南蛇藤提取物抑制肝癌淋巴管生成的作用及机制研究
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摘要
高转移、易复发是影响原发性肝细胞癌(hepatocellular carcinoma HCC,以下简称肝癌)疗效的首要问题。淋巴转移是肝癌转移的重要途径之一,也是影响患者预后的关键因素。
淋巴管新生是肿瘤淋巴转移过程中的早期事件。血管内皮生长因子C(vascular endothelial growth factor C,VEGF-C)是最重要的促淋巴管内皮生长因子之一,VEGF-C与血管内皮生长因子受体-3(vascular endothelial growth factor receptor3, VEGFR-3)特异结合后,激活下游系列信号转导通路,促进淋巴管内皮细胞的增殖、分化和迁移,形成新生淋巴管,介导肿瘤细胞的淋巴转移。
另有研究认为,肝癌细胞表达的趋化因子受体CXCR4(chemokine receptor4, CXCR4)与淋巴管内皮细胞分泌的基质细胞衍生因子1(stromal-derived factor-1, SDF-1)相互作用,诱导肝癌细胞向淋巴管趋化迁移；转移至淋巴管的肿瘤细胞通过分泌VEGF-C促进更多数目的淋巴管生成。同时,VEGF-C促进淋巴管内皮细胞分泌产生趋化因了SDF-1,并上调CXCR4在肿瘤细胞中的表达水平以募集更多的淋巴管内皮细胞,诱导其迁移并形成新生淋巴管。
我国传统中草药南蛇藤(Celastrus orbiculatus thunb.,卫矛科,南蛇藤属)以往主要用于抗炎、抗风湿治疗。本课题组前期研究显示,南蛇藤茎乙酸乙酯提取物(C.orbiculatus ethyl acetate extracts, COE)有显著的抗肿瘤作用,但其机制还未完全阐明。
本研究首次结合体内外实验对南蛇藤提取物抗肝癌淋巴管生成的作用及相关机制进行研究。体外研究显示,COE能抑制人肝癌细胞的增殖,通过线粒体凋亡通路诱导肝癌细胞凋亡,并降低其体外迁移的能力；COE能明显降低人肝癌细胞中VEGF-C的mRNA及蛋白表达,显著下调人肝癌细胞中CXCR4的蛋白表达水平,调控MAPK及PI3K/AKT信号转导通路。另外,COE明显抑制人淋巴管内皮细胞(human lymphatic endothelial cells, HLECs)的增殖,降低HLECs在Matrigel上形成管样结构的能力。
体内研究证实,COE可抑制裸鼠人肝癌细胞皮下移植瘤的生长,降低荷瘤裸鼠血清VEGF-C的浓度,减少移植瘤组织中的淋巴管新生和微淋巴管密度,降低移植瘤内VEGF-C和CXCR4的表达,下调AKT及ERK1/2蛋白的磷酸化表达水平。
综上分析,COE能抑制人肝癌细胞的生长、诱导其凋亡、降低其体外迁移能力,同时COE能有效抑制裸鼠人肝癌皮下移植瘤的生长和淋巴管的生成。一方面,COE通过下调肝癌细胞VEGF-C的表达直接抑制肝癌淋巴管的新生；另一方面,COE可抑制肝癌细胞CXCR4的表达(或通过抑制VEGF-C的分泌来下调CXCR4的表达),从而阻遏肝癌细胞向淋巴管趋化迁移,间接抑制淋巴管的新生。究其分子机制,我们认为COE可能通过调控ERK1/2及PI3K/AKT信号转导通路,下调移植瘤组织p-ERK1/2及p-AKT蛋白的表达水平,作用于VEGF-C/VEGFR-3和CXCR4/SDF-1生物轴,抑制肝癌淋巴管生成。本研究为进一步阐明南蛇藤提取物的抗肝癌机制及开发新型抗肝癌药物提供了新的策略和理论依据,也为肝癌的治疗提供了新的思路。
研究共分为两部分内容。
第一部分
南蛇藤提取物通过降低VEGF-C的表达抑制肝癌淋巴管生成的体外研究
目的：研究COE对人肝癌细胞增殖、凋亡、迁移及VEGF-C、CXCR4表达水平的影响,分析COE对MAPK和PI3K/AKT信号转导通路的调控作用,并观察COE能否抑制人淋巴管内皮细胞(human lymphatic endothelial cells, HLECs)在Matrigel上形成管样结构的能力,初步探讨COE抑制肝癌淋巴管生成的机制。
方法：人肝癌HepG2, HCCLM6细胞分别经不同浓度(10,20,40,80及160μ/mL)的COE处理后,MTT法观察增殖能力的改变;TUNEL法及流式细胞术检测细胞的凋亡,Western Blot分析线粒体凋亡通路相关分子Cyt-c、Bax、Bcl-2、Caspase-3及PARP的表达水平；Transwell小室检测体外迁移能力的变化；酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)测定细胞培养上清中VEGF-C的浓度,细胞免疫组化、Westernblot及实时荧光定量(realtime quantitative) PCR法分别检测VEGF-C蛋白及mRNA的表达量;同时以Western blot方法分析CXCR4蛋白的表达水平以及MAPK及PI3K/AKT信号转导通路中ERKI/2、JNK、p38MAPK和Akt蛋白的磷酸化表达水平。以不同浓度(10,20,40,80及160μg/mL)的COE作用HLECs, MTT法观察COE对其增殖能力的影响,成管试验观察HLECs在Matrigel基质胶上形成淋巴管样结构的影响。
结果：不同浓度(10,20,40,80,160μg/mL) COE作用24和48h,对HepG2和HCCLM6细胞的增殖均有抑制作用,并呈明显的时间和剂量依赖性。药物作用24h时,HepG2细胞在20～160μg/mL、HCCLM6细胞在40～160μg/mL浓度组的生长存活率与本细胞对照组相比均有统计学差异(P<0.05或P<0.01)；药物作用48h,各浓度COE均对两组细胞的增殖均呈现出显著抑制作用,与对照组相比差异显著(P<0.05或P<0.01),其中COE对HepG2细胞的增殖抑制率分别为11.7%、20.75%、27.9%、35.6%、57.2%,对HCCLM6细胞增殖抑制率分别为12.1%、27.5%、30.3%、37.9%、51.4%。COE对HepG2细胞和HCCLM6细胞的半数抑制浓度(50%concentration of inhibition, IC50)分别为131.948μg/mL和158.289μg/mL。
不同浓度(10,20,40,80及160μg/mL) COE作用24h后,TUNEL法检测HepG2细胞显示,随着药物作用浓度的加大,凋亡细胞数目逐渐增多,凋亡形态也越趋明显。各组平均凋亡指数为12.82±0.41%,18.74±0.66%,24.27±1.24%,34.23±2.10%,44.83±2.76%,与阴性对照组4.444±0.33%相比,差异具有显著性(P<0.01)。流式细胞术检测显示,HCCLM6细胞早期凋亡率分别为12.0±2.3%,13.9±1.5%,15.3±2.8%,17.2±3.4%及21.7±5.6%,与阴性对照组5.2±1.7%相比,具有显著差异(P<0.05或P<0.01)。结果说明COE能诱导HepG2和HCCLM6细胞的凋亡,并呈剂量依赖性。以Western blot进一步检测COE对HCCLM6细胞中与线粒体凋亡通路相关的分子蛋白表达水平。结果可见随着COE作用浓度的增加,细胞质内细胞色素c (Cyt-c)蛋白表达逐渐增多,Bax蛋白表达水平也逐渐升高,而Bcl-2蛋白表达水平逐渐降低,Cleaved Caspase-3及PARP蛋白水平也呈上升趋势。
Transwell实验结果显示,不同浓度(10,20,40,80及160μg/mL) COE作用24h后,HepG2及HCCLM6迁移细胞数均明显减少,呈剂量依赖性,与本细胞对照组相比差异显著(P<0.01),COE10～160μg/mL组HepG2细胞穿膜数量分别下降了28.17±2.86%,41.33±1.72%,51.72±1.50%,69.04±1.58%,72.49±2.18%,相应浓度的COE各组HCCLM6细胞穿膜数量分别下降了28.41±5.20%,37.29±6.82%,48.35±3.86%,63.57±2.46%,74.22±2.27%。
