RESUMO
BACKGROUND: The present study aimed to elucidate the potential anticancer activity and mechanism of P. harmala's alkaloid extract, harmine (HAR), and harmaline (HAL) in HCT-116 colorectal cancer cells. METHODS AND RESULTS: P. harmala's alkaloid was extracted from harmala seeds. HCT-116 cells were treated with P. harmala's alkaloid extract, HAR and HAL. Cytotoxicity was determined by MTT assay, apoptotic activity detected via flow cytometry and acridine orange (AO)/ethidium bromide (EB) dual staining, and cell cycle distribution analyzed with flow cytometry. The mRNA expression of Bcl-2-associated X protein (Bax) and glycogen synthase kinase-3 beta (GSK3ß) was measured by real-time PCR. Furthermore, the expression of Bax, Bcl-2, GSK3ß and p53 proteins, were determined by western blotting. The findings indicated that, P. harmala's alkaloids extract, HAR and HAL were significantly cytotoxic toward HCT116 cells after 24 and 48 h of treatment. We showed that P. harmala's alkaloid extract induce apoptosis and cell cycle arrest at G2 phase in the HCT116 cell line. Downregulation of GSK3ß and Bcl-2 and upregulation of Bax and p53 were observed. CONCLUSION: The findings of this study indicate that the P. harmala's alkaloid extract has anticancer activity and may be further investigated to develop future anticancer chemotherapeutic agents.
Assuntos
Apoptose , Neoplasias do Colo , Glicogênio Sintase Quinase 3 beta , Harmina , Peganum , Sementes , Humanos , Peganum/química , Células HCT116 , Apoptose/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Sementes/química , Harmina/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Alcaloides/farmacologia , Harmalina/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proliferação de Células/efeitos dos fármacosRESUMO
BACKGROUND: Mutations and polymorphisms of the GSK3ß gene have been associated with several diseases including Alzheimer disease, diabetes and cancer; however, to date, no variants of this gene have been associated with colorectal cancer (CRC). This study aims to explore, for the first time, the association of the GSK3ß rs334558 and rs6438552 polymorphisms with CRC. METHODS: Genomic DNA from 330 CRC patients and healthy blood donors were analyzed. Identification of polymorphisms was made by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. Association was calculated by the odds ratio (OR) test. RESULTS: Patients carrying the C/T genotype for the rs334558 (T>C) polymorphism showed an increased risk for CRC (OR = 1.71, 95% CI: 1.05-2.79, P = 0.039); this association was also observed for TNM stage and tumor location. For the rs6438552 (T>C) polymorphism, the OR analysis showed that patients carrying C/T and C/C genotypes have a decreased risk for CRC (OR = 0.44, 95% CI: 0.27-0.70, P = 0.001 and OR = 0.24, 95% CI: 0.10-0.64, P = 0.001, respectively); this decreased risk was also evident in the stratified analysis by TNM stage and tumor location. Haplotype analysis of these 2 loci of GSK3ß (rs334558 and rs6438552) showed differential distribution. The T-T and C-C haplotype was associated with a decreased risk of CRC, while the T-C haplotype was associated with an increased risk of CRC. CONCLUSION: Our results denote that GSK3ß gene polymorphisms play a significant role in promoting or preventing CRC. Additionally, variations in this gene are associated with the tumor site and the tumor-node-metastasis (TNM) stage in these patients.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Glicogênio Sintase Quinase 3 beta/genética , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Genótipo , Haplótipos , Humanos , Irã (Geográfico) , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: The GSK3ß has been associated to pathological functions in neurodegenerative diseases. This kinase is involved in hyperphosphorylation of microtubule-associated tau protein, leading to aggregation andformation of NFTs. It has clearly been shown that GSK3ß is regulated at posttranslational level: phosphorylation at Tyr216 activates kinase, while phosphorylation at Ser9 is essential to inhibit its activity. OBJECTIVES: At present, there are contradictory findings about the possibility that GSK3ß may be regulated at gene level. Previous data showed overexpression of GSK3ß mRNA in hypomethylating conditions, pointing out to the existence of epigenetic mechanisms responsible for GSK3ß gene regulation. Analysis of human GSK3ß promoter through bisulphite modification, both in neuroblastoma cells and in postmortem frontal cortex from AD patients (AD patients both at Braak stages I-II and at stages V-VI) , allowed us to characterize the methylation pattern of a putative CpG islands in human GSK3ß 5'- flanking region. RESULTS: The analysis evidenced overall hypomethylation of CpG and non-CpG cytosine residues both in cells and in human brain (AD patients and control subjects). We found that GSK3ß mRNA was overexpressed only in patients with initial AD, with no effect on the levels of the protein. On the other hand, we unexpectedly observed the decrease of the inactive GSK3ß in cortex from AD patients at Braak stages I-II, whereas considerable increase was observed in AD patients at stages V-VI compared to the control subjects. CONCLUSIONS: These results point out that GSK3ß hyperactivity, and then NFTs formation, could come into function at an early stage of the disease and then turn off at the last stages.