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.

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.

Background Medicinal herbs in Malaysia like Clinacanthus nutans (CN) traditionally are used in the treatment of various diseases and cancers. The present research was conducted to determine the effects of C. nutans leaf different solvent extracts on the human breast cancer cell lines (MCF-7). The antiproliferative growth and survival effects of dichloromethane CN leaf extracts (CNDCM), as well as the short- and long-term effects through metastasis, apoptosis and cell cycle effects, were observed. The chemical profiles were done to evaluate the properties of the CNDCM. Results The evaluation of GC–MS identified 16 major phytochemical compounds present in this extract with biological activities. Antiproliferative assay used is the SRB assay, which showed the CNDCM induced strong antiproliferative property compared with the other extracts, so its IC50 dose was selected for further testing with value 108 µg/mL at 72 h after exposure on MCF-7 and MCF-10A cell lines. The clonogenic survival effects of CNDCM in various concentrations (31.25, 62.5, 125, 250 and 500 µg/mL) inhibited the ability of MCF-7 cells to form colonies, and the metastasis result was indicated in an image of wound healing assay. Moreover, the CNDCM extract significantly induced apoptosis in all the cell cycle phases. Finally, the experiments with various extract concentrations on normal human breast cell lines showed no antiproliferative effects for all the extracts tested. Conclusion Among all the extracts of CN, the CNDCM extracts demonstrated the highest antiproliferative activity and survival against the MCF-7 cell lines tested.

Background: Recent years have witnessed major development of novel therapeutic agents like chemotherapy, targeted therapy and immune checkpoint inhibitors for cervical cancer. However, cervical cancer remains prevalent, leading to a large number of deaths worldwide. A better understanding of the cervical cancer biology and signaling pathways might lead to the development of targeted therapies in reducing the incidence and mortality rate. Methods: In this study, the RNA-Seq reads of HeLa cells treated with C. nutans were compared to the untreated sample. The reads of these two sample groups were firstly aligned to the human reference genome. The results in BAM files format that were generated were then sorted before being assembled. The output of assembly which was in coverage table form was ready for downstream statistical analyses for differential expression. Differentially expressed genes were obtained and the cell-death related pathway were identified by canonical pathway, QIAGEN Ingenuity Pathway Analysis (IPA). The verification of significant genes was carried out using qRT-PCR by including GAPDH as a housekeeping gene Results: With this, we identified a total of 668 upregulated and 479 downregulated analysis-ready genes across observations upon cut-off setting log2FoldChange at 0.5 and P-value 0.05. A total of 28 cell-death related canonical pathways and 4 activation of cell-death related functions were identified. Upon analyses, we identified four significant genes (Casp9, KAI1, REL and FOXO4) that hold important role in promoting cell death. These findings were also verified against the quantification using qRT-PCR by including GAPDH as a housekeeping gene. Conclusions: This study provides an insight on the potential role of DCM fraction of C. nutans in activating Casp9, KAI1, REL and FOXO4 genes in mediating apoptosis in cervical cancer cells.