采用实时荧光定量(realtime quantitative) PCR法及Western blot检测不同浓度(10,20,40,80及160μg/mL) COE作用HepG2细胞24h后对VEGF-C mRNA及蛋白表达水平的影响,结果提示COE能明显抑制VEGF-C mRNA及蛋白的表达水平,且抑制作用呈剂量依赖性。分别采用细胞免疫组化及ELISA的方法,检测相应浓度COE作用24h后HCCLM6细胞中及其培养上清中VEGF-C蛋白的表达量。结果显示,随着药物作用浓度的增高,VEGF-C蛋白的表达量均逐渐下降,与对照组相比,除了浓度为10μg/mL组无差异外,其余各组细胞胞浆中和培养上清中VEGF-C蛋白的表达强度与对照组比较均有显著差异(P <0.01)。Western blot进一步分析COE对HepG2细胞CXCR4蛋白的影响,结果发现COE呈剂量依赖性抑制CXCR4蛋白的表达水平。
不同浓度(10,20,40,80及160μg/mL) COE作用24h后,Western blot分析COE对HCCLM6细胞中ERKI/2, JNK和p38MAPK及Akt蛋白磷酸化表达水平的影响。结果提示,当COE浓度为增至40μg/mL时,可检测出磷酸化JNK和p38MAPK蛋白的表达,且表达水平且随着药物浓度的加大逐渐上调。相反,磷酸化AKT蛋白的表达水平则逐渐下降。各组均可检测出磷酸化ERK1/2蛋白的表达,但未呈现明显的量效关系。
不同浓度(10,20,40,80及160μg/mL)的COE作用HLECs24h,40μg/mLCOE开始有效抑制HLECs的增殖,随着药物浓度加大,抑制作用逐渐增强,40,80,160μg/mL COE组细胞增殖抑制率分别为19.85±2.5%,38.08±6.11%,70.40±10.11%,与阴性对照组相比差异著(P<0.01)。将HLECs接种于Matrigel基质胶上,当COE作用18h时,10μg/mLCOE组HLECs形成的管样结构数目虽然与阴性对照组接近,但管腔变粗大,且有管壁断开现象；COE20μg/mL和40μg/mL组管样结构数目明显减少,至浓度为80μg/mL时,部分HLECs能够形成长条状或梭状,但不能形成完整的管样结构；COE浓度为160μg/mL时,HLECs难以连接形成连续的条索状结构,或仅见散在的簇状细胞团。
结论：南蛇藤提取物可抑制人肝癌细胞增殖,通过线粒体凋亡通路诱导肝癌细胞凋亡,并降低其体外迁移的能力,下调VEGF-C和CXCR4的表达水平,调控MAPKP信号通路及PI3K/AKT信号通路,抑制人淋巴管内皮细胞形成管样结构。
第二部分
南蛇藤提取物抑制肝癌裸鼠移植瘤淋巴管生成的作用及机制研究
目的：研究COE对裸鼠人肝癌移植瘤及淋巴管生成的影响,并分析COE对移植瘤组织VEGF-C、CXCR4蛋白表达水平及ERK1/2、AKT蛋白磷酸化表达水平的影响。
方法：BALB/c裸鼠56只,随机选择8只进入正常组,其余48只均于右侧背部皮下接种人肝癌细胞株HepG2(2.0×105)0.2mL/只。待瘤块约3mm3大小时,将裸鼠随机分为阴性对照组(生理盐水)、溶媒对照组(1%DMSO)、COE高、中、低剂量(40,20,10mg/kg)组及阳性对照5-Fu组(10mg/kg),每组8只。分组当天开始给药,连续21d,末次药24h后,检测肿瘤体积及重量,末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸盐缺口标记法(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, TUNEL)检测移植瘤组织内肿瘤细胞的凋亡,ELISA检测血清中VEGF-C的浓度,免疫组化检测移植瘤组织内微淋巴管密度及VEGF-C、CXCR4及p-ERK1/2和p-AKT蛋白的表达。
结果：与阴性对照组相比,COE各治疗组肿瘤生长速度明显减缓,瘤体积显著减小(P<0.05或P<0.01),且存在剂量依赖性。COE各组瘤重均低于阴性对照组(1.57±0.58g),其中低、中剂量组平均瘤重分别为1.06±0.29g和0.93±0.28g,抑瘤率为32.38%、40.46%(P<0.05),高剂量组平均瘤重为0.71±0.38g,抑瘤率达54.46%,显著抑制肿瘤生长(P<0.01),抑制作用具有剂量依赖性。阳性对照5-Fu组平均瘤重为0.69±0.20g,抑制率为56.26%,与COE高剂量组抑制作用接近。
肝癌移植瘤组织切片内,COE低、中、高剂量及5-Fu组平均凋亡细胞数目逐渐增多,分别为32.88±2.90、42.75±3.06、73.13±3.04、79.38±5.32,与阴性对照组7.63±0.52相比,差异显著(P<0.01)
ELISA检测荷瘤裸鼠血清VEGF-C浓度,COE低、中、高剂量组及阳性对照5-Fu组分别为205.85±56.33pg/mL,144.31±16.78pg/mL,129.93±16.94pg/mL,136.01±21.62pg/mL,与阴性对照组316.18±15.01pg/mL相比,均有统计学差异(P<0.05或P<0.01),提示COE能够明显抑制肝癌荷瘤裸鼠血清中VEGF-C的含量,且抑制作用呈剂量依赖性。
免疫组化分别检测肝癌移植瘤组织切片内VEGF-C、CXCR4、p-ERK1/2及p-AKT蛋白的表达,结果提示COE低、中、高剂量组及5-Fu组肿瘤细胞VEGF-C、CXCR4、p-ERK1/2及p-AKT阳性染色细胞数及染色强度均较阴性对照组显著下降(P<0.05或P<0.01)。表明COE能有效抑制移植瘤组织中VEGF-C、CXCR4的表达,并对p-ERKl/2及p-AKT的表达水平有抑制作用。
抗VEGFR-3及抗LYVE-1特异性抗体分别对移植瘤组织微淋巴管密度分析,结果均提示COE各治疗组及5-Fu组的MLVD较阴性对照组均有显著下降,且差异显著(P<0.05或P<0.01)。其中以抗VEGFR-3抗体标记的COE低、中、高剂量组、5-Fu组及阴性对照组MLVD分别为15.08±1.49、13.01±1.31、7.33±1.64、12.01±1.62、19.96±2.61,以抗LYVE-1抗体标记的相应各组MLVD分别为10.13±1.04、6.62±1.13、4.88±0.91、6.29±0.95、15.83±1.65。Pearson相关分析结果显示,LYVE-1与VEGFR-3标记的MLVD呈正相关(r=0.901,P=0.000)。表明COE能有效抑制肝癌移植瘤组织内淋巴管的生成,降低微淋巴管密度。
结论：COE能抑制裸鼠肝癌移植瘤的生长,并减少淋巴管的生成,其机制可能与促进肿瘤细胞凋亡、抑制肿瘤细胞VEGF-C和CXCR4的表达水平以及干扰ERK1/2及PI3K/AKT信号转导通路相关。
Metastasis and recurrence are the main obstacles to the improvement of treatment efficacy of hepatocellular carcinoma. Metastasis spread of the hepatocellular carcinoma via lymphatic system often represents the key step of dissemination and serves as a major prognostic indicator for the progression.
