Wang Zheng, Fan Jia, Zhou Jian, Wu Zhi-quan, Qiu Shuang-jian, Yu Yao, Huang Xiao-wu, Tang Zhao-you
Liver Cancer Institute, Fudan University, Shanghai 200032, China.
Zhonghua Yi Xue Za Zhi. 2006 Jun 27;86(24):1666-70.
To investigate the effects of rapamycin (RPM) in inhibiting the growth and metastasis of hepatocellular carcinoma (HCC).
Human HCC cells of the line MHCC97H with a high potential of metastasis were divided into 3 groups to be cultured with cyclosporine A (CsA) 100 ng/ml, RPM 10 ng/ml, or CsA + RPM for 48 hours. Flow cytometry was used to examine the apoptosis and cell cycle MTT method was used to examine the effect of RMP on the proliferation of the MHCC97H cells. RT-PCR was used to detect the mRNA expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hypoxia-inducible factor-1alpha (HIF-1alpha), and transforming growth factor b (TGFb). Another MHCC97H cells were cultured in complete medium without RPM for 48 hours, then the protein expression of VEGF in the supernatant was detected by ELISA. Twenty-eight nude LCI-D20 mice were inoculated with human HCC cells and then divided into 4 groups to be fed with CsA (25 mg/kg), RPM (2 mg/kg), CsA + RPM, and normal saline (0.2 ml, as control group) for 35 days. Then the mice were killed to take the weight of inoculated tumor, measure the blood drug concentration, calculate the lung metastasis rate and number of metastatic foci, and observe pathology of the lung.
CsA showed no effect on the cycle of the MHCC97H cells. The MHCC97H cells of the RPM and CsA + RPM groups arrested at the stage G(0)/G(1) (both P = 0.000). MMT method also showed that the proliferation of the MHCC97H cells in the RPM and CsA + RPM groups were blocked (P = 0.003 and P = 0.002). However, CsA did not influence the proliferation of the MHCC97H cells. Flow cytometry showed that RPM did not promote the apoptosis of the MHCC97H cells. RT-PCR showed that RPM down-regulated the mRNA expression of VEGF and HIF-1alpha (both P < 0.05), however, did not influence the mRNA expression of bFGF, TGFb, and TGFb. The VEGF protein level in the supernatant of the culture fluid of MHCC97H cells of the RPM group was (890.3 +/- 25.1) pg/ml, significantly lower than that of the control group, (1583.7 +/- 17.3) pg/ml (P = 0.000). The tumor inhibiting rate of the RPM group was 63.7%, not significantly different from that of the RPM + CsA group (80.9%, P = 1.000). The metastatic rate of the CsA and control groups were both 100% with a higher number of metastatic tumors in the CsA group (P = 0.046).
RPM significantly inhibits the growth and metastasis of HCC. RPM-based immunosuppressive regimen may be of value in HCC patients receiving liver transplantation.
探讨雷帕霉素（RPM）对肝癌（HCC）生长和转移的抑制作用。
将具有高转移潜能的人肝癌MHCC97H细胞分为3组，分别用100 ng/ml环孢素A（CsA）、10 ng/ml RPM或CsA + RPM培养48小时。采用流式细胞术检测细胞凋亡和细胞周期，MTT法检测RMP对MHCC97H细胞增殖的影响。采用RT-PCR检测血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）、缺氧诱导因子-1α（HIF-1α）和转化生长因子β（TGFβ）的mRNA表达。另将MHCC97H细胞在无RPM的完全培养基中培养48小时，然后用ELISA法检测上清液中VEGF的蛋白表达。将28只裸鼠接种人肝癌细胞，然后分为4组，分别给予CsA（25 mg/kg）、RPM（2 mg/kg）、CsA + RPM和生理盐水（0.2 ml，作为对照组），连续喂养35天。然后处死小鼠，称取接种肿瘤的重量，测量血药浓度，计算肺转移率和转移灶数量，并观察肺组织病理学。
CsA对MHCC97H细胞周期无影响。RPM组和CsA + RPM组的MHCC97H细胞停滞于G(0)/G(1)期（P均 = 0.000）。MTT法也显示RPM组和CsA + RPM组的MHCC97H细胞增殖受到抑制（P = 0.003和P = 0.002）。然而，CsA不影响MHCC97H细胞的增殖。流式细胞术显示RPM不促进MHCC97H细胞凋亡。RT-PCR显示RPM下调VEGF和HIF-1α的mRNA表达（P均 < 0.05），但不影响bFGF、TGFβ和TGFβ的mRNA表达。RPM组MHCC97H细胞培养液上清中VEGF蛋白水平为（890.3 ± 25.1）pg/ml，显著低于对照组的（1583.7 ± 17.3）pg/ml（P = 0.000）。RPM组的抑瘤率为63.7%，与RPM + CsA组的80.9%无显著差异（P = 1.000）。CsA组和对照组的转移率均为100%，CsA组的转移瘤数量较多（P = 0.046）。
RPM显著抑制HCC的生长和转移。基于RPM的免疫抑制方案可能对接受肝移植的HCC患者有价值。
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