摘要
背景与目的：
随着人类对抗癌症的科学研究的深入，淋巴管新生在决定肿瘤预后方面的重要作用已渐为人熟知。胃癌在极早期即可发生淋巴管转移，是严重影响胃癌治疗效果及术后生存率乃至术后复发的关健。面对淋巴管转移在肿瘤发展及预后中如此重要作用，研究人员为了阐明淋巴管转移具体机制而不懈努力。近年，人们发现Akt在肿瘤的发生、发展及预后中起着重要的作用，同时发现其与肿瘤淋巴管转移或肿瘤淋巴管新生中有着密切的联系。不少报道已证实Akt信号通路参与肿瘤淋巴管转移或淋巴新生。但目前有关活化Akt在促进胃癌淋巴管新生或淋巴管转移作用及具体机制尚未见报道。鉴于VEGF信号途径在淋巴管新生方面中也起着不可代替的作用。我们研究拟通过分析原位胃癌p-Akt、p-mTOR两个Akt信号通路上核心蛋白、淋巴管新生因子VEGF-C、VEGF-D与微淋经巴管密度三者之间的关系，并通过抑制Akt信号转导通路对淋巴管新生因子的影响，来探讨Akt/mTOR信号通路介导VEGF-C/-D来促进肿瘤淋巴管新生的作用，进而明确Akt/mTOR/VEGF-C/-D信号转导通路在胃癌中的存在。
方法：
1、原位胃癌组织实验：收集南昌大学第一附属医院2011年4月到11月份胃癌患者手术新鲜标本55例，患者手术前未接受任何放、化疗及免疫治疗。其中，男42例，女13例；通过免疫组化分别检测原位胃癌中p-Akt、p-mTOR、VEGF-C、VEGF-D、D2-40蛋白表达水平，观察各指标在胃癌中的阳性表达率，并分析它们之间的相关性关系。
2、胃癌SGC7901细胞体外实验：分LY294002与Rapamycin两大药物干预组。（1）MTT法：利用Akt活化抑制剂LY294002和mTOR活化抑制剂Rapamycin分别分0μM、12.5μM、25μM、50μM和0nM、25nM、50nM、100nM四个不同浓度作用于SGC7901细胞，并在24h，48h及72h三个不同时间观察SGC7901胃癌细胞存活率的变化。（2）根据MTT结果选取合适药物浓度，利用上述两药物分别抑制PI3K/Akt信号通路的不同靶点，在药物作用24h、48h、72h提示细胞蛋白。通过Western Blot检测分析两药物对Akt、p-Akt、mTOR、p-mTOR、VEGF-C、VEGF-D蛋白表达的影响。
结果：
1、原位胃癌中p-Akt、p-mTOR、VEGF-C、VEGF-D表达之间关系
胃癌p-Akt、p-mTOR、VEGF-C、VEGF-D的阳性表达率分别是74.5%，85.45%，72.73%和58.18%。结果还显示p-Akt和p-mTOR、VEGF-C、VEGF-D的表达水平呈正相关（P＜0.01）。p-mTOR与VEGF-C、VEGF-D的表达水平亦存在正相关关系（P＜0.01）。同时我们还发现p-mTOR、VEGF-C与VEGF-D表达水平在p-Akt的阴性组、阳性组、强阳性组中存在组间差异(P＜0.05或＜0.01)。VEGF-C与VEGF-D表达水平在p-mTOR的阴性组、阳性组、强阳性组中也存在组间差异(P＜0.05或＜0.01)。
2、p-Akt、p-mTOR、VEGF-C、VEGF-D表达与胃癌淋巴管密度的关系
原位胃癌组织平均LVD为94.18±72.965，范围0-263，而癌旁正常组织中平均LVD为23.31±21.569，范围为0-95,两部位LVD计数存在显著性差异（P＜0.001）。胃癌组织中p-Akt、p-mTOR、VEGF-C和VEGF-D蛋白表达水平与LVD之间呈正相关关系（P＜0.05或＜0.01）。LVD在p-Akt或p-mTOR阴性组、阳性组、强阳性组中呈逐级递增的表现，三组间存在差异性（P＜0.05或P＜0.01）。
3、Akt活化抑制剂LY294002和mTOR活化抑制剂Rapamycin对胃癌SGC7901细胞的生长的影响
不同浓度的LY294002和Rapamyci n对胃部SGC7901细胞均有不同程度的抑制作用，其抑制率随着药物浓度和药物作用时间的延长而升高，各组间存在着显著性差异，P均＜0.001。
4、LY294002和Rapamycin对胃癌SGC7901细胞Akt信号通路及淋巴管新生子的影响
在LY294002干预组中，随着药物作用时间的延长p-Akt、p-mTOR、VEGF-C、VEGF-D各蛋白的表达水平逐渐下降，而且下降的程度与未干预组相比均有统计学意义（P＜0.05或＜0.01）；在组间比较中也存在显著性差异（P＜0.05）。Akt、mTOR表达水平在干预前后无明显变化，统计学上无差异（P＞0.05）。p-Akt与p-mTOR、VEGF-C、VEGF-D的表达呈一致性变化，统计学上呈正相关（P均＜0.05）。p-mTOR的表达与VEGF-C、VEGF-D表达变化同样也存在正相关性关系（P均＜0.05）。在Rapamycin组中，结果表示随着药物作用时间的延长p-mTOR、VEGF-C、VEGF-D各蛋白的表达逐渐下降，而且下降的程度与未干预组相比均有统计学意义（P＜0.05）；组间比较存在显著性差异（P＜0.05）。另外， Akt、p-Akt、mTOR的表达干预前后无明显变化，统计学上无差异（P＞0.05）；此外，p-mTOR与VEGF-C、VEGF-D表达变化呈正相关关系（P均＜0.05）。
结论：
p-Akt、p-mTOR、VEGF-C、VEGF-D蛋白表达水平均与胃癌淋巴管密度密切相关。通过阻断Akt和mTOR的活化可使淋巴管新生因子VEGF-C及VEGF-D表达下调。故此，胃癌中存在一条Akt/mTOR/VEGF-C/-D信号通路调控淋巴管新生乃至淋巴管转移。
Background and objective:
With the progress of basic anticancer research, lymphangiogenesis has beenshown to play more and more important role in the cancer prognosis. Gastric cancercan have a metastasis to lymphnode even in a very early stage which leading to a verypoor outcome of these patients. So nowadays, more and more insights have beenfocused to the mechanism of the lymphnode metastasis and lymphangiogenesis ofgastric cancer. Previous studies revealed that protein kinase B (Akt) played a key rolein tumor progress and prognosis and it has a close correlation with tumor lymphnodemetastasis. But the role of Akt played in the lymphangiogenesis of stomach cancer isstill unknown. In the current study, we analysed the relationship of the proteinexpression of Akt/mTOR signaling pathway with lymphangiogenic factorVEGF-C/-D and also with lymph vessel density (LVD) in situ and in vitro andeventually to clarify whether a Akt/mTOR/VEGF-C/-D signaling pathway exist inthe gastric cancer.