Lymphangiogenesis, the generation of new lymphatic vessels, often occurs at an early stage of lymphatic metastasis in human malignant tumors. Among several prolymphangiogenic factors secreted by tumor cells, the most potent one is vascular endothelial growth factor (VEGF)-C, which bind to a tyrosine kinase receptor, VEGF receptor (R)-3, expressed on the lymphatic endothelium. The VEGF-C/VEGFR-3axis plays a key role in activating resultant signaling pathway, then promoting endothelial cell proliferation, migration, and survival, significantly increasing tumor-associated lymphangiogenesis and the incidence of lymphatic metastasis.
Recent evidence indicates that the chemokine receptors and their corresponding chemokine ligands directly contribute to lymphatic metastasis of tumor cells. CXC chemokine receptor-4(CXCR4) is widely expressed in various types of tumor cells including hepatocarcinoma cells. Stromal-derived factor-1(SDF-1) is a unique ligand of the CXCR4, which can be expressed on lymphatic endothelial cells (LECs). Activation of the SDF-1/CXCR4axis can contribute to chemoattraction between tumor cells and LECs including inducing the activation and tubule formation of LECs, promoting tumor lymphangiogenesis and chemotaxis of tumor cells to lymphatic vessels. Further studies show that, in addition to mediating tumor lymphangiogenesis, VEGF-C may also induce the secretion of SDF-1by LECs and up-regulate the expressing of chemokine receptors in tumor cells and thereby active tumor invasion of lymphatics, which indirectly promote tumor lymphangiogenesis.
The Celastraceae plant Celastrus orbiculatus has traditionally been used as an effective remedy for arthritis and other inflammatory diseases in China. Our previous studies revealed that ethyl acetate extract from C. orbiculatus stem exerts potent antitumor activity. However, the mechanisms underlying this activity remain unclear, and further investigations are needed to explore the potential of COE for clinical use.
In the present study, we have examined, for the first time, the inhibitory effects of COE on hepatocellular carcinoma lymphangiogenesis and investigated the underlying associated molecular mechanisms. Results of in vitro study indicated that COE can significantly inhibit proliferation, migration and induced Mitochondria-mediated apoptosis of hepatoma cells in a dose-dependent manner. More importantly, COE treatment can effectively down-regulate the protein and gene expression of VEGF-C, suppressing the protein expression of CXCR4. Furthermore, activation of extracellular signal-regulated kinase (ERK), p38kinase, and c-Jun N-terminal kinase (JNK) phosphorylation, and down-regulation of Akt phosphorylation was observed. Simultaneously, it has been observed that COE can inhibit proliferation and the tube-like structure formation of HLECs.
In in vivo studies, COE can attenuate growth and decrease lymphangiogenesis of the subcutaneous tumor in hepatocellular carcinoma xenograft model, and thereby reduce lymphatic vessel density. Meanwhile, COE strongly suppresses VEGF-C and CXCR4protein expression in tumor sections, and moreover efficiently down-regulates of the phosphorylation of extracellular signal-regulated kinase1/2and AKT.
Taken together, the C. orbiculatus extract acts on hepatocellular carcinoma and tumor lymphangiogenesis by inhibiting cellular proliferation, inducing apoptosis, down-regulating of the VEGF-C and CXCR4, and moreover by modulating the extracellular signal-regulated kinase1/2and the phosphatidylinositol3-kinase/AKT signaling pathway. Our study may help elucidate the mechanism of antitumor activity of COE and provide further evidence for the development of a new type of antitumor drug.
The findings will be described in two parts as follows.
