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1.
Mol Cancer Ther ; 12(10): 2121-34, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23960095

RESUMO

Despite recent advances in the treatment of colorectal cancer (CRC), tumor resistance is a frequent cause of chemotherapy failure. Therefore, new treatment options are needed to improve survival of patients with irinotecan-refractory CRCs, particularly those bearing KRAS mutations that preclude the use of anti-EGFR therapies. In this study, we investigated whether sorafenib could reverse irinotecan resistance, thereby enhancing the therapeutic efficacy of routinely used irinotecan-based chemotherapy. We used both in vitro (the HCT116, SW48, SW620, and HT29 colon adenocarcinoma cell lines and four SN-38-resistant HCT-116 and SW48 clones) and in vivo models (nude mice xenografted with SN-38-resistant HCT116 cells) to test the efficacy of sorafenib alone or in combination with irinotecan or its active metabolite, SN-38. We have shown that sorafenib improved the antitumoral activity of irinotecan in vitro, in both parental and SN-38-resistant colon adenocarcinoma cell lines independently of their KRAS status, as well as in vivo, in xenografted mice. By inhibiting the drug-efflux pump ABCG2, sorafenib favors irinotecan intracellular accumulation and enhances its toxicity. Moreover, we found that sorafenib improved the efficacy of irinotecan by inhibiting the irinotecan-mediated p38 and ERK activation. In conclusion, our results show that sorafenib can suppress resistance to irinotecan and suggest that sorafenib could be used to overcome resistance to irinotecan-based chemotherapies in CRC, particularly in KRAS-mutated tumors.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Camptotecina/análogos & derivados , Neoplasias Colorretais/tratamento farmacológico , Proteínas de Neoplasias/genética , Niacinamida/análogos & derivados , Compostos de Fenilureia/administração & dosagem , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Camptotecina/administração & dosagem , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Receptores ErbB , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Irinotecano , Camundongos , Niacinamida/administração & dosagem , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Sorafenibe , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética
2.
Autophagy ; 8(7): 1098-112, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22647487

RESUMO

Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.


Assuntos
Autofagia/efeitos dos fármacos , Camptotecina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Supressora de Tumor p53/deficiência , Camptotecina/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Ativação Enzimática/efeitos dos fármacos , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Irinotecano , Proteína Supressora de Tumor p53/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/ultraestrutura
3.
Mol Cancer ; 10: 64, 2011 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-21619602

RESUMO

BACKGROUND: Topoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecan's active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance. METHODS: We previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of TOP1 mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with γH2AX staining and replication activity with molecular combing. RESULTS: In SN38 resistant HCT116 clones we identified three new TOP1 mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these TOP1 mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells. CONCLUSIONS: These results indicate that the TOP1 mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , DNA Topoisomerases Tipo I/genética , Resistencia a Medicamentos Antineoplásicos/genética , Mutação/genética , Sequência de Bases , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Neoplasias Colorretais/enzimologia , Quebras de DNA de Cadeia Dupla , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , Células HCT116 , Humanos , Estrutura Secundária de Proteína , Inibidores da Topoisomerase I/farmacologia
4.
Cancer Res ; 71(3): 1041-9, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21159664

RESUMO

Despite recent advances in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. To better elucidate the molecular mechanisms involved in resistance to irinotecan (and its active metabolite SN38), we established SN38-resistant clones derived from HCT-116 and SW48 cell lines. These clones show various levels (6- to 60-fold) of resistance to SN-38 and display enhanced levels of activated MAPK p38 as compared with the corresponding parental cells. Because four different isoforms of p38 have been described, we then studied the effect of p38 overexpression or downregulation of each isoform on cell sensivity to SN38 and found that both α and ß isoforms are involved in the development of resistance to SN38. In this line, we show that cell treatment with SB202190, which inhibits p38α and p38ß, enhanced the cytotoxic activity of SN38. Moreover, p38 inhibition sensitized tumor cells derived from both SN38-sensitive and -resistant HCT116 cells to irinotecan treatment in xenograft models. Finally, we detected less phosphorylated p38 in primary colon cancer of patients sensitive to irinotecan-based treatment, compared with nonresponder patients. This indicates that enhanced level of phosphorylated p38 could predict the absence of clinical response to irinotecan. Altogether, our results show that the p38 MAPK pathway is involved in irinotecan sensitivity and suggest that phosphorylated p38 expression level could be used as a marker of clinical resistance to irinotecan. They further suggest that targeting the p38 pathway may be a potential strategy to overcome resistance to irinotecan-based chemotherapies in colorectal cancer.


Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/enzimologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Camptotecina/análogos & derivados , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Camptotecina/administração & dosagem , Camptotecina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Fluoruracila/administração & dosagem , Células HCT116 , Humanos , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Imuno-Histoquímica , Irinotecano , Isoenzimas , Leucovorina/administração & dosagem , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Nus , Fosforilação , Piridinas/administração & dosagem , Piridinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
5.
Genes Chromosomes Cancer ; 33(2): 141-9, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11793440

RESUMO

It has been proposed that women carrying heterozygous mutations of the ATM gene could be at increased risk of developing breast cancer. However, data in the literature are contrasting and no firm conclusion has been reached. Our aim was to verify whether ATM inactivation could play a role in breast tumor development. Following the classical tumor suppressor inactivation scheme, tumors showing loss of heterozygosity (LOH) at the ATM locus should present an increased proportion of mutated ATM forms. We screened a cohort of 173 nonselected primary breast tumors for LOH in a 4 cM region at 11q23 spanning the ATM gene. We analyzed 25 tumors presenting LOH within the ATM locus for mutations in the ATM coding sequence using an RT-PCR-SSCP approach. Five patients were found to bear a coding missense variant, out of which four corresponded to a frequent polymorphism in exon 39. One patient presented a previously unreported variant in exon 19 (2614C>T) resulting in a nonconservative change (Pro>Ser) at aa 872. This variant was not found in any of the other 172 patients nor in 63 healthy controls tested, indicating that it is a rare ATM variant. LOH involved the ATM wild-type allele in the tumor presenting variant 2614. However, because the ATM gene presents a relatively large number of rare coding polymorphism it is difficult, in the absence of familial data, to be conclusive on the significance of this variant. Searching for further variants in exons 19 and 39 in the whole set of 173 breast tumors, we found one tumor showing an acquired deletion of four bases in the ATM gene. Somatic mutations affecting the ATM gene thus seem rare in breast cancer. In our cohort of breast cancer patients, tumors presenting LOH at the ATM locus did not show an increased frequency of sequence variants. Furthermore, allelic imbalance profiles in a 4-cM region of chromosome arm 11q spanning the ATM locus revealed that hot spots of LOH were more likely to correspond to a region localized telomeric to the gene. Therefore, these data suggest that other target genes for genetic inactivation exist in the 11q23 region.


Assuntos
Ataxia Telangiectasia/genética , Neoplasias da Mama/genética , Variação Genética/genética , Mutação de Sentido Incorreto/genética , Proteínas Serina-Treonina Quinases/genética , Alelos , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 11/genética , Análise Mutacional de DNA , Proteínas de Ligação a DNA , Feminino , Marcadores Genéticos/genética , Humanos , Perda de Heterozigosidade/genética , Pessoa de Meia-Idade , Polimorfismo Genético/genética , Proteínas Supressoras de Tumor
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