NS-398对人卵巢癌细胞株SW626细胞Snail、MMP-2表达的影响
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摘要
背景与目的：卵巢恶性肿瘤在妇科恶性肿瘤中发病率居第三位,死亡率居第一位,其中60%～90%是卵巢上皮性癌(ovarian epithelial carcinoma,简称卵巢癌)。卵巢癌发展快,容易发生淋巴通道及血行通道的转移,预后差。目前,卵巢癌的死亡率在妇科恶性肿瘤中居于榜首,由于发病其隐匿,一般在确诊的时候,大部分已经属于是晚期了,即使给予手术、放疗及化疗后,一部分病人虽然已经达临床缓解,但仍然有70%左右的患者最终还要复发。曾有研究表明：侵袭与转移不仅是恶性肿瘤的重要生物学特征,也是造成肿瘤患者死亡的主要原因。在肿瘤细胞发生侵袭和转移的过程中,参与细胞间相互作用的细胞黏附分子是肿瘤细胞恶性行为的基础,也是许多致癌因素综合作用的最终靶位点。相关研究提示COX-2在妇科恶性肿瘤的细胞凋亡、新生血管形成、肿瘤浸润方面存在某些致瘤机制。用非甾体类抗炎药(NSAID)阻断COX-2的作用在恶性肿瘤的综合治疗研究领域中有着非常重要的地位,选择性COX-2抑制剂在恶性肿瘤的综合治疗中已经成为研究热点。本研究采用选择性COX-2抑制剂(NS-398)作用于体外培养的人卵巢腺癌、高转移的细胞株SW626,观察其对人卵巢癌细胞株侵袭、转移方面的影响,初步探讨选择性COX-2抑制剂对人卵巢腺癌高转移的细胞株SW626的Snail以及MMP-2mRNA及蛋白水平表达的影响,以探索肿瘤细胞侵袭转移的恶性行为的可能的分子生物学机制。
方法：本实验选择人卵巢腺癌高转移细胞株SW626体外常规培养；以不同浓度的COX-2抑制剂(NS-398) 0、50、100、200umol/L四个浓度组,每个浓度组选取24h、48h、72h三个作用时间点,MTT法检测其生长情况,计算抑瘤率；以不同浓度COX-2抑制剂(NS-398) 0、50、100、200umol/L四个浓度组,每个浓度组选取24h、48h、72h三个作用时间点采用RT-PCR半定量对不同时间点、不同浓度进行检测SW626细胞Snail、MMP-2基因mRNA水平表达的影响。以不同浓度COX-2抑制剂(NS-398) 50、100、200umol/L,于72h作用的时间点行免疫细胞化学(SABC)观察蛋白水平的表达情况。
结果：MTT法测定结果显示：NS-398对人卵巢腺癌细胞株SW626的生长抑制具有时间和浓度的依赖性,与对照组比较有统计学意义(P<0.05)。RT-PCR半定量结果显示：NS-398作用对人卵巢癌细胞株SW626细胞的Snail mRNA的表达情况,随着NS-398药物作用时间的延长及浓度的增加Snail mRNA表达均下调,有统计学意义(P<0.05)。NS-398能显著抑制人卵巢癌细胞株SW626细胞MMP-2因子的mRNA的表达情况,随着NS-398药物作用时间的延长及浓度的增加MMP-2mRNA表达均下调,有统计学意义(P<0.05)。免疫细胞化学结果显示；随着NS-398药物作用浓度的增加,Snail以及MMP-2在蛋白水平的表达均减弱,表达呈浓度依赖性,镜下观察见有显著差异。
结论：选择性COX-2抑制剂NS-398能够抑制人卵巢癌细胞株SW626的Snail蛋白和MMP(?)表达,使Snail蛋白及MMP-2的]mRNA及蛋白水平表达下调,这可能是其抑制人(?)巢癌细胞株SW626肿瘤细胞侵袭力的可能机制之一。
Background and objectives:Ovarian cancer incidence in gynecological cancer ranks third in the mortality rate ranks first, with 60% to 90% of epithelial ovarian cancer (ovarian epithelial carcinoma, referred to as ovarian cancer).The rapid development of Epithelial ovarian cancer is likely to result in the transfer of lymphatic channels and blood line and the prognosis is poor, For the moment, ovarian cancer ranks first among all the gynecological malignancies in terms of mortality. Because it is hard to detect the ovarian cancer, when diagnosed, most are already at an advanced stage. with the treatment of surgery, radiotherapy and chemotherapy, some patients have completely recovered clinically, but about 70% of them will ultimately relapse. studies show that invasion and metastasis are not only important biological characteristics of malignant tumors, but also the leading cause for the death of those patients. In the course of invasion and spread of the tumor cells, adhesion molecules which involve in the interplay of cells form