Part I:Extracts from Celastrus Orbiculatus Inhibit Hepatocellular Carcinoma Lymphangiogenesis by down-regulating the expression of VEGF-C in vitro
Objective:To determin the antitumor function of Celastrus orbiculatus Exacts (COE) and explore the mechanisms preliminarily by investigating its effects on proliferation, apoptosis, migration and vascular endothelial growth factor-C (VEGF-C), CXC chemokine receptor-4(CXCR4) expression, the MAPK and PI3K/AKT signaling transduction pathwaws in human hepatoma cells and the anti-lymphangiogenesis of HLECS.
Methods:Human hepatocellular carcinoma cells were treated with COE at different concentrations (10,20,40,80,160mg/L). The effect of COE on cell viability was examined using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Cellular apoptosis following COE treatment was assessed by TUNEL and flow cytometry assay. The migration ability of cells was assayed by Transwell chamber experiment. VEGF-C production in the conditioned medium was analyzed with the human VEGF-C ELISA kit. The mRNA expressions of VEGF-C were detected by realtime quantitative PCR. The protein expressions of VEGF-C, Cyt-c、Bax、Bcl-2、Caspase-3, PARP, CXCR4, ERKI/2、JNK、p38MAKP and Akt were detected by Western blot analysis. The anti-lymphangiogenesis effects of COE were analyzed using an in vitro tube-formation assay.
Results:COE significantly inhibited the proliferation and induced the apoptosis of HepG2and HCCLM6cells in a dose-dependent manner. The inhibition rates of HepG2cells were11.7%、20.75%、27.9%、35.6%and57.2%for10to160μg/mL of COE, respectively. While the corresponding inhibition rates of HCCLM6cells were12.1%,27.5%,30.3%,37.9%,51.4%. After exposure to COE at doses of10to160μg/mL for24h, apoptosis index of HepG2cells were12.82±0.41%,18.74±0.66%,24.27±1.24%,34.23±2.10%,44.83±2.76%, respectively. And the percentage of the early apoptotic HCCLM6cells (Annexin V+PI-) were12.0±2.3%,13.9±1.5%,15.3±2.8%,17.2±3.4%and21.7±5.6%, respectively. Meanwhile, COE increased Bax expression, decreased Bcl-2expression, promoted the release of cytochrome c, activated cleavage of caspase-3and poly (ADP-ribose) polymerase (PARP). COE significantly inhibited VEGF-C expression at both mRNA and protein levels, suppressed the protein expression of CXCR4in a dose-dependent manner. COE activated the extracellular signal-regulated kinase (ERK), p38kinase, and c-Jun N-terminal kinase (JNK) phosphorylation, and down-regulated the Akt phosphorylation. COE significantly inhibited proliferation and the tube-like structure formation of HLECs on Matrigel in a dose-dependent manner.
Conclusion:COE acts on hepatocellular carcinoma and lymphangiogenesis by inhibiting cellular proliferation, inducing apoptosis, down-regulating of the VEGF-C and CXCR4, and modulating the extracellular signal-regulated kinase1/2and the phosphatidylinositol3-kinase/AKT signaling pathway.
Part Ⅱ:Inhibition of Lymphangiogenesis by Extracts from Celastrus orbiculatus in a xenograft model of Hepatocellular Carcinoma and Associated mechanisms
Objective:To investigate the effect of Celastrus orbiculatus ethyl acetate extracts (COE) on tumor lymphangiogenesis in hepatocellular carcinoma xenograft model, and further explore its potential mechanisms.
Methods:HepG2cells (2.0×105) suspended in0.2mL of serum-free DMEM were inoculated s.c. in the right flank of each nude mouse. When the average tumor volume reached3mm3, mice were randomly divided into six groups (eight mice per group) as follows:untreated control, solvent vehicle control (DMSO), different dosages COE-treated groups and5-Fu treated group. The animals received200μL of a vehicle [1%DMSO and99%saline] or COE at different dosages (10,20or40mg/kg/d) by gavage. In the5-Fu treated group, mice were injected intraperitoneally with10mg/kg/d of5-Fu. Mice received21doses and24h after the last dose, they were sacrificed and the tumors were removed and weighed. Apoptosis in tumor tissue were detected by the histologic TUNEL staining. ELISA was used to detect the expression of vascular endothetial growth factor-C (VEGF-C) in blood serum. And MLVD, VEGF-C, CXCR4, p-ERK1/2, p-AKT expression were detected by immunohistochemisty.
Results:COE has been shown to significantly inhibit the growth of subcutaneous tumor in hepatocellular carcinoma xenograft model in a dose-dependent manner. The mean endpoint tumor masses of10,20, and40mg/kg/d COE groups were1.06±0.29g,0.93±0.28g (P<0.05) and0.71±0.38g (P<0.01), compared with1.57±0.58g of the untreated control. Apoptosis in tumor tissue induced by COE showed dose-dependent increase in10,20and40mg/kg/d COE groups, the mean apoptotic cells32.88±2.90(P<0.01)、42.75±3.06(P<0.01)、73.13±3.04(P<0.01) and79.38±5.32(P<0.01), compared with the untreated control (7.63±0.52). VEGF-C protein expression in the serum of10,20, and40mg/kg/d COE were respectively down-regulated to205.85±56.33pg/mL (P<0.05),144.31±16.78pg/mL (P<0.01),129.93±16.94pg/mL(P<0.01), compared with the untreated control(316.18±15.01pg/mL). Immunohistochemisty revealed that the percentages and the intensity of VEGF-C, CXCR4, p-ERK1/2and p-AKT positive-staining cells were respectively decreased when compared to the untreated group (P<0.05or P<0.01).VEGFR-3positive staining MLVD were respectively15.08±1.49,13.01±1.31and7.33±1.64in10,20and40mg/kg/d COE groups, which were significantly lower compared to untreated group (19.96±2.61)(P<0.01). Similar results were found when comparing LYVE-1positive staining MLVD in each COE treated group to untreated group (P<0.05or P<0.01).
Conclusion:COE exerts the effective anti-tumor action via induction tumor cell apoptosis and inhibition of lymphangiogenesis in hepatocellular carcinoma xenograft model. The effect is possibly due to the down-regulation of VEGF-C and CXCR4expression by COE in hepatoma cells, which may be through modulation of extracellular signal-regulated kinase1/2and the phosphatidylinositol3-kinase/AKT signaling pathway. All these results provide evidence that COE may play an important role in down-regulating the tumor lymphangiogenesis and it may be a valuable therapeutic strategy for hepatocellular carcinoma in the future.
