancer effects of UFT and S-1 in FPGS shRNA HCT-15. This was in keeping with the results that LV did not increase the ternary complex of TS, FdUMP and reduced folate. In conclusion, the present results provide in vivo evidence that intratumor FPGS plays an important role in the efficacy of oral fluoropyrimidine plus LV therapy for colorectal cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/enzimologia , Peptídeo Sintases/metabolismo , Administração Oral , Animais , Linhagem Celular Tumoral , Regulação para Baixo/genética , Fluoruracila/administração & dosagem , Ácido Fólico/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Leucovorina/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Peptídeo Sintases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The combination of oral tegafur-uracil (UFT) with leucovorin (LV) is used to treat patients with stage II to III colon cancer based on the results of postoperative randomized studies in which UFT/LV treatment showed an equivalent efficacy to intravenous 5-FU plus LV therapy. However, whether the addition of LV to UFT can elevate the antitumor activity of UFT in colorectal tumors with high expression levels of thymidylate synthase (TS), which affects 5-FU efficacy, remains to be clarified. This study investigated the effect of LV on the antitumor activity of UFT and/or 5-FU prodrugs in low folate diet-fed nude mice using human colorectal cancer xenografts with various expression levels of TS. The addition of LV to UFT resulted in a 55-79% inhibition of tumor growth among 11 types of colorectal tumor xenograft, whereas UFT alone showed 23-67% antitumor activity. Although there was an inverse relationship between the antitumor effect of UFT alone and UFT plus LV and tumoral TS activity, UFT plus LV appeared to have a more potent antitumor effect than UFT alone on colorectal tumors such as Co-3 and KM12C/5-FU with high expression levels of TS. This finding was confirmed by the significant positive correlation between the relative inhibition ratio of UFT/LV to UFT alone and TS levels in tumors. To investigate the reason for the higher efficacy of UFT/LV on colorectal cancer xenografts with high TS activity, intratumoral levels of reduced folates and a ternary complex of TS after oral UFT with or without LV were measured using Co-3 xenografts. Elevated levels of reduced folates and an increased ternary complex of TS in LV-treated tumors were noted. Our results indicate that a combined therapy of UFT with LV may contribute to the treatment of colorectal cancer patients with low and high expression levels of tumoral TS by increased formation of the ternary complex of TS leading to potentiated antitumor efficacy of UFT.
RESUMO
The oral fluoropyrimidine S-1 has marked efficacy in treating metastatic colorectal cancer patients. In the present study, we explored the therapeutic potential of combined in vivo treatment in the human colon cancer cell lines COL-1, KM12C and KM20C with consecutive oral S-1 and leucovorin (LV) in 2-week therapeutic periods. This combination had increased anticancer activity compared to S-1 alone in the xenografts tested. Moreover, oral S-1/LV treatment showed a more potent anticancer effect on COL-1 xenografts than infusional 5-fluorouracil (5-FU)/LV, with comparable loss of body weight. The reduced folate level in the tumors was initially low, but rapidly rose and persisted for a long period of over 24 h after a single LV administration. This resulted in the formation of much higher levels of the ternary complex with thymidylate synthase (TS) and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) derived from 5-FU, leading to a prolonged inhibition of TS activity in combined administration with oral S-1. These results suggest that the co-administration of LV with S-1 might improve therapeutic efficacy in the treatment of colorectal cancer patients.
RESUMO
Biological activity of thioaurones was not tested so far and the group constitute completely unexplored source of new molecules of pharmacological interest. We report synthesis and evaluation of cytotoxic activity of thioaurone derivatives bearing p-hydroquinone system in ring A. Their activity was found to depend strongly on substitution pattern, so eventually both the activity and pharmacokinetic parameters of the molecules could be tailored by further structural modifications.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/farmacologia , Tiofenos/síntese química , Tiofenos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Espectroscopia de Ressonância Magnética , Relação Estrutura-AtividadeRESUMO
A methanol extract of the ripe fruits of Melia azedarach collected in Curitiba, Parana, Brazil, afforded seven new ring C-seco limonoids (1-7) together with three known limonoids (8-10). The structures of the new compounds were elucidated by NMR and MS analysis and comparison of spectral data with those of previously known compounds. Compounds 4 and 5 exhibited significant inhibitory activity against HeLa S3 cancer cells, whereas 1, 2, 3, and 8 showed weak cytotoxicity.