组蛋白去乙酰化酶抑制剂DWP0016对肺癌A549细胞和小鼠Lewis肺癌的抑制作用

目的 探讨新型组蛋白去乙酰化酶(HDAC)抑制剂DWP0016对肺癌的抑制作用及可能机制。方法 1 体外实验:DWP0016 0.625~10 μmol·L^-1作用A549细胞48 h,MTT法检测细胞存活,实时荧光定量PCR检测10号染色体同源丢失性磷酸酶张力蛋白(PTEN)mRNA水平;Western蛋白质印迹法测定总组蛋白H3, 乙酰化H3(Ac-H3), 细胞周期调控因子p21,PTEN,总Akt和磷酸化Akt(p-Akt)的表达。2 在体实验:小鼠腋下接种0.2 ml Lewis肿瘤细胞悬液制备肺癌模型,7 d后按照分组分别ip给予DWP0016 12.5, 25和50 mg·kg^-1及伏林司他(SAHA)50 mg·kg^-1,每天1次,连续8 d。取肿瘤组织称重,计算肿瘤抑制率;免疫组化法检测肿瘤组织中Ac-H3和PTEN的表达。结果 1 体外实验:MTT结果显示,DWP0016抑制A549细胞增殖的IC₅₀为 2.51 μmol·L^-1,SAHA IC₅₀为6.03 μmol·L^-1。与正常对照组相比, DWP0016 2.5 μmol·L^-1组细胞组蛋白H3乙酰化及p21蛋白表达显著上升(P<0.05),PTEN的转录水平和蛋白水平上调, Akt的磷酸化水平下调(P<0.05)。2 在体实验:与模型组比较,给予DWP0016 12.5 mg·kg^-1移植瘤小鼠的瘤质量显著降低(P<0.05),抑瘤率达42.0%,给予DWP0016 50 mg·kg^-1肿瘤抑制率高于SAHA 50 mg·kg^-1(P<0.05);与模型组比较,肿瘤组织中Ac-H3和PTEN的表达增加。结论 DWP0016能明显抑制A549细胞增殖和Lewis肺癌模型小鼠的肿瘤生长,其机制与诱导Ac-H3的表达、激活p21、促进抑癌因子PTEN的转录及下调Akt的磷酸化水平有关。

Antitumor activity of histone deacetylases inhibitor, DWP0016, in lung carcinoma A549 cells and xenograft mouse model

OBJECTIVE To study the effect and the underlying molecular mechanisms of a novel histone deacetylases (HDAC) inhibitor named DWP0016 in lung carcinoma A549 cells and xenografts. METHODS 1 In vitro: A549 cells were treated with DWP0016 0.625-10 μmol·L^-1 for 48 h before cell viability was detected by MTT assay. The effect of DWP0016 on mRNA level of phosphatase and tensin homolog deleted on chromosome 10(PTEN) was detected by real-time PCR. The protein expression of total H3, acetylated-H3(Ac-H3), p21, PTEN, total-Akt and phosphorylated-Akt(p-Akt) was detected by Western blotting. 2 In vivo: Lewis lung cancer animal models were constructed by injecting the axillae with 0.2 ml tumor suspension. After 7 d, the xenografts groups were ip given DWP0016 12.5, 25 and 50 mg·kg^-1 and positive drug vorinostat(SAHA) 50 mg·kg^-1, once daily, for 8 d. After treatment, the animals were sacrificed to get the tumors. The tumor specimens were weighed and the tumor growth inhibition was calculated. The expression of Ac-H3 and PTEN in the specimen was detected by immunohistochemical assay. RESULTS 1 In vitro: MTT results showed that IC₅₀ of DWP0016 was 2.51 μmol·L^-1 and IC₅₀ of SAHA was 6.03 μmol·L^-1. Compared with normal control, DWP0016 significantly up-regulated the protein expression of Ac-H3 and p21 at the concentration of 2.5 μmol·L^-1(P< 0.05). DWP0016 promoted the mRNA level and protein expression of PTEN while decreasing the expression of p-Akt concentration-dependently(P<0.05). 2 In vivo: compared with model group, the mean mass of the specimens from DWP0016 12.5 mg·kg^-1 significantly decreased(P<0.05) with a 42.0% tumor growth inhibition. The tumor growth inhibition in DWP0016 50 mg·kg^-1 group was higher than in SAHA 50 mg·kg^-1 group(P<0.05). Compared with the model group, the expression of Ac-H3 and PTEN in tumor specimens was up-regulated by DWP0016. CONCLUSION DWP0016 effectively inhibits the proliferation in A549 cells and the tumor growth in xenografts. The molecular mechanisms are associated with the induction of Ac-H3,the activation of p21, PTEN and down-regulation of p-Akt. DWP0016 is a potent compound to be developed as an anti-tumor agent for clinic application in the future.