嵌合分子VEGI~+的构建、表达及其抗血管生成和抗肿瘤活性

血管内皮细胞生长抑制因子VEGI是肿瘤坏死因子超家族新成员,通过诱导内皮细胞凋亡而抑制肿瘤生长。通过PCR方法将CTTHWGFTLC与VEGI23-174氨基酸相连,构成嵌合分子VEGI+。原核表达的VEGI+经过纯化后,以非重折叠的沉淀形式进行活性实验,VEGI+能够抑制鸡胚尿囊膜血管新生,对于Lewis肺癌小鼠移植瘤,5mgL组抑瘤率99.7%,10mgL组抑瘤率96%,25mgL组抑瘤率83%。实验说明VEGI+通过抑制血管新生对移植瘤的早期发展起抑制作用。     

嵌合分子VEGI~+的构建、表达及其抗血管生成和抗肿瘤活性

血管内皮细胞生长抑制因子VEGI是肿瘤坏死因子超家族新成员,通过诱导内皮细胞凋亡而抑制肿瘤生长。通过PCR方法将CTTHWGFTLC与VEGI23-174氨基酸相连,构成嵌合分子VEGI+。原核表达的VEGI+经过纯化后,以非重折叠的沉淀形式进行活性实验,VEGI+能够抑制鸡胚尿囊膜血管新生,对于Lewis肺癌小鼠移植瘤,5mgL组抑瘤率99.7%,10mgL组抑瘤率96%,25mgL组抑瘤率83%。实验说明VEGI+通过抑制血管新生对移植瘤的早期发展起抑制作用。     

人血管生成抑制素抑瘤研究进展

肿瘤的发展和转移需要新生血管的形成。人血管生成抑制素是近年发现的能够专一性抑制血管内皮细胞的内皮抑制因子。大量研究表明,在体外用血管生成抑制素处理血管内皮细胞可以抑制新生血管的形成,在体内单独使用血管生成抑制素,或者将血管生成抑制素与其他物质如基质金属蛋白酶、尿激酶联合处理荷瘤小鼠,可以降低小鼠体内肿瘤组织新生血管密度,抑制肿瘤的生长和肿瘤细胞的迁移。简要综述了血管生成抑制素抑制肿瘤生长和转移及其作用机理。     

用腺相关病毒载体介导人血管内皮生长因子受体KDR胞外区基因的表达

新生血管大量形成在实体瘤的生长和转移中起着关键的作用。血管内皮生长因子(VEGF)是介导肿瘤血管生成的最主要因素。从原代培养的人脐静脉血管内皮细胞(HUVEC)提取细胞总RNA,采用逆转录PCR(RT-PCR)方法得到VEGF受体KDR胞外区cDNA片段。将获得的受体基因克隆到AAV基因治疗载体pSNAV中,得到重组质粒pSNAV/KDR。重组质粒转染BHK细胞,加入辅助病毒后,获得了表达目的蛋白的重组AAV。重组病毒表达的KDR在体外实验中具有与VEGF结合的活性。在体内实验中,重组AAV感染的黑色素瘤细胞在小鼠中形成肿瘤的血管化程度明显低于对照组。     

用腺相关病毒载体介导人血管内皮生长因子受体KDR胞外区基因的有达

新生血管大量形成在实体瘤的生长和转移中起着关键的作用。血管内皮生长因子 (VEGF)是介导肿瘤血管生成的最主要因素。从原代培养的人脐静脉血管内皮细胞 (HUVEC)提取细胞总RNA ,采用逆转录PCR(RT PCR)方法得到VEGF受体KDR胞外区cDNA片段。将获得的受体基因克隆到AAV基因治疗载体pSNAV中 ,得到重组质粒pSNAV KDR。重组质粒转染BHK细胞 ,加入辅助病毒后 ,获得了表达目的蛋白的重组AAV。重组病毒表达的KDR在体外实验中具有与VEGF结合的活性。在体内实验中 ,重组AAV感染的黑色素瘤细胞在小鼠中形成肿瘤的血管化程度明显低于对照组。     

