Tumstatin185～191对人肺腺癌细胞的抑制作用及其与蛋白激酶B和细胞外调节蛋白激酶活性的关系

目的 观察Tumstatin185～191对肺腺癌细胞增生与凋亡的影响,探讨其与蛋白激酶B(Akt)及细胞外调节蛋白激酶(ERK)活性的关系.方法 以人肺腺癌细胞株A549为研究对象,分别给予不同浓度的Tumstatin185～191、顺铂及Tumstatin185～191联合顺铂进行干预,以四唑盐比色法测定A549细胞的增殖情况;流式细胞仪检测分析A549细胞凋亡的变化;Western blot检测细胞内磷酸化的Akt(p-Akt)及磷酸化的ERK(p-ERK)蛋白表达水平.结果 Tumstatin185～191对A549细胞的半数抑制浓度(IC_(50))为73.7μmol/L,顺铂对A549细胞的IC_(50)为5.2μmol/L,当顺铂与20μmol/L或40μmol/L的Tumstatin185～191联合应用时,顺铂对A549细胞的IC_(50)分别下降为3.5 μmol/L和1.4μmol/L;两药联合使用时,A549细胞的早期凋亡率为(19.34±0.97)%,较顺铂(12.5±2.1)%]和Tumstatin185～191单用(9.6±1.6)%]的早期凋亡率显著增加(F=5.74,P<0.01);Western blot检测显示p-Akt及p-ERK在未经干预的A549细胞内均呈高表达状态,Tumstatin185～191可显著抑制p-Akt及p-ERK的表达,顺铂则无明显影响;Tumstatin185～191与顺铂联合使用并不增加Tumstatin185～191对p-Akt及p-ERK的抑制效应.结论 Tumstatin185～191单药或联合顺铂对于A549细胞均有显著抑制作用,Tumstatin185～191能够增加A549细胞对顺铂的敏感性;Tumstatin185～191可能通过下调A549细胞内p-Akt及p-ERK水平发挥其抑制细胞生长、促进细胞凋亡的作用.

The effects of Tumstatin185 -191 on lung adenocarcinoma cell lines and the association with protein kinase B and extracellularregulated protein kinase activation

Objective To investigate the antitumor effects of tumstatin 185-191 as a single agent or combination with cisplatin(DDP) on non-small lung cancer(NSCLC) cell lines A549. In addition, the changes of the protein kinase B(Akt) and extracelluarregulated protein kinase(ERK) in cultured NSCLC cells treated by tumstatin 185-191 and cisplatin were evaluated. Methods A549 cells were treated with tumstatin185 -191 and cisplatin. Cell viability was assessed using the modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was measured by flow cytometry. The activation of Akt and Erk were evaluated by Western blotting. Results Tumstatin185 - 191 inhibited the proliferation of A549 and the IC_(50) values of tumstatin185 - 191 was 73. 7 μmol/L. After cotreatment with 20 μmol/L tumstatin185 - 191, IC_(50) values of cisplatin in A549 cells reduced from 5.2 μmol/L to 3. 5 μmol/L, while 40 μmol/L tumstatin185 - 191 reduced from 5.2 μmol/L to 1.4 μmol/L. The early apoptesis rate was (19.34± 0.97)% in the cotreatment group, (12.5±2.1)% in cisplatin group and (9.6±1.6)% in tumstatin185 -191 group (F = 5.74, P <0.01). The levels of phospho-Akt (p-Akt) and phospho-ERK (p-ERK) in the A549 cells were remarkably lower after being treated with tumstatin 185-191, while tumstatin 185 - 191 treatment whether alone, or in combination with cisplatin, had the similar effects on the protein levels of p-Akt and p-ERK in A549 cells. Conclusion Our data suggest that tumstatin185 - 191 might enhance the sensitivity of A549 cells to cisplatin. The effects of promoting apoptosis and downregulation of proliferation induced by tumstatin185 - 191 may be mediated through inactivation of the Akt and ERK pathways.