Metformin Induces a Caspase 3-Unrelated Apoptosis in Human Colorectal Cancer Cell Lines HCT116 and SW620.

Purpose: To study the effects of metformin on the proliferation and growth of human colorectal cancer cell lines HCT116 and SW620. Materials and Methods: The antiproliferative effect of metformin was assayed using an MTS reagent and its ability to inhibit colony formation was demonstrated using a clonogenic assay. Flow cytometry using YO-PRO-1/PI was performed to examine the effects of metformin on apoptosis and cell death of HCT116 and SW620. Caspase 3 activities were measured in caspase-3 activity tests using a caspase-3 activity kit. Furthermore, Western blots were performed with anti-PARP1, anti-caspase 3, and anti-cleaved caspase 3 to confirm whether caspase activation was present or not. Results: Both MTS proliferation assays and clonogenic assays showed that metformin inhibited the proliferation and growth of HCT116 and SW620 cells in a concentration-dependent manner. Flow cytometric analysis identified early apoptosis and metformin-induced cell death in both cell lines. However, caspase 3 activity could not be detected. Cleavage of both PARP1 and pro-caspase 3 was not observed in the Western blot, confirming the absence of caspase 3 activations. Conclusion: This present study suggests a caspase 3-unrelated apoptosis mechanism of metformin-induced cell death in human colorectal cancer cell lines HCT116 and SW620.

Clinicopathological associations and prognostic values of IDH1 gene mutation, MGMT gene promoter methylation, and PD-L1 expressions in high-grade glioma treated with standard treatment.

INTRODUCTION: the objective was to evaluate the impact of IDH1 R132H mutation, MGMT methylation and PD-L1 expression in high grade glioma that received standard therapy (surgery, radiation and chemotherapy) to overall survival (OS). METHODS: this is a retrospective study of 35 high grade glioma cases. Genotyping of IDH1 gene alteration on the mutation hotspot R132 (Sanger sequencing method with Applied Biosystems 3500 Genetic Analyzer), EZ DNA Methylation-Gold kit (Zymo Research) is used to study the methylation, Cell line BT549 (ATCC HTB-122) and HCT-116 (ATCC CCL-247) were used as unmethylated control and partially methylated control respectively. Anti-human PD-L1 antibody clone E1L3N®from Cell Signalling Technology (USA) and Rabbit XP®were used to see PDL-1 expression. RESULTS: anaplastic astrocytoma cases had more MGMT promoter methylation (50%) than glioblastoma multiforme (GBM) (20%), more IDH1 R132H mutation (42%) than GBM (4.3%). Immunohistochemistry tumor proportion score method (TPS) identified 17% and 8.7% were PD-L1 positive in AA and GBM groups, respectively. Cases with IDH1 R132H mutation and MGMT methylation still showed better OS although with high PD-L1 expression. CONCLUSION: IDH1 R132H mutation and MGMT methylation were good prognostic markers. High expression of PD-L1 apparently might not indicate poor overall survival in the presence of IDH1 R132 mutation and MGMT methylation.