摘要
目的神经酰胺(ceramide, Cer)、鞘氨醇(Sphingosine, SP)、1-磷酸鞘氨醇(Sphingosine-phospate, S1P)均参与肿瘤浸润、增殖、血管生成等的调节,是重要的细胞信号转导分子。鞘氨醇激酶-1(Sphingosine kinasel, SPK1)是产生S1P的关键酶,亦是调节Cer／S1P动态平衡的关键酶,通过磷酸化Cer的产物SP生成S1P。目前已经证实利用N,N,一二甲基鞘氨醇(N,N-Dimethyl sphingosine,DMS)干涉SPK1／S1P信号途径后,HepG2细胞生长受到抑制,并可能发生凋亡,对人肝癌细胞血管内皮生长因子(Vascular endothelial growth factor, VEGF)的分泌具有明显的下调作用。但SPK1／S1P信号途径是否具有调节人肝癌耐药细胞株BEL-FU凋亡、侵袭力及耐药特性的作用目前国内外均未见报道。本课题研究利用DMS干涉SPK1／S1P信号后对人肝癌耐药细胞株BEL-FU的凋亡、侵袭能力及耐药特性的影响。
方法人肝癌耐药细胞株BEL-FU培养于含10%小牛血清、100u／L青霉素、100μg／L链霉素、20000ng／mL5-FU的RPMI1640培养基中,置于37℃、5%CO2孵箱中培养,0.25%胰蛋白酶消化,倒置显微镜下观察细胞生长状况,采用对数生长期的细胞进行实验。利用不同浓度的DMS处理人肝癌耐药细胞株BEL-FU,倒置相差显微镜及荧光显微镜观看凋亡形态变化,流式细胞仪检测凋亡发生；并用transwell小室模型测定侵袭力；Western blot杂交检测多药耐药相关蛋白1(Multidrug resistance-related proteinl, MRP1)表达的变化。
结果1.SPK1／S1P信号途径对人肝癌耐药细胞株BEL-FU凋亡的影响。①流式结果显示：5-20μmoL／L浓度范围的DMS对细胞均有显著凋亡作用,并且具有剂量依赖效应。浓度组与对照组比较,及各浓度组间比较差异均有统计学意义(P<0.01),且呈剂量依赖效应。②形态学观察：倒置相差显微镜观察：正常细胞呈克隆性生长,细胞呈梭形或多角形。药物作用后主要表现为细胞内部颗粒、空泡增多,体积变小、形态变圆,较多细胞从贴壁状态脱落,悬浮于培养液中。随培养时间的延长和药物浓度的增大,变化渐趋明显。荧光显微镜观察：对照组仅有极少数早、晚期凋亡细胞；与对照组比较,各浓度组早、晚期凋亡细胞均增加,随着药物浓度的升高晚期凋亡细胞逐渐增加,一部分凋亡细胞细胞核出现碎裂,核碎片清晰可见。
2.SPK1／S1P信号途径对人肝癌耐药细胞株BEL-FU侵袭力的影响。侵袭实验结果显示：5-20μmoL／L浓度范围的DMS均可使各浓度组的穿膜细胞数减少、侵袭力减弱,与对照组比较及组间比较差异均有统计学意义(P<0.01),且呈剂量依赖效应。
3.SPK1／S1P信号途径对人肝癌耐药细胞株BEL-FU MRP1蛋白表达的影响。Western blot结果显示：对照组、10μmoL／L组及20μmoL／L组均有MRPl蛋白的表达,且10μmoL／L组及20μmoL／L组MRP1表达量均少于对照组,20μmoL／L组MRP1表达量少于10μmoL／L组。10μmoL／L组及20μmoL／L组光密度比值均小于对照组,差异具有统计学意义(P<0.05),但10μmoL／L组与20μmoL／L组比较光密度比值无统计学意义(P>0.05)
结论1、DMS干涉SPK1／S1P信号后,可以引起人肝癌耐药细胞株BEL-FU的凋亡,且呈剂量依赖效应。
2、DMS干涉SPK1／S1P信号后,可以降低人肝癌耐药细胞株BEL-FU的侵袭力,且呈剂量依赖效应。
3、DMS干涉SPK1／S1P信号后,人肝癌耐药细胞株BEL-FU的MRP1蛋白表达量减少,从而说明了干涉SPK1／S1P信号后可以克服其耐药特性。
Objective Ceramide (Cer),Sphingosine (Sp) and Sphingosine-1-phospate (SIP) are signal transduction molecules involved in invasion, proliferation, blood vessel formation of tumors. Sphingosine kinasel (SPK1) regulates the production of S1P, and is a key lipid signal moleculer regulates the balance of Cer/S1P. It has been proved that the SPK1/S1P signal pathway interfered by Dimethyl sphingosine (DMS)can cause the apoptosis and inhibit the expression of VEGF of hepatoma cells. However very little is known about whether it can cause the apoptosis, reduce invasiveness and inhibit the expression of multidrug resistance-related protein of human hepatocellular carcinoma multidrug resistant (MDR) cells. The objective of this study is to elucidate the roles of SPK1/S1P signal in human hepatocellular carcinoma MDR cell strain BEL-FU in terms of apoptosis, invasiveness and multidrug resistance.