淋巴管新生是肿瘤淋巴转移过程中的早期事件。血管内皮生长因子C(vascular endothelial growth factor C,VEGF-C)是最重要的促淋巴管内皮生长因子之一,VEGF-C与血管内皮生长因子受体-3(vascular endothelial growth factor receptor3, VEGFR-3)特异结合后,激活下游系列信号转导通路,促进淋巴管内皮细胞的增殖、分化和迁移,形成新生淋巴管,介导肿瘤细胞的淋巴转移。
另有研究认为,肝癌细胞表达的趋化因子受体CXCR4(chemokine receptor4, CXCR4)与淋巴管内皮细胞分泌的基质细胞衍生因子1(stromal-derived factor-1, SDF-1)相互作用,诱导肝癌细胞向淋巴管趋化迁移；转移至淋巴管的肿瘤细胞通过分泌VEGF-C促进更多数目的淋巴管生成。同时,VEGF-C促进淋巴管内皮细胞分泌产生趋化因了SDF-1,并上调CXCR4在肿瘤细胞中的表达水平以募集更多的淋巴管内皮细胞,诱导其迁移并形成新生淋巴管。
我国传统中草药南蛇藤(Celastrus orbiculatus thunb.,卫矛科,南蛇藤属)以往主要用于抗炎、抗风湿治疗。本课题组前期研究显示,南蛇藤茎乙酸乙酯提取物(C.orbiculatus ethyl acetate extracts, COE)有显著的抗肿瘤作用,但其机制还未完全阐明。
本研究首次结合体内外实验对南蛇藤提取物抗肝癌淋巴管生成的作用及相关机制进行研究。体外研究显示,COE能抑制人肝癌细胞的增殖,通过线粒体凋亡通路诱导肝癌细胞凋亡,并降低其体外迁移的能力；COE能明显降低人肝癌细胞中VEGF-C的mRNA及蛋白表达,显著下调人肝癌细胞中CXCR4的蛋白表达水平,调控MAPK及PI3K/AKT信号转导通路。另外,COE明显抑制人淋巴管内皮细胞(human lymphatic endothelial cells, HLECs)的增殖,降低HLECs在Matrigel上形成管样结构的能力。
体内研究证实,COE可抑制裸鼠人肝癌细胞皮下移植瘤的生长,降低荷瘤裸鼠血清VEGF-C的浓度,减少移植瘤组织中的淋巴管新生和微淋巴管密度,降低移植瘤内VEGF-C和CXCR4的表达,下调AKT及ERK1/2蛋白的磷酸化表达水平。
综上分析,COE能抑制人肝癌细胞的生长、诱导其凋亡、降低其体外迁移能力,同时COE能有效抑制裸鼠人肝癌皮下移植瘤的生长和淋巴管的生成。一方面,COE通过下调肝癌细胞VEGF-C的表达直接抑制肝癌淋巴管的新生；另一方面,COE可抑制肝癌细胞CXCR4的表达(或通过抑制VEGF-C的分泌来下调CXCR4的表达),从而阻遏肝癌细胞向淋巴管趋化迁移,间接抑制淋巴管的新生。究其分子机制,我们认为COE可能通过调控ERK1/2及PI3K/AKT信号转导通路,下调移植瘤组织p-ERK1/2及p-AKT蛋白的表达水平,作用于VEGF-C/VEGFR-3和CXCR4/SDF-1生物轴,抑制肝癌淋巴管生成。本研究为进一步阐明南蛇藤提取物的抗肝癌机制及开发新型抗肝癌药物提供了新的策略和理论依据,也为肝癌的治疗提供了新的思路。
研究共分为两部分内容。
第一部分
南蛇藤提取物通过降低VEGF-C的表达抑制肝癌淋巴管生成的体外研究
目的：研究COE对人肝癌细胞增殖、凋亡、迁移及VEGF-C、CXCR4表达水平的影响,分析COE对MAPK和PI3K/AKT信号转导通路的调控作用,并观察COE能否抑制人淋巴管内皮细胞(human lymphatic endothelial cells, HLECs)在Matrigel上形成管样结构的能力,初步探讨COE抑制肝癌淋巴管生成的机制。
方法：人肝癌HepG2, HCCLM6细胞分别经不同浓度(10,20,40,80及160μ/mL)的COE处理后,MTT法观察增殖能力的改变;TUNEL法及流式细胞术检测细胞的凋亡,Western Blot分析线粒体凋亡通路相关分子Cyt-c、Bax、Bcl-2、Caspase-3及PARP的表达水平；Transwell小室检测体外迁移能力的变化；酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)测定细胞培养上清中VEGF-C的浓度,细胞免疫组化、Westernblot及实时荧光定量(realtime quantitative) PCR法分别检测VEGF-C蛋白及mRNA的表达量;同时以Western blot方法分析CXCR4蛋白的表达水平以及MAPK及PI3K/AKT信号转导通路中ERKI/2、JNK、p38MAPK和Akt蛋白的磷酸化表达水平。以不同浓度(10,20,40,80及160μg/mL)的COE作用HLECs, MTT法观察COE对其增殖能力的影响,成管试验观察HLECs在Matrigel基质胶上形成淋巴管样结构的影响。
结果：不同浓度(10,20,40,80,160μg/mL) COE作用24和48h,对HepG2和HCCLM6细胞的增殖均有抑制作用,并呈明显的时间和剂量依赖性。药物作用24h时,HepG2细胞在20～160μg/mL、HCCLM6细胞在40～160μg/mL浓度组的生长存活率与本细胞对照组相比均有统计学差异(P<0.05或P<0.01)；药物作用48h,各浓度COE均对两组细胞的增殖均呈现出显著抑制作用,与对照组相比差异显著(P<0.05或P<0.01),其中COE对HepG2细胞的增殖抑制率分别为11.7%、20.75%、27.9%、35.6%、57.2%,对HCCLM6细胞增殖抑制率分别为12.1%、27.5%、30.3%、37.9%、51.4%。COE对HepG2细胞和HCCLM6细胞的半数抑制浓度(50%concentration of inhibition, IC50)分别为131.948μg/mL和158.289μg/mL。
不同浓度(10,20,40,80及160μg/mL) COE作用24h后,TUNEL法检测HepG2细胞显示,随着药物作用浓度的加大,凋亡细胞数目逐渐增多,凋亡形态也越趋明显。各组平均凋亡指数为12.82±0.41%,18.74±0.66%,24.27±1.24%,34.23±2.10%,44.83±2.76%,与阴性对照组4.444±0.33%相比,差异具有显著性(P<0.01)。流式细胞术检测显示,HCCLM6细胞早期凋亡率分别为12.0±2.3%,13.9±1.5%,15.3±2.8%,17.2±3.4%及21.7±5.6%,与阴性对照组5.2±1.7%相比,具有显著差异(P<0.05或P<0.01)。结果说明COE能诱导HepG2和HCCLM6细胞的凋亡,并呈剂量依赖性。以Western blot进一步检测COE对HCCLM6细胞中与线粒体凋亡通路相关的分子蛋白表达水平。结果可见随着COE作用浓度的增加,细胞质内细胞色素c (Cyt-c)蛋白表达逐渐增多,Bax蛋白表达水平也逐渐升高,而Bcl-2蛋白表达水平逐渐降低,Cleaved Caspase-3及PARP蛋白水平也呈上升趋势。
Transwell实验结果显示,不同浓度(10,20,40,80及160μg/mL) COE作用24h后,HepG2及HCCLM6迁移细胞数均明显减少,呈剂量依赖性,与本细胞对照组相比差异显著(P<0.01),COE10～160μg/mL组HepG2细胞穿膜数量分别下降了28.17±2.86%,41.33±1.72%,51.72±1.50%,69.04±1.58%,72.49±2.18%,相应浓度的COE各组HCCLM6细胞穿膜数量分别下降了28.41±5.20%,37.29±6.82%,48.35±3.86%,63.57±2.46%,74.22±2.27%。
