| Objective: To observe the role of BRD4 and PI3K-AKT double inhibitor SF2523 in vitro and in vivo anti-renal cell carcinoma(RCC)cells and to analyze the molecular mechanism of its role in RCC's molecular targeted therapy to provide a useful exploration.Methods:1.The human RCC cell lines(786-O and A489),the two lines of primary human RCC cells(RCC-1,RCC-2)were treated with CCK-8 colorimetry,Trypan blue staining,cell colony test,Brdu enzyme-linked immunosorbent assay(ELISA)and other methods were used to detect the survival and cell proliferation of RCC cells at 48 h and 72 h after treatment with different doses of SF2523.At the same time,compared with the control group,SF2523 was used to analyze the survival and cell proliferation influences.The effect of SF2523 on the survival and proliferation of HK-2 renal tubular epithelial cell line and primary cultured renal epithelial cell(Renal Epi)was observed by using Brdu enzyme-linked immunosorbent assay(ELISA)and CCK-8 method.2.The human RCC cell lines(786-O and A489),the two lines of primary human RCC cells(RCC-1,RCC-2)were used to detect the activity of Caspase-3/9,and the expression of Annexin V was detected by spectrophotometry.The expression of Caspase-3/9 was detected by TUNEL nuclear staining.(FACS)and single chain-DNA(ssDNA)ELISA were used to detect the apoptosis of RCC cells treated with SF2523,and the effect of SF2523 on RCC cell apoptosis was analyzed.3.The cell cycle of the human RCC cell line 786-O and the primary human RCC-1 was further treated with SF2523 for 24 h,and the cell cycle progression was measured by pyridine-FACS method.The results were quantitatively analyzed.The effect of SF2523 on cell cycle of human RCC was observed.4.The human RCC cell line 786-O cell line was treated with 1.0 ?M SF2523 for 24 hours.Transwell method and cell orbital tracing experiment were used to observe the migration and invasion ability of the human RCC cell line.Compared with the control group,the effect of SF2523 on the migration ability of RCC cells was analyzed.5.The expression of P-P85,P85,P-AKT S473,P-AKT T308,AKT1/2,PI3K-AKT-mTOR signaling pathways were observed by Western blotting after 7825-O cells treated with SF2523.P-S6K1,P-S6 and so on.The expression of BRD4 regulatory protein Bcl-2 and Myc was observed and the effect of SF2523 on PI3K-AKT-mTOR and BRD4 signaling pathway in human RCC cells was analyzed.The expression of p-AKT S473,p-AKT T308,AKT1 / 2,p-S6K1,p-S6 and Bcl-2 and Myc of the primary cultured renal epithelial cells(Renal Epi)after SF2523 treatment was observed.The effect of SF2523 on renal epithelial cells was analyzed.The effects of SF2523 and JQ1(BRD4 inhibitor)and Wortmannin(PI3K inhibitor)on human RCC 786-O cells were compared.6.Establishment of SCID murine tumor model,female SCID mice injected 786-O RCC cells into the abdomen of the left flank to establish a tumor xenograft model.The tumor-bearing SCID mice were randomly divided into three groups and given solvent control or SF2523(15/50 mg / kg body weight)respectively.The mice were injected intraperitoneally and treated every other day for 15 consecutive days.The tumor growth was observed daily for 6 Daily record of tumor volume and body weight of mice.At the end of the experiment("Day-36"),tumors from each group were isolated and weighed.Tumor bearing SCID mice were randomly divided into 2 groups(n = 3 in each group),and were given solvent control and SF2523(50 mg / kg)respectively.The mice were injected intraperitoneally and treated every other day.Two groups of three 786-O xenografts("# 1/2/3")were each isolated on day 6 after the first dose.The tumor tissue was lysed,and the expression of p-AKT Ser-473,Myc and Bcl-2 was observed by Western Blotting assay.The mechanism of SF2523 in anti-RCC cells was analyzed.Results:1.SF2523 significantly inhibited the survival and proliferation of human RCC cells(786-O and A489)and primary human RCC cell(RCC-1,RCC-2).SF2523 had no significant toxic effect on normal human renal epithelial cells of HK-2 renal tubular epithelial cell line and primary cultured renal epithelial cells(Renal Epi).2.SF2523 induced apoptosis of human RCC cells.SF2523 treated human RCC cells significantly increased the content of ss DNA,increased the activity of Caspase-3 and Caspase-9,and significantly increased the number of Annexin V and TUNEL positive cells.3.SF2523 can lead to RCC cell cycle arrest,G2-M cycle block.The percentage of G1 phase in RCC cells treated with SF2523 was significantly decreased,and the percentage of cells in the G2 and S phases increased.4.SF2523 inhibits RCC cell migration.SF2523 treatment significantly inhibited the number of "migrated" 786-O cells(at the bottom of Transwell),and the results of cell orbital tracing showed significant inhibition of 786-O cell invasion,infiltration,and quantitative analysis of 10 cell orbital tracer experiments Found that the role of SF2523 is very significant.5.SF2523 also blocked PI3K-AKT-mTOR in RCC cells and down-regulated the expression of BRD4 regulatory protein Bcl-2 and Myc.The expression of p-p85,P-AKT S473,P-AKT T308,p-S6K1 and p-S6 in 786-O cells was significantly inhibited,while the expression of p85,AKT,S6K1 and S6 did not change.BRD4 regulatory protein(Bcl-2 and Myc)were also significantly down-regulated.SF2523 inhibits RCC cell survival and induces apoptosis more effectively than JQ1(BRD4 inhibitor)and Wortmannin(a PI3 K inhibitor).The levels of phosphorylated p-p85,p-AKT1,p-S6 and p-S6K1 and Bcl-2/Myc in renal epithelial cells were very low,and the inhibitory effect of SF2523 was not obvious.Bcl-2/Myc expression was downregulated without significant RCC cells.6.SF2523 could inhibit the growth of 786-O tumor in SCID mice,and the PI3K-AKT and Bcl-2/Myc expression were down-regulated.SF2523 was significantly lighter in the SF2523 treatment group than in the control control tumor.SF2523 showed a dose-dependent response to 786-O tumor growth in vivo,and 50 mg/kg SF2523 was significantly better than the 15 mg/kg of the dose.The expression of AKT(p-AKT Ser-473)and Myc and Bcl-2 were significantly decreased in SF2523 treated tumors.Conclusions:1.SF2523 can significantly inhibit the survival and proliferation of human RCC cells,can induce apoptosis,and lead to cell cycle arrest,inhibition of RCC cell migration.SF2523 had no significant toxic effect on human normal renal epithelial cells.2.SF2523 has the effect of simultaneously blocking PI3K-AKT-mTOR in RCC cells and down-regulating the expression of BRD4 regulatory protein Bcl-2 and Myc,which is superior to single channel inhibitor in RCC,and more effectively induces RCC cell death and apoptosis Death.3.SF2523 has significant in vitro and in vivo anti-RCC cell action,its mechanism may be related to its inhibition of PI3K-AKT-mTOR and BRD4 signal,is an effective double inhibitor,may become a better choice for the treatment of RCC. |
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