RESUMO
Estramustine administered orally as estramustine phosphate (EMP) remains a major tool in hormone refractory prostate cancer chemotherapy. The presence of estramustine binding protein, prostatin, in prostate tissue may be a determinant of response to treatment. Lipophilins are secretory proteins with homology to prostatin. Reverse transcription-polymerase chain reaction was performed to estimate expression patterns of lipophilins A to C in human biopsies and cell lines resistant to estramustine. Although lipophilin A was not expressed in prostate tissue, both lipophilins B and C were expressed in normal and tumor prostate without significant differences. For lipophilin C, a somatic mutation (T to C transition at positions 409 and 412) was found in human tumor samples and absent in normal prostate tissue. No consistent response to EMP was observed in enhanced green fluorescent protein (EGFP)-tagged lipophilin C-transfected PC3 cells compared with parental controls. Among these EGFP-lipophilin C clones, no direct correlation between response to EMP treatment (IC50 values) and EGFP expression was observed (p = 0.73). Lipophilin C mRNA levels did not vary significantly between wild-type and estramustine-resistant cells in prostate (DU145 and PC3) and ovarian (SKOV3) cancer cell lines. Overall, these results suggest that lipophilins are not specific determinants of estramustine efficacy.
Assuntos
Antineoplásicos Hormonais/farmacologia , Estramustina/farmacologia , Proteínas de Neoplasias/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Antineoplásicos Hormonais/uso terapêutico , Biópsia , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Estramustina/uso terapêutico , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Masculino , Mutação , Proteínas de Neoplasias/classificação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND: ABCA2 is a member of the ATP binding cassette transporter family with functional roles in cholesterol homeostasis and drug resistance. MATERIALS AND METHODS: In order to characterize its ATPase activity, we transfected HEK293 cells with an ABCA2 mammalian expression system and isolated ABCA2-enriched membranes. RESULTS: We found no measurable ATPase activity of ABCA2 in isolated membranes, except in the presence of the methyl-beta-cyclodextrin. However, competitive binding of a pseudo-substrate, 8-azido-[alpha-32P]-ATP, was demonstrated. CHO cells transfected with ABCA2 did not have a higher rate of endogenous ATP hydrolysis when compared to the mock-transfected cells. CONCLUSION: Overall, we conclude that, while ABCA2 may have low levels of ATPase activity that can be substrate-stimulated, it is more likely to have a regulatory role in cell physiology.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Regulação Enzimológica da Expressão Gênica , Células 3T3/efeitos dos fármacos , Células 3T3/enzimologia , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Animais , Células CHO/efeitos dos fármacos , Células CHO/enzimologia , Clonagem Molecular , Cricetinae , Cricetulus , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/enzimologia , Isoenzimas , Rim/efeitos dos fármacos , Rim/embriologia , Rim/enzimologia , Camundongos , Coelhos , Transfecção , beta-Ciclodextrinas/farmacologiaRESUMO
Intracellular polyamine pools are homeostatically maintained by processes involving biosynthesis, catabolism, and transport. Although most polyamine-based anticancer strategies target biosynthesis, we recently showed that activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase-1 (SSAT) suppresses tumor outgrowth in a mouse prostate cancer model. Herein, we examined the effects of differential SSAT expression on intestinal tumorigenesis in the Apc(Min/+) (MIN) mouse. When MIN mice were crossed with SSAT-overproducing transgenic mice, they developed 3- and 6-fold more adenomas in the small intestine and colon, respectively, than normal MIN mice. Despite accumulation of the SSAT product, N(1)-acetylspermidine, spermidine and spermine pools were only slightly decreased due to a huge compensatory increase in polyamine biosynthetic enzyme activities that gave rise to enhanced metabolic flux. When MIN mice were crossed with SSAT knock-out mice, they developed 75% fewer adenomas in the small intestine, suggesting that under basal conditions, SSAT contributes significantly to the MIN phenotype. Despite the loss in catabolic capability, tumor spermidine and spermine pools failed to increase significantly due to a compensatory decrease in biosynthetic enzyme activity giving rise to a reduced metabolic flux. Loss of heterozygosity at the Apc locus was observed in tumors from both SSAT-transgenic and -deficient MIN mice, indicating that loss of heterozygosity remained the predominant oncogenic mechanism. Based on these data, we propose a model in which SSAT expression alters flux through the polyamine pathway giving rise to metabolic events that promote tumorigenesis. The finding that deletion of SSAT reduces tumorigenesis suggests that small-molecule inhibition of the enzyme may represent a nontoxic prevention and/or treatment strategy for gastrointestinal cancers.
