Metformin Effect on Endocan Biogenesis in Human Endothelial Cells Under Diabetic Condition.

BACKGROUND AND AIM: Endocan is a novel endothelium-derived proteoglycan and may play a role in endothelial cells activity under diabetic conditions. Here, we evaluated the effect of high glucose concentration (30 mmol glucose) on endocan level in presence or absence of metformin in human umbilical vein endothelial cells (HUVECs). METHODS: Cells were incubated with 30 mmol glucose for 72 h. High glucose content, metformin (2.5 to 500 mmol) and compound C (10 mmol) effects were assessed on cell viability. HUVECs migration was studied by scratch test. The changes in endocan expression and protein level were evaluated by RT-PCR, ELISA and flow cytometry assays. Griess reaction was used to measure NO levels. Functional activity of endothelial cells was monitored related to lipoprotein lipase activity using Dil-Ac-LDL uptake. p-AMPK/AMPK ratio was assessed by western blotting. RESULTS: Cells viability significantly was reduced under high glucose condition (p <0.05). 30 mmol glucose inhibited HUVECs migration, whereas these features were improved by 50 mmol metformin (p <0.05). Endocan transcription and protein levels were increased in diabetic HUVECs exposed to metformin (p <0.05). Metformin increased NO production in HUVECs under high glucose condition (p <0.001). Metformin increased LDL uptake capacity under high glucose condition (p <0.05). The addition of compound C blunted these effects. Western blot analysis confirmed the increase of p-AMPK/AMPK ratio in metformin-treated cells. CONCLUSION: Data demonstrated that metformin could promote angiogenic potential of endothelial cells which its reduction is a main cause in the development of diabetic foot ulcer, probably by the regulation of endocan dynamics under high glucose condition.

The Impact of Thymidylate Synthase and Methylenetetrahydrofolate Reductase Genotypes on Sensitivity to 5-Fluorouracil Treatment in Colorectal Cancer Cells.

5-fluorouracil (5-FU) is one of the major components of many standard regimens for chemotherapy of colorectal cancer (CRC) and some other malignancies. Given the known relationship between thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) activity and 5-FU metabolism, this study investigated the impact of selected functional polymorphisms of the TS and MTHFR genes on chemotherapy resistance in 5 human CRC cell lines. HCT116, SW1116, HT29/219, LS180, and Caco-2 CRC cells were cultured as monolayer and their chemosensitivity to 5-FU, oxaliplatin, and irinotecan was determined by MTT assay. Genomic DNA was extracted from the cultured cells, and a 6-bp insertion or deletion (6-bp ins/del) polymorphism in 3´-UTR of the TS gene was determined by the PCR-RFLP method. Genotyping of MTHFR 677 C/T and 1298A/C single nucleotide polymorphism (SNP) was also performed by MS-PCR and PCR-RFLP, respectively. Caco-2 with the homozygous TS 6-bp ins/ins and MTHFR 677 T/T and 1298 C/C genotype, was the most 5-FU resistant cell line. HCT116 with the homozygous TS 6-bp del/del and MTHFR 1298 A/A and heterozygous MTHFR 677 C/T genotype was the least 5-FU resistant cell. LS180, the second most 5-FU resistant cell line, was heterozygous for all three polymorphic sits. HT29/219 and SW1116 cells with homozygous TS 6-bp ins/ins and heterozygous MTHFR 677 C/T and 1298 A/C genotypes had intermediate 5-FU sensitivity. The results indicate that TS 3´-UTR 6-bp insertion and MTHFR 677T and 1298C alleles increase 5-FU resistance in CRC cells. No relationship was observed between TS and MTHFR genotypes and oxaliplatin or irinotecan sensitivity in these cells.