POS0044 T315 SUPPRESSES OSTEOGENIC DIFFERENTIATION IN SAOS-2 CELLS BY INHIBITING PHOSPHORYLATION OF AKT

Background: New bone formation is common in the late stage of various inflammatory arthritis, while osteoblasts play a vital role this process. Activation PI3K/ Akt pathway promotes differentiation and enhances function [1]. T315 novel small molecule drug, which may induce apoptosis suppress expression cellular markers chronic lymphocytic leukemia cells by disrupting [2]. However, lack study focuses on influence T31T other cells, except tumor cell lines. Objectives: We aimed to assess effect human osteoblast-like Saos-2 its potential mechanism was evaluated as well. Methods: (1) stimulated with an osteogenic reagent contained L-ascorbic acid, β-glycerophosphoric dexamethasone. The concentration adjusted 0μg/ml, 1μg/ml, 2μg/ml culture medium. (2) Alizarin red stain alkaline phosphatase (ALP) were performed at d0, d7, d14, d21 after being treated T315. (3) Cellular protein extracted d3, d6 T315, then ALP activity tested based recommendation from manufacturer kit. (4) Collagen type 1α2 Chain (Col1α2) osteocalcin (OCN), two markers, measured through western blot, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) endogenous control. (5) Phospho-phosphoinositide 3-kinase (pPI3K), phospho-protein kinase B (pAkt), runt-related transcription factor 2 (Runx2) blot GAPDH or (Akt) chosen internal reference appropriate. (6) Analysis variance least significant difference used analyze data. A P <0.05 considered statistically significant. Results: higher related less relative mineralized area positive ALP, longer incubation time decreased these regions well (Figure 1A-C). reduced accordingly 1D). suppressed Col1α2 OCN dose-dependent time-dependent manner 1E, F). did not alter pPI3K, but it inhibited phosphorylation 1G, H). Runx2 because greater dose 1). Conclusion: inhibits inhibiting Akt. Surprisingly, pPI3K seldom changes process, so detail should be investigated further. References: [1]Exp Biol Med (Maywood) 2020;245(6):552-561. [2]Blood 2015;125(2):284-295. Figure 1. Effect PI3K/Akt pathway. (A) d21. (B) Relative stain. (C) Positive (D) activity. Western analysis bands for (E), (F), (G), pAkt (H), (I). Results normalized ALP: Alkaline phosphatase; Col1α2: Chain; GAPDH: Glyceraldehyde dehydrogenase; OCN: Osteocalcin; pPI3K: 3-kinase; pAkt: Phospho-protein B; Akt: Protein kinase; Runx2: Runt-related 2. Compared d3 same <0.05. b d0 c 1μg/ml time, d Disclosure Interests: None declared.