康莱特抑制结肠癌细胞转移的作用及其分子机制研究

Roles of the inhibition mediated by Kang-lai-te and molecular mechanism on metastasis of human colon cancer cells

目的以康莱特(KLT)、环磷酰胺(CTX)抑制癌细胞LoVo细胞增殖、迁移,观察对骨桥蛋白(OPN)、基质金属蛋白酶-9(MMP-9)和尿激酶型纤溶酶原激活物(uPA)表达的影响。方法培养人结肠癌LoVo细胞,取处于对数生长期细胞悬液200μL(细胞浓度为5×104/mL)接种,加入不同体积浓度的KLT或CTX培养,以MTT法检测细胞毒副作用;以Transwell小室检测细胞迁移能力;以Western blotting法分析OPN、MMP-9和uPA变化。结果 KLT、CTX抑制LoVo细胞增殖呈时间、剂量依赖性,其IC50 KLT为20μL/mL,CTX为2μM。在CTX、KLT组细胞存活率分别为57%、63%,比对照组均显著降低(x2值分别为54.78、45.90,P均<0.05)。CTX+KLT组细胞存活率为38%,比CTX、KLT组显著降低(x2值分别为7.29、12.50,P均<0.05)。细胞迁移力:空白组细胞数为117±3.5,CTX组为45±1.3,KLT组为67±2.1,比对照组明显减少(t值分别为43.12、27.89,P均<0.05)。CTX+KLT组为21±0.9,比CTX、KLT组显著减少(t值分别为33.94、45.02,P均<0.05)。KLT下调LoVo细胞OPN、MMP-9和uPA表达,并呈现剂量依赖性。与空白对照组、KLT组及CTX组比,KLT联合CTX组抑制LoVo细胞OPN、MMP-9和uPA的表达更明显。结论 KLT可能通过下调OPN、MMP-9及uPA表达,抑制LoVo细胞增殖和转移,且与CTX联合具有协同作用。

Objective To investigate the proliferation inhibition and migration alteration of human LoVo ceils in vitro by Kang-lai-te （KLT） , eyclophosphamide （CTX） or both combination to observe the expression of osteopontin （OPN） , matrix metallo-proteinase-9 （MMP-9） and urokinase-type plasminogen activator （uPA） for exploring the metastasis mechanism of colon cancer cells at the molecular level. Methods The colon cancer LoVo cell lines were cultured in vitro and taken 200 μL of cells （5 × 10^4/mL） at logarithmic growth phase into 96 well plates with a different doses of KLT, CTX or both. The proliferation inhibition and cytotoxicity effects of the cells were detected by MTT assay at 24 h, 48 h, and 72 h. The cell migration and the expression of OPN, MMP-9 and uPA were analyzed by Transwell test and Western Blotting respectively. Results After the LoVo cells were treated with different doses of KLT, CTX, or both in vitro. The proliferation inhibition of the LoVo cells by KLT and CTX were on time- and dose-dependent manner and IC50 were 20 μL/mL in KLT, and 2 μM in CTX. The ceils viability at48 h was 57% in CTX, 63% in KLT, and 38% in KLT plus CTX, respectively. Significant difference was found between the control group and the CTX group （x^2 = 54.78, P〈0.05） or the CTX group （x^2 =45.90, P〈0.05） or that of the CTX plus KLT group （x^2 =89.86, P〈 0.05）. The inhibition effect of the CTX plus KLT group was superior to that of the CTX group （x^2 = 7.29, P 〈 0.05） or the KLT group （x^2 = 12.50, P 〈 0.05）. The average migration cell number was （117 ± 3.5 ） in the control group and significantly lower than that in CTX （45 ±1.3, t =43. 12, P 〈0.05）, KLT （67 ±2. 1, t =27. 89, P 〈0.05）, and the CTX plus KLT group （21± 0.9, t = 59.40, P 〈 0.05） , respectively. The average migration number of the CTX plus KLT group was superior to that of the CTX group （t=33.94, P〈0.05） or the KLT group （t=45.02, P〈 0. 05）. The expression of OPN, MMP-9, and uPA protein in the LoVo cells were significantly down-regulated by KLT inhibition with a dose-dependent manner. Compared with the control groups or the KLT group or the CTX group, the strongest inhibition of OPN, MMP-9 and uPA expression were found in the plus KLT group. Conclusion The KLT by the molecular mechanism of down-regulated OPN, MMP-9, and uPA expression inhibit the proliferation and metastasis of LoVo cells, and a synergy effect with CTX.