RESUMO
Salinomycin (SAL) is a monocarboxylic polyether ionophore antibiotic isolated from Streptomyces albus. It exhibits an effective antitumor potential against numerous human cancer cells. This study aimed to assess the antiproliferative effects of SAL in human hepatocellular carcinoma HepG2/C3a cell line. We investigated the effects of SAL on cell growth, DNA damage induction, cell cycle changes and apoptosis; and relative changes in expression of cell cycle-related, apoptosis-related, and CYP450 genes. SAL induced cell cycle arrest in the G2/M phase, upregulation of CDKN1A and GADD45A and downregulation of cyclin genes including CCNB1 and CCNA2. SAL effectively suppressed mRNA levels of CTNNB1 gene, an important oncogene that promotes tumorigenesis. The decrease of HepG2/C3A cells' survival can also be due to downregulation of antiapoptotic BCL-2 expression, thus promoting the induction of apoptosis by SAL. This study also demonstrated the ability of SAL in modulating hepatic cytochrome P450 (CYP) mRNA expression, such that SAL caused the upregulation of CYP1A members and CYP3A5; and downregulation of CYP3A4. Taken together, these data contribute to the understanding of the mechanism of action of SAL, highlighting that metabolizing enzymes modulated by SAL can interfere with chemotherapy treatment and it must be considered in associated treatments.
Assuntos
Apoptose , Neoplasias Hepáticas , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sistema Enzimático do Citocromo P-450/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Piranos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND/AIMS: Compared with non-obese individuals, obese individuals commonly store more vitamin D in adipose tissue. VDR expression in adipose tissue can influence adipogenesis and is therefore a target pathway deserving further study. This study aims to assess the role of 1,25(OH)2D3 in human preadipocyte proliferation and differentiation. METHODS: RTCA, MTT, and trypan blue assays were used to assess the effects of 1,25(OH)2D3 on the viability, proliferation, and adipogenic differentiation of SGBS cells. Cell cycle and apoptosis analyses were performed with flow cytometry, triglycerides were quantified, and RT-qPCR was used to assess gene expression. RESULTS: We confirmed that the SGBS cell model is suitable for studying adipogenesis and demonstrated that the differentiation protocol induces cell maturation, thereby increasing the lipid content of cells independently of treatment. 1,25(OH)2D3 treatment had different effects according to the cell stage, indicating different modes of action driving proliferation and differentiation. In preadipocytes, 1,25(OH)2D3 induced G1 growth arrest at both tested concentrations without altering CDKN1A gene expression. Treatment with 100 nM 1,25(OH)2D3 also decreased MTT absorbance and the lipid concentration. Moreover, increased normalized cell index values and decreased metabolic activity were not induced by proliferation or apoptosis. Exposure to 100 nM 1,25(OH)2D3 induced VDR, CEBPA, and CEBPB expression, even in the preadipocyte stage. During adipogenesis, 1,25(OH)2D3 had limited effects on processes such as VDR and PPARG gene expression, but it upregulated CEBPA expression. CONCLUSIONS: We demonstrated for the first time that 1,25(OH)2D3 induces changes in preadipocytes, including VDR expression and growth arrest, and increases the lipid content in adipocytes treated for 16 days. Preadipocytes are important cells in adipose tissue homeostasis, and understanding the role of 1,25(OH)2D3 in adipogenesis is a crucial step in ensuring adequate vitamin D supplementation, especially for obese individuals.
