塞来昔布联合氟伐他汀对人肝癌裸鼠皮下移植瘤生长及细胞凋亡的影响

Effects of celecoxib combined with fluvastatin on tumor growth and cell apoptosis in a xenograft model of hepatocellar carcinoma

目的探讨塞来昔布联合氟伐他汀对实验性人肝癌裸鼠皮下移植瘤生长及细胞凋亡的影响。方法32只实验裸鼠左腋窝皮下接种BEL-7402肝癌细胞株，随机分为对照组、塞来昔布组、氟伐他汀组及塞来昔布和氟伐他汀联合用药组。实验结束时，留取移植瘤标本，流式细胞术及原位缺口末端标记法检测肿瘤细胞凋亡率，Westernblot检测Akt、磷酸化Akt（p-Akt）和survivin蛋白的表达情况。数据比较采用析因设计多因素方差分析及多个样本均数间多重比较的SNK-q检验。结果联合用药组肿瘤生长明显被抑制，塞来昔布组、氟伐他汀组抑瘤率分别为34．％和25．0％，联合用药组抑瘤率为72．2％。对照组细胞凋亡指数为3．5％±0．8％，联合用药组为19．4%±3．0％，塞来昔布组和氟伐他汀组分别为8．5％±1．4％和9．4％±1．7％，联合用药组肿瘤细胞凋亡明显高于塞来昔布及氟伐他汀单药组（P值均〈0．01）。流式细胞术检测结果显示，移植瘤细胞凋亡率对照组为4．1％±1．6％，塞来昔布组为9．1％±2．1％，氟伐他汀组为10．1％±2．3％，联合用药组为23．6％±5．8％，各单药用药组均高于对照组（P值均〈0．05），其中联合用药组高于对照组及各单药组（P值均〈0．01）。Western blot检测结果显示，联合用药组较对照组明显下调p—Akt（0．23±0．08比1．12±0．07）和survivin蛋白（0．50±0．07比1．47±0．19）的表达（P值均〈0．01）。结论与单用塞来昔布或氟伐他汀相比，联合用药能更有效地抑制肝癌细胞生长。

Objective To evaluate effects of celecoxib （a selective cox-2 inhibitor）combined with fluvastatin （a HMG-CoA reductase inhibitor） on tumor growth and cell apoptosis in hepatocellular carcinoma xenograft in nude mice. Methods Hepatocellular carcinoma BEL-7402 cells were inoculated subcutaneously into the left armpit of nude mice, the mice （n = 32） were then randomly divided into 4 groups： the control group, the celecoxib group,the fluvastatin group and the combination group. At the end of the study, Tumor Tissues were collected for analysis. Cell apoptosis was determined by flow cytometry analysis and TUNEL assay. Akt, p-Akt and survivin protein levels were measured by Western blot. Statistical comparisons were made using factorial analysis of variance （ANOVA） and multiple comparisons between each two groups were calculated using SNK-q test. Results The combination of Cclecoxib and fluvastatin resulted in a greater inhibition of tumor growth than either agent alone, tile tumor inhibitory rate was 34.0% in the Celecoxib group, 25.0% in the fluvastatin group and 72.2% in the combination group. The percentages of TUNEL-positive cancer cells in the celecoxib and fluvastatin alone treatment groups were 8.5% ± 1.4% and 9.4% ± 1.7% respectively as compared to the control group which was 3.5% ± 0.8%, Combination therapy showed a significantly greater increase in tumor cell apoptosis in comparison with the control and single-therapy groups （apoptotic index： 19.4% ± 3.0%; P 〈 0.01 versus celecoxib or fluvastatin groups）. The results of flow cytometry analysis also showed the same tendency, a small number of apoptotic cells were detected in the control tumours （4.1% ± 1.6%）, whereas a large number of apoptotic cells were detected in tumours treated with celecoxib （9.1% ± 2.1%） or fluvastatin （10.1% ± 2.3 %） alone; and the combination therapy resulted in even more apoptotic cells （23.6% ± 5.8%; P 〈 0.01 versus celecoxib or fluvastatin groups）. Western blot analysis demonstrated that the combination of celecoxib and fluvastatin significantly down-regulated p-Akt （0.23 ± 0.08 versus 1.12 ± 0.07 and surviving （0.50 ± 0.07 versus 1.47 ± 0.19） in BEL-7402 tumours compared with the control （P 〈 0.01 for all）. Conclusion The present study provided evidence that treatment with celecoxib in combination with fluvastatin resulted in the inhibition of HCC tumour growth in an in vivo mouse model.