N-甲基环丙沙星氟喹诺酮查尔酮类衍生物对人胰腺癌BxPC-3细胞凋亡和自噬的影响

Effect of N-methylciprofloxacin-based fluoroquinolone-chalcone derivative on apoptosis and autophagy of human pancreatic carcinoma Bx PC-3 cells

目的研究N-甲基环丙沙星氟喹诺酮查尔酮类衍生物1环丙基-6-氟-7-(4-甲基-哌嗪-1-基)-3-[2-(3,4,5-三甲氧苯甲酰基)-乙烯-1-基]-喹啉-4-(1H)-酮(HGQ7)对人胰腺癌Bx PC-3细胞凋亡和自噬的诱导作用。方法 MTT法检测细胞增殖;AnnexinⅤ-FITC/PI双染法和TUNEL法测定细胞凋亡率;用自噬抑制剂氯喹处理细胞,验证HGQ7对细胞增殖和凋亡的影响;间接免疫荧光技术和Western blot法检测细胞自噬标志性蛋白LC3的表达。结果 HGQ7对Bx PC-3细胞增殖有明显抑制作用,24、48 h的IC50分别为4.952、4.564μmol·L^(-1);各组HGQ7作用24 h后,细胞凋亡率高于对照组(P<0.01);HGQ7处理导致Bx PC-3细胞LC3-Ⅱ表达量增加,6～24 h达到最高,之后明显下降;自噬抑制剂氯喹增强低剂量HGQ7(0.312 5～2.5μmol·L^(-1))对BxPC-3细胞的增殖抑制作用,并进一步提高HGQ7(1.25μmol·L^(-1))诱导的细胞凋亡率;而在高剂量HGQ7(5～10μmol·L^(-1))处理Bx PC-3细胞时,HGQ7联合氯喹组的细胞生长抑制率明显低于HGQ7单独处理组,HGQ7(5μmol·L^(-1))联合氯喹组细胞凋亡率则高于HGQ7单独处理组。结论 HGQ7能够明显诱导Bx PC-3细胞凋亡和自噬,低剂量HGQ7作用于Bx PC-3细胞时,自噬对细胞具有保护作用,高浓度HGQ7作用时,自噬作用则降低细胞增殖率,促进细胞凋亡。

Aim To study the effect of N-methylciprofloxacin-based fluoroquinolone-chalcone derivative 1-cyclopropyl-6-fluoro-7-（4-methyl-piperazin-1-yl）-3-[2-（3,4,5-trimethoxy-benzoyl）-vinyl-1-yl]-quinolin-4（1 H）-one（HGQ7） on apoptosis and autophagy of pancreatic carcinoma Bx PC-3 cells in vitro. Methods Cell viability was analyzed by MTT assay. Cell apoptosis was determined by flow cytometry（FCM） with Annexin V-FITC conjugated propidium iodide（PI）staining and TUNEL assay, in which chloroquine（CQ,an autophagy inhibitor） was used to inhibit autophagy. The expressions of autophagy marker protein microtubule associated protein 1 light chain 3（LC3）were detected by indirect immunofluorescence and Western blot. Results HGQ7 showed significant proliferation inhibition activity on Bx PC-3 cells. IC50 of 24 h and 48 h was 4. 952 μmol·L-1 and 4. 564 μmol·L-1,respectively. Treatment of Bx PC-3 cells with different concentrations of HGQ7 for 24 h increased the percentage of the apoptotic cells（P〈0. 01）. With the increase of time of exposure to HGQ7,the expressions of autophagy-related proteins LC3-Ⅱ of Bx PC-3 cells increased,reaching peaks during 6 ~ 24 h,but decreased afterwards. The autophagy inhibitor CQ further decreased the viability of cells treated by the low dose HGQ7（0. 312 5 -2. 5 μmol·L-1） and increased the percentage of the apoptotic cells. But CQ with high dose HGQ7（5 ~ 10 μmol·L-1） reversed the proliferation inhibition and pre-apoptosis activity of HGQ7 on Bx PC-3 cells. Conclusions HGQ7 could induce apoptosis and autophagy of Bx PC-3 cells. The autophagy might play a protective role in low dose HGQ7 exposure of Bx PC-3 cells. But with high dose HGQ7 group,the autophagy may further decrease the viability and increase the percentage of the apoptotic Bx PC-3 cells.