a basis for malignant tumors and the final target sites as well where many carcinogenic factors interact.Related research revealed that COX-2 contributes to the formation of tumors in the areas of cell apoptosis in gynecologic tumor, angiogenesis and tumor-infiltrating. Blocking COX-2 with NSAID has played an important role in the comprehensive treatment of gynecological tumors and the election of COX-2 inhibitors has become the latest research topic。In this study, the election of COX-2 inhibitor, (NS-398) will be applied to the highly metastatic cell line SW626 which is cultivated in vitro ovarian adenocarcinoma, observing its effects on the invasion and metastasis of ovarian cancer cell line. Exploring how the election of COX-2 inhibitor impacts the Snail protein of SW626 as well as MMP-2 expression with a purpose to probe into the molecular biological mechanism which may lead to the invasion and metastasis of tumor cells.
Methods:Apply different concentrations of NS-398 (0,50, 100and 200 umol/L) to act on human ovarian cancerous cells, using MTT to determine how the concentration of NS-398 restrains the growth of SW626 cells in 24,48,72h time points, using real-time fluorescence quantitative PCR method to detect the expression NS-398 acts on the transcription level of Snail&MMP-2 mRNA expression in different concentrations (0、50,100& 200 umol/L) and different periods of time(24h,48h,72h). With different concentrations of COX-2 inhibitor (NS-398) 50,100&200 umol/L three concentrations and the time points 72h immunohistochemistry (SABC) Qualitative detection of Snail&MMP-2 at the protein level.
Results:MTT method showed that:NS-398 inhibited the growth of SW626 was time and concentration dependent, and the control group (P<0.05). RT-PCR semi-quantitative results shows that NS-398 can greatly reduce Snail expression with a dose gradient descent and concentration-dependent (P<0.05),tendency which rises with increased concentration and prolonged time.NS-398 can reduce the expression of MMP-2 with duration protraction and concentration increase. (P<0.05). immunocyte chemistry result shows that,if the concentration of NS-398 increased,Snail and MMP-2 expreession will be decreased in protein level,the expression level based on the concentration,a significant difference will be found through microscope.
Conclusion:Selective COX-2 inhibitors can inhibit the Snail & MMP-2 expression, and thereby can bring down Snail&MMP-2 mRNA and protein expression, which may be one of the mechanisms to reduce tumor cell invasion.
