RESUMO
Introduction: Microsatellite instability (MSI) is a hallmark of defective DNA mismatch repair (MMR) of genes especially MLH1 and MSH2. It is frequently involved in the carcinogenesis of various tumours including gastric cancer (GC). However, MSI in GCs have not been reported in Malaysia before. Objective: This study was conducted to determine the microsatellite instability (MSI) status in gastric cancer by microsatellite analysis, sequencing, its association with MLH1 and MSH2 protein expression and H.pylori infection by immunohistochemistry. Method: A total of 60 gastric cancer cases were retrieved. DNA was extracted from paired normal and tumour tissues while MLH1 and MSH2 protein expression as well as H. pylori status were determined by IHC staining. For microsatellite analysis, polymerase chain reaction (PCR) was performed for paired tissue samples using a panel of five microsatellite markers. MSI-positive results were subjected for DNA sequencing to assess mutations in the MLH1 and MSH2 genes. Results: Microsatellite analysis identified ten MSI positive cases (16.7%), out of which only six cases (10.3%) showed absence of MLH1 (n=3) or MSH2 (n=3) protein expression by IHC. The most frequent microsatellite marker in MSI positive cases was BAT26 (90%). Nine of ten MSI positive cases were intestinal type with one diffuse and all were located distally. H. pylori infection was detected in 13 of 60 cases (21.7%) including in three MSI positive cases. All these results however were not statistically significant. Our sequencing data displayed novel mutations. However these data were not statistically correlated with expression levels of MLH1 and MSH2 proteins by IHC. This may be due to small sample size to detect small or moderately sized effects. Conclusion: The frequency of MSI in this study was comparable with published results. Determination of affected MMR genes by more than two antibodies may increase the sensitivity of IHC to that of MSI analysis.
Assuntos
Biomarcadores Tumorais/metabolismo , Mutação em Linhagem Germinativa , Instabilidade de Microssatélites , Proteína 1 Homóloga a MutL/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Prognóstico , Estudos Retrospectivos , Neoplasias Gástricas/patologia , Taxa de SobrevidaRESUMO
Objective: This study was conducted to investigate the antiproliferative activity of extracts of Clinacanthus nutans leaves against human cervical cancer (HeLa) cells. Methods: C. nutans leaves were subjected to extraction using 80% methanol or water. The methanol extract was further extracted to obtain hexane, dichloromethane (DCM), and aqueous fractions. The antiproliferative activity of the extracts against HeLa cells was determined. The most cytotoxic extract was furthered analyzed by apoptosis and cell cycle assays, and the phytochemical constituents were screened by gas chromatography-mass spectrometry (GC-MS). Results: All of the extracts were antiproliferative against HeLa cells, and the DCM fraction had the lowest IC50 value of 70 µg/mL at 48 h. Microscopic studies showed that HeLa cells exposed to the DCM fraction exhibited marked morphological features of apoptosis. The flow cytometry study also confirmed that the DCM fraction induced apoptosis in HeLa cells, with cell cycle arrest at the S phase. GC-MS analysis revealed the presence of at least 28 compounds in the DCM fraction, most of which were fatty acids. Conclusion: The DCM fraction obtained using the extraction method described herein had a lower IC50 value than those reported in previous studies that characterized the anticancer activity of C. nutans against HeLa cells.