采用实时荧光定量(realtime quantitative) PCR法及Western blot检测不同浓度(10,20,40,80及160μg/mL) COE作用HepG2细胞24h后对VEGF-C mRNA及蛋白表达水平的影响,结果提示COE能明显抑制VEGF-C mRNA及蛋白的表达水平,且抑制作用呈剂量依赖性。分别采用细胞免疫组化及ELISA的方法,检测相应浓度COE作用24h后HCCLM6细胞中及其培养上清中VEGF-C蛋白的表达量。结果显示,随着药物作用浓度的增高,VEGF-C蛋白的表达量均逐渐下降,与对照组相比,除了浓度为10μg/mL组无差异外,其余各组细胞胞浆中和培养上清中VEGF-C蛋白的表达强度与对照组比较均有显著差异(P <0.01)。Western blot进一步分析COE对HepG2细胞CXCR4蛋白的影响,结果发现COE呈剂量依赖性抑制CXCR4蛋白的表达水平。
不同浓度(10,20,40,80及160μg/mL) COE作用24h后,Western blot分析COE对HCCLM6细胞中ERKI/2, JNK和p38MAPK及Akt蛋白磷酸化表达水平的影响。结果提示,当COE浓度为增至40μg/mL时,可检测出磷酸化JNK和p38MAPK蛋白的表达,且表达水平且随着药物浓度的加大逐渐上调。相反,磷酸化AKT蛋白的表达水平则逐渐下降。各组均可检测出磷酸化ERK1/2蛋白的表达,但未呈现明显的量效关系。
不同浓度(10,20,40,80及160μg/mL)的COE作用HLECs24h,40μg/mLCOE开始有效抑制HLECs的增殖,随着药物浓度加大,抑制作用逐渐增强,40,80,160μg/mL COE组细胞增殖抑制率分别为19.85±2.5%,38.08±6.11%,70.40±10.11%,与阴性对照组相比差异著(P<0.01)。将HLECs接种于Matrigel基质胶上,当COE作用18h时,10μg/mLCOE组HLECs形成的管样结构数目虽然与阴性对照组接近,但管腔变粗大,且有管壁断开现象；COE20μg/mL和40μg/mL组管样结构数目明显减少,至浓度为80μg/mL时,部分HLECs能够形成长条状或梭状,但不能形成完整的管样结构；COE浓度为160μg/mL时,HLECs难以连接形成连续的条索状结构,或仅见散在的簇状细胞团。
结论：南蛇藤提取物可抑制人肝癌细胞增殖,通过线粒体凋亡通路诱导肝癌细胞凋亡,并降低其体外迁移的能力,下调VEGF-C和CXCR4的表达水平,调控MAPKP信号通路及PI3K/AKT信号通路,抑制人淋巴管内皮细胞形成管样结构。
第二部分
南蛇藤提取物抑制肝癌裸鼠移植瘤淋巴管生成的作用及机制研究
目的：研究COE对裸鼠人肝癌移植瘤及淋巴管生成的影响,并分析COE对移植瘤组织VEGF-C、CXCR4蛋白表达水平及ERK1/2、AKT蛋白磷酸化表达水平的影响。
方法：BALB/c裸鼠56只,随机选择8只进入正常组,其余48只均于右侧背部皮下接种人肝癌细胞株HepG2(2.0×105)0.2mL/只。待瘤块约3mm3大小时,将裸鼠随机分为阴性对照组(生理盐水)、溶媒对照组(1%DMSO)、COE高、中、低剂量(40,20,10mg/kg)组及阳性对照5-Fu组(10mg/kg),每组8只。分组当天开始给药,连续21d,末次药24h后,检测肿瘤体积及重量,末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸盐缺口标记法(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, TUNEL)检测移植瘤组织内肿瘤细胞的凋亡,ELISA检测血清中VEGF-C的浓度,免疫组化检测移植瘤组织内微淋巴管密度及VEGF-C、CXCR4及p-ERK1/2和p-AKT蛋白的表达。
结果：与阴性对照组相比,COE各治疗组肿瘤生长速度明显减缓,瘤体积显著减小(P<0.05或P<0.01),且存在剂量依赖性。COE各组瘤重均低于阴性对照组(1.57±0.58g),其中低、中剂量组平均瘤重分别为1.06±0.29g和0.93±0.28g,抑瘤率为32.38%、40.46%(P<0.05),高剂量组平均瘤重为0.71±0.38g,抑瘤率达54.46%,显著抑制肿瘤生长(P<0.01),抑制作用具有剂量依赖性。阳性对照5-Fu组平均瘤重为0.69±0.20g,抑制率为56.26%,与COE高剂量组抑制作用接近。
肝癌移植瘤组织切片内,COE低、中、高剂量及5-Fu组平均凋亡细胞数目逐渐增多,分别为32.88±2.90、42.75±3.06、73.13±3.04、79.38±5.32,与阴性对照组7.63±0.52相比,差异显著(P<0.01)
ELISA检测荷瘤裸鼠血清VEGF-C浓度,COE低、中、高剂量组及阳性对照5-Fu组分别为205.85±56.33pg/mL,144.31±16.78pg/mL,129.93±16.94pg/mL,136.01±21.62pg/mL,与阴性对照组316.18±15.01pg/mL相比,均有统计学差异(P<0.05或P<0.01),提示COE能够明显抑制肝癌荷瘤裸鼠血清中VEGF-C的含量,且抑制作用呈剂量依赖性。
免疫组化分别检测肝癌移植瘤组织切片内VEGF-C、CXCR4、p-ERK1/2及p-AKT蛋白的表达,结果提示COE低、中、高剂量组及5-Fu组肿瘤细胞VEGF-C、CXCR4、p-ERK1/2及p-AKT阳性染色细胞数及染色强度均较阴性对照组显著下降(P<0.05或P<0.01)。表明COE能有效抑制移植瘤组织中VEGF-C、CXCR4的表达,并对p-ERKl/2及p-AKT的表达水平有抑制作用。
抗VEGFR-3及抗LYVE-1特异性抗体分别对移植瘤组织微淋巴管密度分析,结果均提示COE各治疗组及5-Fu组的MLVD较阴性对照组均有显著下降,且差异显著(P<0.05或P<0.01)。其中以抗VEGFR-3抗体标记的COE低、中、高剂量组、5-Fu组及阴性对照组MLVD分别为15.08±1.49、13.01±1.31、7.33±1.64、12.01±1.62、19.96±2.61,以抗LYVE-1抗体标记的相应各组MLVD分别为10.13±1.04、6.62±1.13、4.88±0.91、6.29±0.95、15.83±1.65。Pearson相关分析结果显示,LYVE-1与VEGFR-3标记的MLVD呈正相关(r=0.901,P=0.000)。表明COE能有效抑制肝癌移植瘤组织内淋巴管的生成,降低微淋巴管密度。
结论：COE能抑制裸鼠肝癌移植瘤的生长,并减少淋巴管的生成,其机制可能与促进肿瘤细胞凋亡、抑制肿瘤细胞VEGF-C和CXCR4的表达水平以及干扰ERK1/2及PI3K/AKT信号转导通路相关。
Metastasis and recurrence are the main obstacles to the improvement of treatment efficacy of hepatocellular carcinoma. Metastasis spread of the hepatocellular carcinoma via lymphatic system often represents the key step of dissemination and serves as a major prognostic indicator for the progression.
Lymphangiogenesis, the generation of new lymphatic vessels, often occurs at an early stage of lymphatic metastasis in human malignant tumors. Among several prolymphangiogenic factors secreted by tumor cells, the most potent one is vascular endothelial growth factor (VEGF)-C, which bind to a tyrosine kinase receptor, VEGF receptor (R)-3, expressed on the lymphatic endothelium. The VEGF-C/VEGFR-3axis plays a key role in activating resultant signaling pathway, then promoting endothelial cell proliferation, migration, and survival, significantly increasing tumor-associated lymphangiogenesis and the incidence of lymphatic metastasis.