小干扰RNA靶向VEGF基因体内外抑制乳腺癌细胞MCF-7的增殖

 血管生成与肿瘤生长、侵袭、转移密切相关.血管内皮生长因子能特异地促进内皮细胞分裂、增殖及迁移,在肿瘤新生血管生成过程中起着至关重要的作用.通过RNAi抑制VEGF表达的抗血管生成疗法可有效应用于肿瘤治疗.本研究采用化学修饰的siRNA在体内外抑制VEGF基因表达,探讨化学修饰的siRNA介导的RNA干扰技术在乳腺癌基因治疗的可行性和特异性.选用阳离子脂质体Lipofectamine^TM2000作为转染试剂,将针对人VEGF基因的小干扰RNA(small interfering RNA,siRNA)转染人类乳腺细胞株MCF-7和裸鼠移植瘤,在体内外诱导RNAi.采用四甲基偶氮唑蓝（MTT）法,逆转录聚合酶链反应(RT-PCR),蛋白印迹实验等检测siRNA治疗组和对照组VEGF基因表达及细胞增殖变化.体外实验结果显示：靶向VEGF基因siRNA转染乳腺癌MCF-7细胞后,细胞生长抑制率达52.5%;VEGF的mRNA和蛋白表达水平显著降低（P<0.01）;裸鼠体内实验结果显示：siRNA治疗组瘤组织的增长受到明显抑制;RT-PCR结果同时表明治疗组VEGF表达下调.体内外对照组各指标无显著变化.化学修饰的siRNA介导的RNAi在体内外均能成功下调靶基因VEGF的表达,抑制MCF-7细胞增殖,是潜在的肿瘤治疗新方法.     

shRNA靶向干扰COX-2抑制人肝癌裸鼠皮下移植瘤及其血管生成

目的:探讨重组干扰质粒pshRNA-COX-2对人肝癌细胞Hep3B裸鼠皮下移植瘤生长和肿瘤血管生成的抑制作用。方法:重组干扰质粒pshRNA-COX-2转染Hep3B细胞并筛选后,RT-PCR和Western blot检测COX-2mRNA和蛋白表达,RT-PCR检测VEGFmRNA表达。将被成功转染的Hep3B细胞种植于裸鼠皮下,测量肿瘤大小,4周后处死裸鼠,免疫组织化学法检测肿瘤组织中COX-2蛋白表达和肿瘤微血管密度(MVD)。结果:与未转染细胞相比,干扰组COX-2mRNA和蛋白表达抑制率分别为65.3%和52.8%(P<0.05),干扰组VEGFmRNA表达抑制率为56.5%(P<0.05)。干扰组瘤体大小明显小于阴性组和空白组(P<0.01)。干扰组COX-2得分和MVD均明显低于阴性组和空白组(P<0.01)。结论:pshRNA-COX-2通过抑制COX-2表达明显抑制人肝癌细胞Hep3B裸鼠皮下移植瘤生长和肿瘤血管生成。     

骆驼蓬脂转移蛋白基因真核表达载体的构建及其体内外抗瘤效应的研究

目的:构建骆驼蓬脂转移蛋白(lipid transfer protein from Peganum harmala,PhLTP)基因真核表达质粒,并探讨其对黑色素瘤B16细胞在体内外的抗肿瘤作用。方法:将PhLTP基因亚克隆至pcDNA3.1上,获得重组质粒pcDNA3.1-PhLTP;用脂质体转染法将重组质粒及空载体外转染B16细胞,MTT检测其对B16细胞生长的影响。建立B16荷瘤小鼠模型,设重组质粒(pcDNA3.1-PhLTP)、空载(pcDNA3.1)、生理盐水和阳性药物(CTX)组,分别处理小鼠后测量各组肿瘤体积并称瘤重,计算抑瘤率。光镜观察鼠脾、肝等组织变化;免疫组织化学法检测各瘤体中PhLTP、血管内皮生长因子(VEGF)及碱性成纤维细胞生长因子(bFGF)的表达。结果:pcDNA3.1-PhLTP转染B16细胞72 h后,细胞增殖能力明显受到抑制(P0.01)。注射pcDNA3.1-PhLTP组的小鼠肿瘤生长速度明显减慢,肿瘤体积小于空载和生理盐水组(P0.05)。显微镜下可见重组质粒组肿瘤细胞有不同程度的点、片状坏死,而肝、肺等无明显病理损伤。重组质粒组肿瘤组织中有PhLTP蛋白的表达,且VEGF和bFGF的阳性表达指数都低于空载和生理盐水组(P0.01)。结论:成功构建了重组表达质粒pcDNA3.1-PhLTP,体内外实验结果显示其能有效地抑制B16细胞的生长,预示了该重组质粒在治疗黑色素瘤中的潜在应用价值。     

