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目的:观察PI3K/Akt/mTOR通路抑制剂wortmannin或rapamycin对白血病细胞株增殖及其PHLPP(PH domainleucine-rich repeat protein phosphatase)蛋白表达的影响。方法:以不同浓度的wortmannin或rapamycin分别作用于人类髓细胞白血病细胞系K562、HL-60,采用WST-1法检测细胞的增殖活性,AnnexinⅤ-FITC双染流式细胞术检测细胞凋亡,Westernblotting法检测细胞中p-Akt、Akt、PHLPP蛋白的表达。结果:Wortmannin以时间以及剂量依赖方式抑制K562、HL-60细胞的增殖(P<0.05),48 h的IC50值分别为(187.6±48.4)、(185.5±48.1)nmol/L。100 nmol/L wortmannin作用于K562细胞、50nmol/L wortmannin作用于HL-60细胞12和24 h后,细胞凋亡率均较对照细胞显著升高[(12.4±0.7)%、(17.6±2.3)%vs(5.0±0.6)%,P<0.05;(11.0±0.2)%、(17.9±1.6)%vs(6.8±0.4)%,P<0.05]。Wortmannin分别作用于K562、HL-60细胞12、24、36 h后,p-Akt、PHLPP的蛋白表达水平明显降低;rapamycin同样可使K562、HL-60细胞中PHLPP蛋白的表达水平降低。结论:PI3K/Akt/mTOR信号通路抑制剂抑制白血病细胞株增殖的同时降低其PHLPP蛋白的表达。
OBJECTIVE: To investigate the effects of wortmannin or rapamycin, a PI3K / Akt / mTOR pathway inhibitor, on the proliferation of leukemic cell lines and the expression of PH domain peptide binding protein (PHLPP). METHODS: Human myeloid leukemia cell lines K562 and HL-60 were treated with different concentrations of wortmannin or rapamycin, respectively. Cell proliferation was detected by WST-1 assay. Cell apoptosis was detected by flow cytometry with AnnexinⅤ-FITC. Western blotting Method to detect the expression of p-Akt, Akt and PHLPP in cells. Results: Wortmannin inhibited the proliferation of K562 and HL-60 cells in a time and dose-dependent manner (P <0.05). IC50 values at 48 h were (187.6 ± 48.4) and (185.5 ± 48.1) nmol / L, respectively. The apoptosis rate of HL-60 cells treated with 50 nmol / L wortmannin was significantly higher than that of control cells at 12 and 24 h [(12.4 ± 0.7)% vs (17.6 ± 2.3) % vs (5.0 ± 0.6)%, P <0.05; (11.0 ± 0.2)%, (17.9 ± 1.6)% vs (6.8 ± 0.4)%, P <0.05]. The protein expression of p-Akt and PHLPP in K562 and HL-60 cells was significantly decreased after Wortmannin treatment for 12, 24 and 36 h, respectively. However, rapamycin also decreased the expression of PHLPP protein in K562 and HL-60 cells. Conclusion: Inhibitors of PI3K / Akt / mTOR signaling pathway inhibit the proliferation of leukemia cell lines while decreasing the expression of PHLPP protein.