RE-EVALUATION OF LPS CONCENTRATIONS ON THE 293T HUMAN RENAL CELL LINE

Objective: The inflammatory responsiveness of the cells to Lipopolysaccharides (LPS) is commonly used for in vitro experiments. However, the correct dose of the LPS for cell line experiments is elusive. The LPS concentration that gives the maximal response is a critical point of in vitro inflammatory experiments. The aim of this study was to reevaluate the concentration dependent effect of LPS on the 293T human renal cell line.Methods: We evaluated the cell-detachment assay of LPS-stimulated 293T cell line monitored by xCELLigence Real-Time Cell Analyzer (RTCA) system. We applied increasing concentration of LPS followed by Roche xCELLigence Instrument based on the Real-Time Cell Analysis System.Results: Our results demonstrated that the 2, 4 and 8μg of LPS inhibit cell division which diverts cells to a steady-state phase, 1 μg acts as mitogen. Lower concentrations are no effect on cells.Conclusion: These work showed that LPS concentrations had various effects on cell proliferation and need to be estimated for each experiment before carry out the experiments

Impact of calcitriol and an AKT inhibitor, AT7867, on survival of rat C6 glioma cells

Glioma is the most prevalent and lethal type of primary tumor of central nervous system. The regulatory role of calcitriol, the active form of vitamin D3, has been determined in various cellular processes including cell survival and apoptosis. To study the impact of AKT-pathway inhibition with or without calcitriol combination on glioma cell viability, the effects of AT7867 (AKT-pathway inhibitor) and calcitriol on cell viability and apoptosis were investigated in glioma C6 cell-line. Optimal doses of calcitriol and AT7867 were determined by MTT- and xCELLigence-assays. Both agents (alone/in combination) effectively suppressed the proliferation of C6 cells. While AT7867 inhibited glioma cell viability more effectively than calcitriol, this inhibitory action of AT7867 was not significantly increased by calcitriol combination. The expression levels of vitamin D receptor(VDR)-triggered molecules, AKT-pathway components and apoptosis factors were compared between calcitriol-, AT7867- and calcitriol + AT7867-treated and non-treated cells. CYP24A1 gene was upregulated and DBP, CYP1A1, CYP27B1, EGFRvIII, AKT-pathway (AKT1, MTOR, CREB, PTEN), apoptosis (BAD, CYC-C, CASP3, APAF-1, BCL2) and MMP3 genes were downregulated by calcitriol treatment.AT7867 treatment induced CYP27B1 upregulation, reduced VDR and AKT-pathway (PI3K, MTOR, CREB, PTEN) gene expression levels but showed a relatively less pronounced effect on apoptosis-related gene levels. The combination of calcitriol and AT7867 treatment generated an effect that was similar to that induced by calcitriol alone. Our results demonstrate that calcitriol and AT7867 have differing actions on AKT- and apoptotic-pathways of C6 glioma cell-line. Calcitriol has a lesser viability reducing impact than AT7867 putatively due to its apoptosis inhibiting action

Evaluation of Melatonin and Vitamin D’s Effects on Zoledronic Acid Treated Osteoblasts’ Vitality, in Vitro

Objective: In this research, it was aimed to evaluate the effects ofmelatonin and vitamin D on zoledronic acid treated human osteoblasts’ vitality.Our main purpose in this study is try to find an answer to “Can supplement usagesuch as melatonin and vitamin D reduce the severity of MRONJ?”Methods: Human osteoblasts incubated various zoledronic acidconcentrations (10 and 50 μM). Two different concentrations of vitamin D (10and 100 μM) and melatonin (40 μM) were added in several combinations for 24thand 48th hour and 36 groups were created. After that cells are collected and theeffects are tested with the use of the proliferation assay (MTT). Data werecollected and analyzed statistically.Results: There were statistically significant differences between 24th and48th hour’s results.Conclusion: Generally, after 24th hour melatonin and vitamin D additionto zoledronic acid treated human osteoblast can contribute to reset negative effectsof zoledronic acid. Further investigations are needed to suggest melatonin andvitamin d supplements to MRONJ patients both in vivo and in vitro