Methods Human hepatocellular carcinoma MDR cell strain BEL-FU were grown in RPMI1640supplemented with 10% FBS,20000ng/mL 5-FU, 100u/L penicillin and 100μg/L streptomycin at 37℃,5%CO2, and were digested by 0.25% trypsin. Treated with concentration range of 5-20μmoL/L DMS in culture medium, the morphological variations of apoptic cells were observed with invert phase-contrast microscope and with fluorescence microscopy. Meanwhile, cell apoptosis was examined by flow cytometry. The invasion of cells and the expression of multidrug resistance-related protein (MRP1) were detected by transwell chamber assay and Western-blot respectively.
Results 1.The effects of SPK1/S1P signal on survival in human hepatocellular carcinoma MDR cell strain BEL-FU.①The our results demonstrated that 5-20μmoL/L DMS can cause the apoptosis of human hepatocellular carcinoma MDR cell strain BEL-FU and was in dose-dependent pattern. The apoptosis rate of every concentration group increased significantly compared with the control groups (P<0.01), while the apoptosis was significantly different among the concentration groups (P<0.01).②Inverted phase contrast microscope observation:The control group cells were clonally growth and were spindle or polygonal. The cells treated with DMS exhibited characteristics of apoptosis including increased intra-cellular granules, increased vacuoles in size, decreased cytoplasm and condensed nucleus with smaller, rounded cells shape. More cells detached from the adherent state and suspended in culture medium. With the prolongation of treated time and DMS concentration increasing, those characteristics become more evident.The early and late stage apoptotic cells increased in concentration groups compared with control group. The late stage apoptotic cells increased with DMS concentration. Some nucleus fragmentation occurred among the apoptotic cells,the nuclear debris was clearly visible.2. The effects of SPK1/S1P signal on invasion in human hepatocellular carcinoma MDR cell strain BEL-FU. The results of transwell chamber assay indicated that 5～20μmoL/L DMS can reduce its invasiveness. The inhibitory rate of invasion of experimental groups were significantly increased (P<0.01) compared with control group, and the inhibition exhibit dose-dependent relationship among the experimental groups (P<0.01) 3.The effects of SPK1/S1P signal on the expression of multidrug resistance-related protein (MRP1) in human hepatocellular carcinoma MDR cell strain BEL-FU. The results showed that the MRP1 expression was significantly decreased in DMS treated groups compared with control group (P<0.05), but there was no significant difference in the MRP1 expression between 10μmoL/L DMS treated group and 20μmoL/L DMS treated group (P>0.05).
Conclusion 1.The SPK1/S1P signal pathway interfered by DMS can cause the apoptosis of human hepatocellular carcinoma MDR cell strain BEL-FU.2. The SPK1/S1P signal pathway interfered by DMS can reduce the invasion of human hepatocellular carcinoma MDR cell strain BEL-FU.3. The SPK1/S1P signal pathway interfered by DMS can inhibit the expression of mutidrug resistance-related protein in human hepatocellular carcinoma MDR cell strain BEL-FU and overcome its multidrug resistance.
引文
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