TnI-fast基因表达抑制卵巢癌移植瘤生长

为探讨利用TnI-fast 基因进行卵巢癌基因治疗的有效性及其机制, 将TnI-fast基因 cDNA转染人卵巢癌细胞系SKOV3. 采用MTT法和流式细胞技术分别检测TnI-fast基因转染、空载体转染和未转染的SKOV3细胞体外生长状态. 收集3种细胞培养上清液, 检测3种培养上清液对人脐静脉内皮细胞增殖抑制效应. 3种细胞分别接种到裸鼠, 观察肿瘤生长、细胞凋亡、肿瘤血管生成和TnI-fast基因局部表达. 体外试验发现, 与空载体转染和未转染的SKOV3细胞比较, TnI-fast基因表达对肿瘤细胞自身的生长无抑制作用, 但可抑制人脐静脉内皮细胞增殖. 动物实验中, TnI-fast基因表达可显著抑制肿瘤生长, 生长抑制率达73%. 其肿瘤细胞增殖率与对照组相当, 但微血管密度显著降低, 细胞凋亡显著增加. 提示, 肿瘤自身血管生成抑制可显著延缓卵巢癌生长. 利用血管生成特异性抑制基因TnI-fast进行抗肿瘤血管生成基因治疗可作为肿瘤治疗的新策略之一.     

血管生成抑制因子SCAIF80及其抗肿瘤效应

从鲨鱼软骨中提取、纯化出一种新的血管生成抑制因子－－鲨鱼软骨血管生成抑制因子80（shark carti-lage-derived angiogenesis inhibitory factor 80,SCAIF80）,SDS－PAGE银染分析显示一条带,分子量为80kD。利用血管内上细胞增殖、迁移抑制实验,证明SCAIF80能显著抑制血管内皮细胞的增殖与迁移,均有剂量依赖性。采用鸡胚绒毛尿囊膜分析,证明SCAIF80能显著抑制新生血管生成,动物抑瘤实验结果表明SCAIF80显著抑制小鼠肿瘤生长,对Lewis肺癌的抑瘤率达93.83%。上结果提示鲨鱼软骨含有一种新的蛋白质,可抑制新生血管生成,并因此抑制肿瘤生长。     

GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G₁ fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40–55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56–85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.     

Vascular basement membrane-derived multifunctional peptide, a novel inhibitor of angiogenesis and tumor growth

Vascular basement membrane-derived multifunctional peptide(VBMDMP)gene(fusion geneof the human immunoglobulin G3 upper hinge region and two tumstatin-derived fragments)obtained bychemical synthesis was cloned into vector pUC19,and introduced into the expression vector pGEX-4T-1 toconstruct a prokaryotic expression vector pGEX-4T-1-VBMDMP.Recombinant VBMDMP produced inEscherichia coli has been shown to have significant activity of antitumor growth and antimetastasis inLewis lung carcinoma transplanted into mouse C57B1/6.In the present study,we have studied the ability ofrVBMDMP to inhibit endothelial cell tube formation and proliferation,to induce apoptosis in vitro,and tosuppress tumor growth in vivo.The experimental results showed that rVBMDMP potently inhibited prolif-eration of human endothelial(HUVEC-12)cells and human colon cancer(SW480)cells in vitro,with noinhibition of proliferation in Chinese hamster ovary(CHO-K1)cells.rVBMDMP also significantly inhibitedhuman endothelial cell tube formation and suppressed tumor growth of SW480 cells in a mouse xenograftmodel.These results suggest that rVBMDMP is a powerful therapeutic agent for suppressing angiogenesisand tumor growth.     

GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G₁ fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40–55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56–85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.     

Schlafen-3 decreases cancer stem cell marker expression and autocrine/juxtacrine signaling in FOLFOX-resistant colon cancer cells

We have previously demonstrated that expression of the novel gene schlafen-3 (Slfn-3) correlates with intestinal epithelial cell differentiation (Patel VB, Yu Y, Das JK, Patel BB, Majumdar AP. Biochem Biophys Res Commun 388: 752-756, 2009). The present investigation was undertaken to examine whether Slfn-3 plays a role in regulating differentiation of FOLFOX-resistant (5-fluorouracil + oxaliplatin) colon cancer cells that are highly enriched in cancer stem cells (CSCs). Transfection of Slfn-3 in FOLFOX-resistant colon cancer HCT-116 cells resulted in increase of alkaline phosphatase activity, a marker of intestinal differentiation. Additionally, Slfn-3 transfection resulted in reduction of mRNA and protein levels of the CSC markers CD44, CD133, CD166, and aldehyde dehydrogenase 1 in both FOLFOX-resistant HCT-116 and HT-29 cells. This was accompanied by decreased formation of tumorosphere/colonosphere (an in vitro model of tumor growth) in stem cell medium and inhibition of expression of the chemotherapeutic drug transporter protein ABCG2. Additionally, Slfn-3 transfection of FOLFOX-resistant HCT-116 and HT-29 cells reduced Hoechst 33342 dye exclusion. Finally, Slfn-3 transfection inhibited the expression of transforming growth factor-α in both FOLFOX-resistant colon cancer cells, but stimulated apoptosis in response to additional FOLFOX treatment. In summary, our data demonstrate that Slfn-3 expression inhibits multiple characteristics of CSC-enriched, FOLFOX-resistant colon cancer cells, including induction of differentiation and reduction in tumorosphere/colonosphere formation, drug transporter activity, and autocrine stimulation of proliferation. Thus Slfn-3 expression may render colon CSCs more susceptible to cancer chemotherapeutics.     

