RESUMO
Biological background data up to 11 weeks of age and tumorigenic susceptibility to xenotransplantation with HeLa cells were compared between severely immuno-deficient NOG and NSG mice. The body weight was lower in NOG mice than in NSG mice. Severe depletion of peripheral blood lymphocytes and lymphoid hypoplasia that are well-known characteristics of these mice were equally observed. No lymphoproliferative lesions developed in any mouse of either strain. The occurrence of ectopic exocrine gland and cyst was a common finding in the thymus of both strains. In addition, minimal spongiotic change was observed in the medulla oblongata and spinal cord in both strains, and its incidence in female NOG mice was a little higher than that in NSG mice. In the adrenal, subcapsular cell hyperplasia that is known as an age-related change in non-genetically modified mice developed earlier and its incidence was higher in NSG mice than in NOG mice. The development of female genital organs of NOG mice was slightly retarded in comparison with that of NSG mice. To evaluate tumorigenic susceptibility to xenotransplantation, female mice were implanted in the dorsal subcutis with 1×103 to 1×106 cells/head of HeLa cells, and were checked up to 16 weeks after implantation. As a result, there was no significant strain difference on tumor formation rate and tumor volume. In conclusion, the present study clearly demonstrated that NOG and NSG mice showed no distinct strain differences in either biological features or biological disadvantages.
Assuntos
Carcinogênese/imunologia , Camundongos Endogâmicos NOD/fisiologia , Camundongos SCID/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos NOD/imunologia , Camundongos SCID/imunologia , Especificidade da Espécie , Transplante HeterólogoRESUMO
Deregulation of cell-cycle control is a hallmark of cancer. Thus, cyclin-dependent kinases (Cdks) are an attractive target for the development of anti-cancer drugs. Here, we report the biological characterization of a highly potent pan-Cdk inhibitor with a macrocycle-quinoxalinone structure. Compound M inhibited Cdk1, 2, 4, 5, 6, and 9 with equal potency in the nM range and was selective against kinases other than Cdks. This compound inhibited multiple events in the cell cycle in vitro, including retinoblastoma protein (pRb) phosphorylation, E2F-dependent transcription, DNA replication (determined by bromodeoxyuridine incorporation), and mitosis completion (assayed by flow cytometry) in the 10 nM range. Moreover, this compound induced cell death, as determined by induction of the subG1 fraction, activated caspase-3, and anexin V. In vivo, Compound M showed anti-tumor efficacy at a tolerated dose. In a nude rat xenograft tumor model, an 8-h constant infusion of Compound M inhibited pRb phosphorylation and induced apoptosis in tumor cells at ~ 30 nM, which led to the inhibition of tumor growth. Immunosuppression was the only liability observed at this dose, but immune function returned to normal after 10 days. Suppression of pRb phosphorylation in tumor cells was clearly correlated with tumor cell growth inhibition and cell death in vitro and in vivo. In vivo, Compound M inhibited pRb phosphorylation in both tumor and gut crypt cells. Rb phosphorylation may be a suitable pharmacodynamic biomarker in both tumors and normal tissues for monitoring target engagement and predicting the efficacy of Compound M.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Compostos Macrocíclicos/farmacologia , Quinoxalinas/farmacologia , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Bromodesoxiuridina/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinases Ciclina-Dependentes/metabolismo , Relação Dose-Resposta a Droga , Feminino , Células HCT116 , Humanos , Contagem de Leucócitos , Compostos Macrocíclicos/efeitos adversos , Compostos Macrocíclicos/química , Quinoxalinas/efeitos adversos , Quinoxalinas/química , Ratos , Ratos Nus , Especificidade por Substrato/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Abnormalities in the p16INK4a/ cyclin-dependent kinase (Cdk)4, 6/ Retinoblastoma (Rb) pathway frequently occur in various human cancers. Thus, Cdk4/6 is an attractive target for cancer therapy. Here we report the biological characterization of a 2-aminothiazole-derived Cdk4/6 selective inhibitor, named Compound A in vitro and in vivo. Compound A potently inhibits Cdk4 and Cdk6 with high selectivity (more than 57-fold) against other Cdks and 45 serine/threonine and tyrosine kinases. Compound A inhibits Rb protein (pRb) phosphorylation at Ser780, inhibits E2F-dependent transcription, and induces cell-cycle arrest at G1 in the T98G human glioma cell line. Among 82 human cells derived from various tissues, cell lines derived from hematological cancers (leukemia/lymphoma) tended to be more sensitive to Compound A in cell proliferation assay. Rb-negative cells tended to be insensitive to Compound A, as we had expected. In a nude rat xenograft model, Compound A inhibited pRb phosphorylation and bromodeoxyuridine (BrdU) incorporation in Eol-1 xenograft tumor at plasma concentration of 510 nM. Interestingly Compound A only moderately inhibited those pharmacodynamic and cell cycle parameters of normal crypt cells in small intestine even at 5 times higher plasma concentration. In F344 rats, Compound A did not cause immunosuppression even at 17 times higher plasma conc. These results suggest that Cdk4/6 selective inhibitors only moderately affects on the cell cycle of normal proliferating tissues and has a safer profile than pan-Cdk inhibitor in vivo.
Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia , Animais , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Fatores de Transcrição E2F/antagonistas & inibidores , Fatores de Transcrição E2F/metabolismo , Fase G1 , Humanos , Masculino , Fosforilação , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Nus , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Tiazóis/química , Transplante HeterólogoRESUMO
Aurora-A kinase is a one of the key regulators during mitosis progression. Aurora-A kinase is a potential target for anticancer therapies because overexpression of Aurora-A, which is frequently observed in some human cancers, results in aberrant mitosis leading to chromosomal instability and possibly tumorigenesis. MK-5108 is a novel small molecule with potent inhibitory activity against Aurora-A kinase. Although most of the Aurora-kinase inhibitors target both Aurora-A and Aurora-B, MK-5108 specifically inhibited Aurora-A kinase in a panel of protein kinase assays. Inhibition of Aurora-A by MK-5108 in cultured cells induced cell cycle arrest at the G(2)-M phase in flow cytometry analysis. The effect was confirmed by the accumulation of cells with expression of phosphorylated Histone H3 and inhibition of Aurora-A autophosphorylation by immunostaining assays. MK-5108 also induced phosphorylated Histone H3 in skin and xenograft tumor tissues in a nude rat xenograft model. MK-5108 inhibited growth of human tumor cell lines in culture and in different xenograft models. Furthermore, the combination of MK-5108 and docetaxel showed enhanced antitumor activities compared with control and docetaxel alone-treated animals without exacerbating the adverse effects of docetaxel. MK-5108 is currently tested in clinical trials and offers a new therapeutic approach to combat human cancers as a single agent or in combination with existing taxane therapies.
Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Ácidos Cicloexanocarboxílicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Taxoides/farmacologia , Tiazóis/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Aurora Quinase A , Aurora Quinase B , Aurora Quinases , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ácidos Cicloexanocarboxílicos/administração & dosagem , Ácidos Cicloexanocarboxílicos/química , Docetaxel , Humanos , Concentração Inibidora 50 , Camundongos , Mitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Ratos , Taxoides/toxicidade , Tiazóis/administração & dosagem , Tiazóis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Previous studies have shown the efficacy of betacellulin (BTC) to promote beta-cell regeneration. Because of its short half-life, however, the effect of BTC may have been underestimated. This study was conducted to assess the effect of continuous administration of BTC on beta-cell regeneration. Adenovirus vectors encoding proBTC (Ad-proBTC) and mature BTC (Ad-mBTC) were prepared, and the efficacy of secretion of BTC was compared in AML12 hepatocytes. When AML12 cells were infected with Ad-proBTC or Ad-mBTC, cells infected with Ad-mBTC secreted considerably larger amount of BTC. We then infused Ad-mBTC into the mouse tail vein. Expression of BTC was detected in the liver for at least 21 days, and serum BTC was maintained at approximately 1 ng/ml for 7 days. When Ad-mBTC was infused immediately after administration of STZ (170 mg/kg), elevation of the plasma glucose induced by STZ was markedly inhibited, and the plasma glucose concentration remained at less than 200 mg/dl for 21 days. The insulin content and the beta-cell mass were significantly increased in Ad-mBTC-infused mice. These results indicate that continuous administration of BTC is quite effective in promoting regeneration of beta-cells.
Assuntos
Hiperglicemia/induzido quimicamente , Células Secretoras de Insulina/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Estreptozocina/farmacologia , Adenoviridae/genética , Animais , Betacelulina , Glicemia/metabolismo , Linhagem Celular , Vetores Genéticos , Hepatócitos/metabolismo , Imuno-Histoquímica , Insulina/sangue , Células Secretoras de Insulina/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Adenovirus-mediated gene transfer of pancreatic duodenal homeobox transcription factor PDX-1, especially its super-active version (PDX-1/VP16), induces the expression of pancreatic hormones in murine liver and reverses streptozotocin-induced hyperglycemia. Histological analyses suggest that hepatocytes are the major source of insulin-producing cells by PDX-1 gene transfer, although the conversion of cultured hepatocytes into insulin-producing cells remains to be elucidated. The present study was conducted to address this issue. Hepatocytes were isolated from adult rats. Then, PDX-1 or PDX-1/VP16 gene was introduced by using adenovirus vector. Two days later, the expression of insulin was detected at mRNA and protein levels. Transfection of PDX-1/VP16 was more efficient in converting hepatocytes to insulin-producing cells. Immunoreactivity of albumin was downregulated in transdifferentiated cells and some of them almost completely lost albumin expression. During the course of transdifferentiation, upregulation of mRNA for CK19 and alpha-fetoprotein was observed. When cultured in collagen-1 gel sandwich configuration, hepatocytes maintained their mature phenotype and did not proliferate. In this condition, transfer of PDX-1/VP16 also induced the expression of insulin. These results clearly indicate that hepatocytes possess a potential to transdifferentiate into insulin-producing cells in vitro.