Methods:
1. In situ gastric cancer tissue experiments:55fresh gastric cancer tissues withmatched normal gastric mucosas from patients with disparate pathological stageswere collected and fresh-frozen in liquid nitrogen after surgical resections performedat the first affiliated hospital of Nanchang University. Of these,42were male and13were female. None of the patients had received chemo-, radio-or immuno-therapybefore resection. Part of each specimen were routinely processed, fixed in10%buffered formalin, and embedded in paraffin for histopathological analysis(hematoxylin and eosin stain) and for immunohistochemical staining. The expressionstatus of p-Akt, p-mTOR, D2-40, VEGF-C and–D was detected byimmunohistochemistry.
2. In vitro experiments: SGC7901stomach cancer cells were divided intoLY294002intervention group and Rapamycin intervention group. MTT method wasused to determine the effects of these two drugs on SGC7901stomach cancer cells surviving. Western blot was used to detected the expression level of Akt, p-Akt,mTOR, p-mTOR, VEGF-C and-D after the above two drugs intervention.Results:
1.Immunohistochemical expression and relationship of p-Akt、 p-mTOR、VEGF-C、VEGF-D in gastric cancer tissue.
The positive expression rates of p-Akt, p-mTOR, VEGF-C, VEGF-D in gastriccaner were74.5%,85.45%,72.73%and58.18%, respectively. The expression level ofp-Akt was correlated with p-mTOR, VEGF-C and-D expression levels, respectively(P <0.05or0.01). At the meanwhile, the expression level of p-mTORwas alsocorrelated with VEGF-C and–D (P <0.05or0.01).
2. Immunohistochemical expression of p-Akt、p-mTOR、VEGF-C、VEGF-D ingastric cancer tissue and their relationships with LVD.
The LVD was deteceted by immunostain of D2-40. The results revealed that themean LVD in gastric cancer was94.18±72.965, ranged from0-263.The mean LVDwas23.31±21.569in normal gastric tissue, ranged from0-95, which has a significantdifference when compared with that of cancer tissue (P <0.001). A closelyrelationship was found between LVD and p-Akt, p-mTOR, VEGF-C, VEGF-D,respetively(P <0.05or0.01). We also found that the expression level of p-mTOR,VEGF-C and VEGF-D were different among the p-Akt’s negative group, positivegroup,and strongly positive group (P <0.05).The expression level of VEGF-C andVEGF-D were also significantly different among the p-mTOR’s negative group,positive group and strongly positive group (P <0.05).
3. The effects of Akt activation inhibitor LY294002and the mTOR activationinhibitor Rapamycin on the growth of gastric cancer SGC7901cells.
LY294002and Rapamycin can effectively inhibit the growth of stomach cancerSGC7901cells. MTT assay indicated that the viability of SGC7901cells wassignificantly decreased in a concentration-and time-dependent manner afterLY294002and Rapamycin treatment, P <0.001.
4.The inhibitory effects of LY294002and Rapamycin on the expression ofVEGF-C and-D in gastric cancer cell line SGC7901.
LY294002and Rapamycin can effectively downregulate the phosphorylation ofAkt and mTOR, respectively. The expression level of VEGF-C and VEGF-D wasalso downregulated either in LY294002intervention group or Rapamycinintervention group.The inhibitory expression level of p-Akt, p-mTOR are postivelyrelated with the downregulatory expression levels of VEGF-C and–D (P <0.05).
Conclusion：
The expression levels of p-Akt、p-mTOR、VEGF-C and–D were all positivelycorrelated with lymph vessel density in gastric cancer. The blockage of Akt and/ormTOR activation could downregulate the expression of VEGF-C and–D. TheAkt/mTOR/VEGF-C/-D signaling pathway might exist in gastric cancer to regulatethe lymphangiogenesis and eventually to regulate the lymphatic metastasis of thecancer.
引文
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