Assuntos
Adipogenia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Vitamina D/análogos & derivados , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , PPAR gama/genética , PPAR gama/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Regulação para Cima/efeitos dos fármacos , Vitamina D/farmacologiaRESUMO
Harpagophytum procumbens (Hp) has been used as antiinflammatory and analgesic agent for the treatment of rheumatic diseases. The principal active component of Hp is harpagoside (HA). We tested the toxicity of this new therapeutic agent in a hepatic cell line (HepG2/C3A). Hp was found to be cytotoxic, and HA was found to decrease the number of cells in S phase, increase the number of cells in G2/M phase and induce apoptosis. Neither Hp nor HA was genotoxic. The expression of CDK6 and CTP3A4 was reduced by Hp, and both HA and Hp caused a significant reduction of CYP1A2 and CYP3A4 expression. It is possible that the cytotoxicity caused by HA and Hp does not involve transcriptional regulation of the cyclins and CDKs tested but is instead related to the inhibition of metabolism. This is evidenced by the results of an MTT assay and changes in the expression of genes related to drug metabolism, leading to cell death. Indeed, the cells exhibited decreased proliferation upon exposure to Hp and HA. The data show that treatment with either Hp or HA can be cytotoxic, and this should be taken into consideration when balancing the risks and benefits of treatments for rheumatic diseases. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Proliferação de Células/efeitos dos fármacos , Glicosídeos/toxicidade , Inibidores do Crescimento/toxicidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Extratos Vegetais/toxicidade , Piranos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Glicosídeos/farmacologia , Inibidores do Crescimento/farmacologia , Harpagophytum/química , Células Hep G2 , Humanos , Extratos Vegetais/farmacologia , Piranos/farmacologia , Medição de Risco , Testes de ToxicidadeRESUMO
Monastrol is an allosteric inhibitor of the mitotic kinesin Eg5 that exhibits an antiproliferative effect against several cell lines. We investigated the antiproliferative effect of monastrol on human breast adenocarcinoma cells (MCF-7) and mammary epithelial cells (HB4a, non-tumoral). Monastrol treatment decreased cell viability only in MCF-7 tumor cells. Real-time cell growth kinetic analysis showed a decrease in the proliferation of MCF-7 cells exposed to monastrol, while in the HB4a cells, only a concentration of 100 µM was able to induce this effect. In a cell cycle analysis, exposure of MCF-7 cells to monastrol led to an increased population of cells in both the G1 and G2/M phases. In HB4a cells, the proportion of cells in the G2/M phase was increased. Monastrol led to an increased mitotic index in both cell lines. Monastrol was not able to induce cell death by apoptosis in any of the cell lines studied. Gene expression analysis was performed to measure the mRNA levels of cell cycle genes, DNA damage indicator gene, and apoptotic related genes. Treatment with monastrol induced in MCF-7 cells a 5-fold increase in the mRNA levels of the CDKN1A gene, an inhibitor of CDKs related with cell cycle arrest in response a stress stimulus, and a 2-fold decrease in CDKN1C mRNA levels in HB4a cells. These results provide evidence that monastrol has a greater antiproliferative effect on MCF-7 tumor cells compared with non-tumor HB4a cells; however, no selective is observed.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Glândulas Mamárias Humanas/efeitos dos fármacos , Pirimidinas/farmacologia , Tionas/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Relação Dose-Resposta a Droga , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Cinética , Células MCF-7 , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Índice Mitótico , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The search for anticancer drugs has led researchers to study salinomycin, an ionophore antibiotic that selectively destroys cancer stem cells. In this study, salinomycin was assessed in two human cell lines, a breast adenocarcinoma (MCF-7) and a non-tumor breast cell line (HB4a), to verify its selective action against tumor cells. Real-time assessment of cell proliferation showed that HB4a cells are more resistant to salinomycin than MCF-7 tumor cell line, and these data were confirmed in a cytotoxicity assay. The half maximal inhibitory concentration (IC50) values show the increased sensitivity of MCF-7 cells to salinomycin. In the comet assay, only MCF-7 cells showed the induction of DNA damage. Flow cytometric analysis showed that cell death by apoptosis/necrosis was only induced in the MCF-7 cells. The increased expression of GADD45A and CDKN1A genes was observed in all cell lines. Decreased expression of CCNA2 and CCNB1 genes occurred only in tumor cells, suggesting G2/M cell cycle arrest. Consequently, cell death was activated in tumor cells through strong inhibition of the antiapoptotic genes BCL-2, BCL-XL, and BIRC5 genes in MCF-7 cells. These data demonstrate the selectivity of salinomycin in killing human mammary tumor cells. The cell death observed only in MCF-7 tumor cells was confirmed by gene expression analysis, where there was downregulation of antiapoptotic genes. These data contribute to clarifying the mechanism of action of salinomycin as a promising antitumor drug and, for the first time, we observed the higher resistance of HB4a non-tumor breast cells to salinomycin.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Glândulas Mamárias Humanas/efeitos dos fármacos , Piranos/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ensaio Cometa , Dano ao DNA , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Células MCF-7 , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
ß-glucan is an important polysaccharide due to its medicinal properties of stimulating the immune system and preventing chronic diseases such as cancer. The aim of the present study was to determine the anticlastogenic effect of ß-glucan in cells exposed to ultraviolet radiation (UV). Chromosome aberration assay was performed in drug-metabolizing cells (HTC) and non drug-metabolizing cells (CHO-K1 and repair-deficient CHO-xrs5), using different treatment protocols. Continuous treatment (UV + ß-glucan) was not effective in reducing the DNA damage only in CHO-xrs5 cells. However, the pre-treatment protocol (ß-glucan before UV exposition) was effective in reducing DNA damage only in CHO-K1 cells. In post-treatment (ß-glucan after UV exposition) did not show significative anticlastogenic effects, although there was a tendency toward prevention. The data suggest that ß-glucan has more than one action mechanism, being capable of exerting desmutagenic as well as bio-antimutagenic action. The findings also suggest that the presence of the xenobiotic metabolizing system can reduce the chemopreventive capacity of ß-glucan. Therefore, these results indicate that ß-glucan from Saccharomyces cerevisiae can be used in the prevention and/or reduction of DNA damage.