Assuntos
Acetiltransferases/fisiologia , Poliaminas Biogênicas/metabolismo , Neoplasias Intestinais/enzimologia , Acetiltransferases/deficiência , Acetiltransferases/genética , Animais , Poliaminas Biogênicas/biossíntese , Feminino , Genes APC , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Perda de Heterozigosidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
The thymidylate synthase (TS) inhibitors raltitrexed (RTX) and 5-fluorouracil (FUra) have shown promising anti-tumor activity in preclinical and clinical settings for the treatment of colorectal cancer. Though the effects of these two agents have been reasonably well-characterized in cell lines, knowledge of their modes of action in vivo is limited. Here, we utilize the Apc(Min/+) mouse, an animal model of intestinal tumorigenesis, to study the effects of RTX treatment alone and in combination with FUra. Rather surprisingly, RTX monotherapy resulted in a dose dependent 4-10-fold increase in tumor number. The majority of these adenomas (74-95%) were rather small (i.e., less than 1 mm in diameter) and exhibited loss of heterozygosity at the Apc locus, suggesting an increase in mutational events leading to tumor development. RTX augmented BrdU-labeling of crypt epithelial cells, and retarded the movement of these cells along the crypt-villus axis. Co-administration of FUra and RTX resulted in a significant reduction in tumor number compared to mice treated with either RTX or FUra alone (P < 0.0001). In addition, FUra abrogated the RTX-mediated increase in BrdU labeling. In all, the results show that RTX increases tumor burden in the Apc(Min/+) mouse, yet enhances the anti-tumor effect of FUra. This is the first illustration of in vivo synergy of RTX and FUra in a genetically predisposed animal model. Possible mechanisms underlying the current observations are discussed.
Assuntos
Proteína da Polipose Adenomatosa do Colo/fisiologia , Neoplasias Intestinais/patologia , Neoplasias Intestinais/prevenção & controle , Adenoma/metabolismo , Adenoma/patologia , Adenoma/prevenção & controle , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Sinergismo Farmacológico , Células Epiteliais/citologia , Feminino , Fluoruracila/administração & dosagem , Neoplasias Intestinais/metabolismo , Perda de Heterozigosidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quinazolinas/administração & dosagem , Tiofenos/administração & dosagem , Carga TumoralRESUMO
5-Fluorouracil (5-FU) has been the foundation of advanced colorectal cancer treatment for over 40 years. The Apc(Min/+) mouse, which is genetically predisposed to intestinal neoplasia, was used to examine the effects of 5-FU in this system and the impact of dietary folic acid on those effects. 5-FU treatment resulted in a 60-80% reduction in tumor number. Clinically relevant toxicities, including myelosuppression and mucositis, are a part of this response. Tumor numbers rebounded completely following termination of 5-FU therapy, indicating that the drug inhibits tumor growth but does not eradicate them. In mice that were fed with a defined diet containing no folic acid (0 ppm), 5-FU not only induced regression of pre-existing tumors, but also inhibited tumor recovery following drug withdrawal. Our data indicate that a dietary folic acid deficiency, in promoting tumor regression and inhibiting tumor recovery, may enhance the therapeutic effects of 5-FU.