方法：本实验选择人卵巢腺癌高转移细胞株SW626体外常规培养；以不同浓度的COX-2抑制剂(NS-398) 0、50、100、200umol/L四个浓度组,每个浓度组选取24h、48h、72h三个作用时间点,MTT法检测其生长情况,计算抑瘤率；以不同浓度COX-2抑制剂(NS-398) 0、50、100、200umol/L四个浓度组,每个浓度组选取24h、48h、72h三个作用时间点采用RT-PCR半定量对不同时间点、不同浓度进行检测SW626细胞Snail、MMP-2基因mRNA水平表达的影响。以不同浓度COX-2抑制剂(NS-398) 50、100、200umol/L,于72h作用的时间点行免疫细胞化学(SABC)观察蛋白水平的表达情况。
结果：MTT法测定结果显示：NS-398对人卵巢腺癌细胞株SW626的生长抑制具有时间和浓度的依赖性,与对照组比较有统计学意义(P<0.05)。RT-PCR半定量结果显示：NS-398作用对人卵巢癌细胞株SW626细胞的Snail mRNA的表达情况,随着NS-398药物作用时间的延长及浓度的增加Snail mRNA表达均下调,有统计学意义(P<0.05)。NS-398能显著抑制人卵巢癌细胞株SW626细胞MMP-2因子的mRNA的表达情况,随着NS-398药物作用时间的延长及浓度的增加MMP-2mRNA表达均下调,有统计学意义(P<0.05)。免疫细胞化学结果显示；随着NS-398药物作用浓度的增加,Snail以及MMP-2在蛋白水平的表达均减弱,表达呈浓度依赖性,镜下观察见有显著差异。
结论：选择性COX-2抑制剂NS-398能够抑制人卵巢癌细胞株SW626的Snail蛋白和MMP(?)表达,使Snail蛋白及MMP-2的]mRNA及蛋白水平表达下调,这可能是其抑制人(?)巢癌细胞株SW626肿瘤细胞侵袭力的可能机制之一。
Background and objectives:Ovarian cancer incidence in gynecological cancer ranks third in the mortality rate ranks first, with 60% to 90% of epithelial ovarian cancer (ovarian epithelial carcinoma, referred to as ovarian cancer).The rapid development of Epithelial ovarian cancer is likely to result in the transfer of lymphatic channels and blood line and the prognosis is poor, For the moment, ovarian cancer ranks first among all the gynecological malignancies in terms of mortality. Because it is hard to detect the ovarian cancer, when diagnosed, most are already at an advanced stage. with the treatment of surgery, radiotherapy and chemotherapy, some patients have completely recovered clinically, but about 70% of them will ultimately relapse. studies show that invasion and metastasis are not only important biological characteristics of malignant tumors, but also the leading cause for the death of those patients. In the course of invasion and spread of the tumor cells, adhesion molecules which involve in the interplay of cells form a basis for malignant tumors and the final target sites as well where many carcinogenic factors interact.Related research revealed that COX-2 contributes to the formation of tumors in the areas of cell apoptosis in gynecologic tumor, angiogenesis and tumor-infiltrating. Blocking COX-2 with NSAID has played an important role in the comprehensive treatment of gynecological tumors and the election of COX-2 inhibitors has become the latest research topic。In this study, the election of COX-2 inhibitor, (NS-398) will be applied to the highly metastatic cell line SW626 which is cultivated in vitro ovarian adenocarcinoma, observing its effects on the invasion and metastasis of ovarian cancer cell line. Exploring how the election of COX-2 inhibitor impacts the Snail protein of SW626 as well as MMP-2 expression with a purpose to probe into the molecular biological mechanism which may lead to the invasion and metastasis of tumor cells.
Methods:Apply different concentrations of NS-398 (0,50, 100and 200 umol/L) to act on human ovarian cancerous cells, using MTT to determine how the concentration of NS-398 restrains the growth of SW626 cells in 24,48,72h time points, using real-time fluorescence quantitative PCR method to detect the expression NS-398 acts on the transcription level of Snail&MMP-2 mRNA expression in different concentrations (0、50,100& 200 umol/L) and different periods of time(24h,48h,72h). With different concentrations of COX-2 inhibitor (NS-398) 50,100&200 umol/L three concentrations and the time points 72h immunohistochemistry (SABC) Qualitative detection of Snail&MMP-2 at the protein level.
Results:MTT method showed that:NS-398 inhibited the growth of SW626 was time and concentration dependent, and the control group (P<0.05). RT-PCR semi-quantitative results shows that NS-398 can greatly reduce Snail expression with a dose gradient descent and concentration-dependent (P<0.05),tendency which rises with increased concentration and prolonged time.NS-398 can reduce the expression of MMP-2 with duration protraction and concentration increase. (P<0.05). immunocyte chemistry result shows that,if the concentration of NS-398 increased,Snail and MMP-2 expreession will be decreased in protein level,the expression level based on the concentration,a significant difference will be found through microscope.
Conclusion:Selective COX-2 inhibitors can inhibit the Snail & MMP-2 expression, and thereby can bring down Snail&MMP-2 mRNA and protein expression, which may be one of the mechanisms to reduce tumor cell invasion.
引文
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