目的 基于信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄活化因子3（STAT3）信號(hào)通路探討三氟甲基化二氫喹喔啉酮類化合物S-3-（三氟甲基）-6，7-雙［4-（三氟甲基）苯基］-3，4-二氫喹啉-2（1H）-酮（2o）對(duì)人結(jié)直腸癌細(xì)胞生長的抑制作用。方法 采用 MTT法檢測(cè)三氟甲基化二氫喹喔啉酮類化合物 2b、2e、2f、2g、2k、2l、2m、2o、2q（20 μmo·L^-1）作 用 48 h 對(duì) 人 結(jié) 直 腸 癌 細(xì) 胞DLD-1 存活率的影響，檢測(cè)化合物2o（0.5、1.0、2.0、5.0、7.5、10.0、12.5、15.0、17.5 μmo·L^-1）作用48 h對(duì)人結(jié)直腸癌細(xì)胞HCT116、RKO和DLD-1存活率的影響；克隆形成實(shí)驗(yàn)檢測(cè)化合物2o（2.5、5.0、10.0 μmo·L^-1）對(duì)RKO、DLD-1、HCT116細(xì)胞增殖的影響；流式細(xì)胞術(shù)和 Hoechst染色檢測(cè)化合物 2o 對(duì)細(xì)胞凋亡的影響；細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)化合物 2o 對(duì) DLD-1 和RKO 細(xì)胞遷移率的影響；Western blotting 法檢測(cè)化合物 2o 對(duì) RKO 細(xì)胞 p-STAT3、STAT3、骨髓白血病細(xì)胞分化蛋白（MCL）-1、生存素（Survivin）、B淋巴細(xì)胞瘤-2（Bcl-2）、Bcl-2相關(guān)X蛋白（Bax）蛋白表達(dá)水平的影響，檢測(cè)化合物2o對(duì)白細(xì)胞介素-6（IL-6）刺激下p-STAT3、STAT3蛋白表達(dá)水平的影響。結(jié)果 化合物2o對(duì)DLD-1細(xì)胞的殺傷能力較其他化 合 物 強(qiáng) ； 化 合 物 2o 對(duì) HCT116、 RKO 和 DLD-1 細(xì) 胞 的 半 數(shù) 抑 制 濃 度 （IC₅₀） 分 別 為 （3.573±0.172）、（8.056±0.458）、（6.226±0.458）μmo·L^-1。與對(duì)照組比較，化合物2o明顯降低了HCT116、RKO和DLD-1細(xì)胞的集落形成能力；顯著降低DLD-1和RKO細(xì)胞遷移率（P＜0.01、0.001）；明顯增加RKO、DLD-1細(xì)胞凋亡率；明顯增加HCT116和RKO細(xì)胞核亮染比例；明顯降低p-STAT3、MCL-1、Survivin、Bcl-2蛋白表達(dá)，明顯升高Bax蛋白表達(dá)，對(duì)STAT3蛋白表達(dá)無明顯影響。與對(duì)照組比較，IL-6刺激的模型組p-STAT3蛋白表達(dá)明顯升高，STAT3蛋白表達(dá)無明顯變化；與模型組比較，隨著化合物2o濃度的升高，p-STAT3蛋白表達(dá)明顯降低，STAT3蛋白表達(dá)無明顯變化。結(jié)論 化合物2o可能通過下調(diào)STAT3信號(hào)通路發(fā)揮抗人結(jié)直腸癌作用。

Objective To investigate the inhibitory effect of S-3- (trifluoromethyl) -6, 7-bis(4- (trifluoromethyl)phenyl) -3, 4- dihydroquinoxalin-2(1H)-one on the occurrence and development of human colorectal cancer cells based on signal transducer and activator of transcription 3 (STAT3) signaling pathway. Methods MTT methods was used to detected the effects of trifluoromethylated dihydroquinoxalinones 2b, 2e, 2f, 2g, 2k, 2l, 2m, 2o, 2q (20 μmo·L^-1) treatment for 48 hours on the survival rate of human colorectal cancer cell line DLD-1, and the effects of compound 2o (0.5, 1.0, 2.0, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5 μmo·L^-1) treatment for 48 h on the survival rates of human colorectal cancer cells HCT116, RKO, and DLD-1. Clonogenesis assay detected effect of compound 2o (2.5, 5.0, 10.0 μmo·L^-1) on the proliferation of RKO, DLD-1, and HCT116 cells. Flow cytometry and Hoechst staining were used to detect the effect of compound 2o on apoptosis. The effect of compound 2o on the migration rate of DLD-1 and RKO cells was detected by cell scratch test. Western blotting was used to detect the effects of compound 2o on the expression of p-STAT3, STAT3, myeloid leukemia cell differentiation protein (MCL)-1, Survivin, B-lymphomatoma-2 (Bcl-2), and Bcl-2 related X protein (Bax) proteins in RKO cells, and to detect the effects of compound 2o on the expression of p-STAT3, STAT3 proteins stimulated by interleukin-6 (IL-6). Results Compound 2o had stronger killing ability on DLD-1 cells than other compounds. The semi maximum inhibitory concentrations (IC₅₀) of compound 2o on HCT116, RKO, and DLD-1 cells were (3.573 ± 0.172), (8.056 ± 0.458), and (6.226 ± 0.458) μmo·L^-1 respectively. Compared with the control group, compound 2o apparently decreased the colony forming ability of HCT116, RKO, and DLD-1 cells, significantly decreased the migration rates of DLD-1 and RKO cells (P＜0.01, 0.001), apparently increased the apoptosis rate of RKO and DLD-1 cells, apparently increased the ratio of HCT116 and RKO nuclei to bright staining, apparently decreased the expression of p-STAT3, MCL-1, Survivin, and Bcl-2 proteins, apparently increased the expression of Bax protein, and had no significant impact on STAT3 protein expression. Compared with the control group, the expression of p-STAT3 protein in the model group stimulated by IL-6 was significantly increased, while the expression of STAT3 protein had no significant change. Compared with the model group, with the increase of compound 2o concentration, the expression of pSTAT3 protein significantly decreased, while the expression of STAT3 protein did not significantly change. Conclusion Our data suggested that compound 2o may play an anti human colorectal cancer role by downregulating STAT3 signaling pathway.

R965

大理藥業(yè)股份有限公司橫向課題（KJHX1603）