Recent evidence indicates that the chemokine receptors and their corresponding chemokine ligands directly contribute to lymphatic metastasis of tumor cells. CXC chemokine receptor-4(CXCR4) is widely expressed in various types of tumor cells including hepatocarcinoma cells. Stromal-derived factor-1(SDF-1) is a unique ligand of the CXCR4, which can be expressed on lymphatic endothelial cells (LECs). Activation of the SDF-1/CXCR4axis can contribute to chemoattraction between tumor cells and LECs including inducing the activation and tubule formation of LECs, promoting tumor lymphangiogenesis and chemotaxis of tumor cells to lymphatic vessels. Further studies show that, in addition to mediating tumor lymphangiogenesis, VEGF-C may also induce the secretion of SDF-1by LECs and up-regulate the expressing of chemokine receptors in tumor cells and thereby active tumor invasion of lymphatics, which indirectly promote tumor lymphangiogenesis.
The Celastraceae plant Celastrus orbiculatus has traditionally been used as an effective remedy for arthritis and other inflammatory diseases in China. Our previous studies revealed that ethyl acetate extract from C. orbiculatus stem exerts potent antitumor activity. However, the mechanisms underlying this activity remain unclear, and further investigations are needed to explore the potential of COE for clinical use.
In the present study, we have examined, for the first time, the inhibitory effects of COE on hepatocellular carcinoma lymphangiogenesis and investigated the underlying associated molecular mechanisms. Results of in vitro study indicated that COE can significantly inhibit proliferation, migration and induced Mitochondria-mediated apoptosis of hepatoma cells in a dose-dependent manner. More importantly, COE treatment can effectively down-regulate the protein and gene expression of VEGF-C, suppressing the protein expression of CXCR4. Furthermore, activation of extracellular signal-regulated kinase (ERK), p38kinase, and c-Jun N-terminal kinase (JNK) phosphorylation, and down-regulation of Akt phosphorylation was observed. Simultaneously, it has been observed that COE can inhibit proliferation and the tube-like structure formation of HLECs.
In in vivo studies, COE can attenuate growth and decrease lymphangiogenesis of the subcutaneous tumor in hepatocellular carcinoma xenograft model, and thereby reduce lymphatic vessel density. Meanwhile, COE strongly suppresses VEGF-C and CXCR4protein expression in tumor sections, and moreover efficiently down-regulates of the phosphorylation of extracellular signal-regulated kinase1/2and AKT.
Taken together, the C. orbiculatus extract acts on hepatocellular carcinoma and tumor lymphangiogenesis by inhibiting cellular proliferation, inducing apoptosis, down-regulating of the VEGF-C and CXCR4, and moreover by modulating the extracellular signal-regulated kinase1/2and the phosphatidylinositol3-kinase/AKT signaling pathway. Our study may help elucidate the mechanism of antitumor activity of COE and provide further evidence for the development of a new type of antitumor drug.
The findings will be described in two parts as follows.
