[SPARC Overexpression Enhances the Sensitivity of SKM-1 Cells to Ara-C by Regulating CPBP/MLKL].

OBJECTIVE: To investigate the effect of SPARC gene overexpression on the chemotherapeutic sensitivity of AML-MDS cell line SKM-1 to Ara-C and to further explore its mechanism. METHODS: Subjects were divided into 6 groups: SKM-1 cells (Control), Negative control (LV-NC), SPARC overexpression (LV-SPARC), SKM-1 cells+30 ng/ml Ara-C (30 ng/ml Ara-C), LV-NC+30 ng/ml Ara-C and LV-SPARC+30 ng/ml Ara-C. Cell activity was detected by CCK-8 assay, cell cycle distribution and apoptosis were detected by flow cytometry, mRNA expression levels of SPARC, CPBP and MLKL were detected by RT-qPCR, and the expression levels of related protein were detected by Western blot. RESULTS: After co-treatment with SPARC overexpression and Ara-C, the cell viability decreased and apoptosis increased significantly, with obvious up-regulation of Bax and down-regulation of BCL-2 (P<0.05). Compared with the control group, the cell cycle of LV-SPARC+30 ng/ml Ara-C group was significantly arrested in S phase with obvious down-regulation of CDK2 and up-regulation of p27KIP1 (P<0.05). Compared with LV-SPARC group and 30 ng/ml Ara-C group, the mRNA and protein expression levels of CPBP and MLKL (p-MLKL) were significantly elevated in LV-SPARC+30 ng/ml Ara-C group (P<0.05). In addition, after co-treatment with SPARC overexpression and Ara-C, the protein expression level of p-AKT decreased and the protein expression level of p53 increased (P<0.05). CONCLUSION: SPARC overexpression enhanced the sensitivity of SKM-1 cells to Ara-C and promoted cell cycle arrest and apoptosis, the mechanism of which may be related to the regulation of CPBP/MLKL pathway.

[Inhibitory Effects of Metformin on Proliferation of SKM-1 Cells and The Related Underlying Mechanisms].

OBJECTIVE: To investigate the effects of metformin on the proliferation of AML-MDS cells (SKM-1 cells) and its related mechanisms. METHODS: CCK-8 was used to test the cell proliferation; Flow cytometry was used to detect the cell apoptosis and cell cycle; Western blot was used to test the expression level of AMPK and cell cycle regulatory proteins. RESULTS: Metformin could inhibit the proliferation of SKM-1 cells, which may be attributed to metformin-induced cell cycle arrest in G0/G1 but not to metformin induced cell apoptosis. The expression levels of G1-related protein CyclinD1 and CDK4 were down-regulated, while the expression levels of P53, P21CIP1 and P27kIP1 were up-regulated. Moreover, the phosphorylation level of AMPK was up-regulated. CONCLUSION: Metformin inhibits the proliferation of SKM-1 cells, which may relate with AMPK-induced cell cycle arrest. However, future studies are necessary to further explore the related mechanisms.

Senescent Cell-Secreted Netrin-1 Modulates Aging-Related Disorders by Recruiting Sympathetic Fibers.

Cellular senescence is implicated in several lines of aging-related disorders. However, the potential molecular mechanisms by which cellular senescence modulates age-related pathologies remain largely unexplored. Herein, we report that the density of sympathetic fibers (SFs) is significantly elevated in naturally aged mouse tissues and human colon adenoma tissues compared to the SFs densities in the corresponding young mouse tissues and human non-lesion colon tissues. A dorsal root ganglion (DRG)-human diploid fibroblast coculture assay revealed that senescent cells promote the outgrowth of SFs, indicating that the senescent cells induce recruitment of SFs in vitro. Additionally, subcutaneous transplantation of 2BS fibroblasts in nude mice shows that transplanted senescent 2BS fibroblasts promote SFs infiltration. Intra-articular senolytic molecular injection can reduce SFs density and inhibit SFs infiltration caused by senescent cells in osteoarthritis (OA), suggesting senescent cells promote the infiltration of SFs in vivo in aged tissues. Notably, the elevated level of SFs contributes to impaired cognitive function in naturally aged mice, which can be reversed by treatment with propranolol hydrochloride, a non-selective ß receptor blocker that inhibits sympathetic nerve activity (SNA) by blocking non-selective ß receptors. Additionally, 6-hydroxydopamine (6-OHDA)-induced sympathectomy improved hepatic sympathetic overactivity mediated hepatic steatosis in high fat diet (HFD)-fed APOE knockout mice (APOE-/- mice) by reducing hepatic SNA. Taken together, this study concludes that senescent cell-secreted netrin-1 mediated SFs outgrowth and infiltration, which contributes to aging-related disorders, suggesting that clearing senescent cells or inhibiting SNA is a promising therapeutic strategy for improving sympathetic nervous system (SNS) hyperactivity-induced aging-related pathologies.

[Effect of vitamine A on mice immune response induced by specific periodontal pathogenic bacteria-immunization].

PURPOSE: The aim of this study was to investigate the effect of vitamine-A deficiency on the induction of specific periodontal pathogenic bacteria A. actinomycetetemcomitans(Aa) immunization. METHODS: BALB/c mice were fed with vitamine A-depleted diet or control regular diet throughout the whole experiment period. After 2 weeks, immunized formalin-killed Aa to build immunized models, 6 weeks later, sacrificed to determine specific antibody-IgG, IgM and sub-class IgG antibody titers in serum, and concentration of IL-10, IFN-γ, TNF-α and RANKL in T cell supernatant were measured by ELISA and T cell proliferation was measured by cintilography. SPSS 11.5 software package was used for statistical analysis. RESULTS: The levels of whole IgG and IgM antibody which were immunized by Aa significantly elevated, non-immune group was unable to produce any antibody. Compared with Aa immunized+RD group, the level of whole IgG in Aa immunized+VAD group was significantly higher (P<0.05); The levels of IgG2a increased obviously, whereas the levels of IgG1 subtype antibody conspicuous decreased, with a significant difference (P<0.05). Aa immunized group could induce body to produce a strong specific T-cell immune response, but Aa immunized+VAD group had a higher T cell proliferate response compared with Aa immunized+RD group, with a statistically significant difference (P<0.05); The expression of RANKL, IFN-γ and TNF-α supernatant increased, while the expression of IL-10 decreased (P<0.05). CONCLUSIONS: The lack of vitamin-A diet can increase the immunized mice's susceptibility to periodontal pathogenic bacteria and trigger or aggravate immune inflammatory response. Adequate vitamin A is an important factor in maintaining body health. Supported by Natural Science Foundation of Liaoning Province (Grant No.20092139) and Science and Technology Program of Shenyang Municipality (Grant No.F10-149-9-32).