Part I:Extracts from Celastrus Orbiculatus Inhibit Hepatocellular Carcinoma Lymphangiogenesis by down-regulating the expression of VEGF-C in vitro
Objective:To determin the antitumor function of Celastrus orbiculatus Exacts (COE) and explore the mechanisms preliminarily by investigating its effects on proliferation, apoptosis, migration and vascular endothelial growth factor-C (VEGF-C), CXC chemokine receptor-4(CXCR4) expression, the MAPK and PI3K/AKT signaling transduction pathwaws in human hepatoma cells and the anti-lymphangiogenesis of HLECS.
Methods:Human hepatocellular carcinoma cells were treated with COE at different concentrations (10,20,40,80,160mg/L). The effect of COE on cell viability was examined using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. Cellular apoptosis following COE treatment was assessed by TUNEL and flow cytometry assay. The migration ability of cells was assayed by Transwell chamber experiment. VEGF-C production in the conditioned medium was analyzed with the human VEGF-C ELISA kit. The mRNA expressions of VEGF-C were detected by realtime quantitative PCR. The protein expressions of VEGF-C, Cyt-c、Bax、Bcl-2、Caspase-3, PARP, CXCR4, ERKI/2、JNK、p38MAKP and Akt were detected by Western blot analysis. The anti-lymphangiogenesis effects of COE were analyzed using an in vitro tube-formation assay.
Results:COE significantly inhibited the proliferation and induced the apoptosis of HepG2and HCCLM6cells in a dose-dependent manner. The inhibition rates of HepG2cells were11.7%、20.75%、27.9%、35.6%and57.2%for10to160μg/mL of COE, respectively. While the corresponding inhibition rates of HCCLM6cells were12.1%,27.5%,30.3%,37.9%,51.4%. After exposure to COE at doses of10to160μg/mL for24h, apoptosis index of HepG2cells were12.82±0.41%,18.74±0.66%,24.27±1.24%,34.23±2.10%,44.83±2.76%, respectively. And the percentage of the early apoptotic HCCLM6cells (Annexin V+PI-) were12.0±2.3%,13.9±1.5%,15.3±2.8%,17.2±3.4%and21.7±5.6%, respectively. Meanwhile, COE increased Bax expression, decreased Bcl-2expression, promoted the release of cytochrome c, activated cleavage of caspase-3and poly (ADP-ribose) polymerase (PARP). COE significantly inhibited VEGF-C expression at both mRNA and protein levels, suppressed the protein expression of CXCR4in a dose-dependent manner. COE activated the extracellular signal-regulated kinase (ERK), p38kinase, and c-Jun N-terminal kinase (JNK) phosphorylation, and down-regulated the Akt phosphorylation. COE significantly inhibited proliferation and the tube-like structure formation of HLECs on Matrigel in a dose-dependent manner.
Conclusion:COE acts on hepatocellular carcinoma and lymphangiogenesis by inhibiting cellular proliferation, inducing apoptosis, down-regulating of the VEGF-C and CXCR4, and modulating the extracellular signal-regulated kinase1/2and the phosphatidylinositol3-kinase/AKT signaling pathway.
Part Ⅱ:Inhibition of Lymphangiogenesis by Extracts from Celastrus orbiculatus in a xenograft model of Hepatocellular Carcinoma and Associated mechanisms
Objective:To investigate the effect of Celastrus orbiculatus ethyl acetate extracts (COE) on tumor lymphangiogenesis in hepatocellular carcinoma xenograft model, and further explore its potential mechanisms.
Methods:HepG2cells (2.0×105) suspended in0.2mL of serum-free DMEM were inoculated s.c. in the right flank of each nude mouse. When the average tumor volume reached3mm3, mice were randomly divided into six groups (eight mice per group) as follows:untreated control, solvent vehicle control (DMSO), different dosages COE-treated groups and5-Fu treated group. The animals received200μL of a vehicle [1%DMSO and99%saline] or COE at different dosages (10,20or40mg/kg/d) by gavage. In the5-Fu treated group, mice were injected intraperitoneally with10mg/kg/d of5-Fu. Mice received21doses and24h after the last dose, they were sacrificed and the tumors were removed and weighed. Apoptosis in tumor tissue were detected by the histologic TUNEL staining. ELISA was used to detect the expression of vascular endothetial growth factor-C (VEGF-C) in blood serum. And MLVD, VEGF-C, CXCR4, p-ERK1/2, p-AKT expression were detected by immunohistochemisty.
Results:COE has been shown to significantly inhibit the growth of subcutaneous tumor in hepatocellular carcinoma xenograft model in a dose-dependent manner. The mean endpoint tumor masses of10,20, and40mg/kg/d COE groups were1.06±0.29g,0.93±0.28g (P<0.05) and0.71±0.38g (P<0.01), compared with1.57±0.58g of the untreated control. Apoptosis in tumor tissue induced by COE showed dose-dependent increase in10,20and40mg/kg/d COE groups, the mean apoptotic cells32.88±2.90(P<0.01)、42.75±3.06(P<0.01)、73.13±3.04(P<0.01) and79.38±5.32(P<0.01), compared with the untreated control (7.63±0.52). VEGF-C protein expression in the serum of10,20, and40mg/kg/d COE were respectively down-regulated to205.85±56.33pg/mL (P<0.05),144.31±16.78pg/mL (P<0.01),129.93±16.94pg/mL(P<0.01), compared with the untreated control(316.18±15.01pg/mL). Immunohistochemisty revealed that the percentages and the intensity of VEGF-C, CXCR4, p-ERK1/2and p-AKT positive-staining cells were respectively decreased when compared to the untreated group (P<0.05or P<0.01).VEGFR-3positive staining MLVD were respectively15.08±1.49,13.01±1.31and7.33±1.64in10,20and40mg/kg/d COE groups, which were significantly lower compared to untreated group (19.96±2.61)(P<0.01). Similar results were found when comparing LYVE-1positive staining MLVD in each COE treated group to untreated group (P<0.05or P<0.01).
Conclusion:COE exerts the effective anti-tumor action via induction tumor cell apoptosis and inhibition of lymphangiogenesis in hepatocellular carcinoma xenograft model. The effect is possibly due to the down-regulation of VEGF-C and CXCR4expression by COE in hepatoma cells, which may be through modulation of extracellular signal-regulated kinase1/2and the phosphatidylinositol3-kinase/AKT signaling pathway. All these results provide evidence that COE may play an important role in down-regulating the tumor lymphangiogenesis and it may be a valuable therapeutic strategy for hepatocellular carcinoma in the future.
引文
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