Anticancer effects of Chinese red yeast rice versus monacolin K alone on colon cancer cells

Chinese red yeast rice (RYR) is a food herb made by fermenting Monascus purpureus Went yeast with white rice. RYR contains a mixture of monacolins, one of which--monacolin K (MK)--is identical to lovastatin (LV). Epidemiological studies show that individuals taking statins have a reduced risk of colon cancer. In the present study, LV decreased cellular proliferation (P<.001) and induced apoptosis (P<.05) in HCT-116 and HT-29 human colon cancer cells. RYR inhibited both tumor cell growths (P<.001) and enhanced apoptosis (P<.05) in HCT-116 cells. Inhibition of proliferation was reversed by mevalonate (MV) in LV-treated cells, since LV is a 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGCR) inhibitor. However, RYR with MV did not reverse the observed inhibition of growth. MK-free RYR did not reverse the observed LV-mediated inhibition of cancer cell growth. These observations suggest that other components in RYR, including other monacolins, pigments or the combined matrix effects of multiple constituents, may affect intracellular signaling pathways differently from purified crystallized LV in colon cancer cells. RYR was purified into two fractions: pigment-rich fraction of Chinese red yeast rice (PF-RYR) and monacolin-rich fraction of Chinese red yeast rice (MF-RYR). The effect of MF-RYR was similar to that of LV, while the effect of PF-RYR was similar to the effect of the whole RYR extract on the proliferation, apoptosis and mRNA level of HMGCR and sterol response element binding protein-2. These results suggest that the matrix effects of RYR beyond MK alone may be active in inhibiting colon cancer growth. RYR with or without MK may be a botanical approach to colon cancer chemoprevention worthy of further investigation.     

人结直肠癌细胞HCT116红色和绿色荧光标记肿瘤模型的建立

目的筛选表达绿色荧光蛋白和红色荧光蛋白基因的人的单克隆结直肠癌细胞系,为体内监测肿瘤的早期生长建立一种新的肿瘤动物模型。方法以脂质体2000介导chickenβ-actin-GFP-neo和chickenβ-actin-DsRed-neo转染人结直肠癌细胞HCT-116,经梯度浓度G418筛选获得稳定表达红色和绿色荧光蛋白的细胞克隆并扩大培养。BALB/CA-nu裸鼠皮下接种1×10^6个发光细胞使其成瘤,活体荧光成像系统观察肿瘤的生长情况。结果获得了稳定表达GFP、DsRed的人结肠癌细胞株,将其接种到裸鼠体内可成瘤,利用活体成像系统观察了肿瘤的生长过程,肿瘤的发光随着观察时间的延长而增加。结论红色和绿色荧光蛋白能够在人结直肠癌细胞HCT-116中长期稳定表达,用红色和绿色荧光蛋白标记的人结直肠癌细胞HCT-116建立的裸鼠肿瘤模型为进一步研究结肠肿瘤和相应的药物筛选提供了一种简便、可行的新方法。     

Betulinic acid induces apoptosis and inhibits metastasis of human colorectal cancer cells in vitro and in vivo

Betulinic acid (BA) is a pentacyclic triterpenoids extracted from birch with a wide range of biological properties. Recent studies have shown that BA has significant cytotoxicity to various types of human cancer cells, and shows potential in cancer treatment. However, the efficacy of BA on human colorectal cancer tumor cells is still unclear. The purpose of our study was to evaluate the anti-cancer activity of BA in human colorectal cancer cells in vitro and in vivo to investigate the possible mechanism. In this experiment, we found that BA inhibited colorectal cancer cell lines in vitro with a time-dependent and dose-dependent manner. Moreover, BA could induce cell apoptosis by upregulating expression of Bax and cleaved caspase-3 and downregulating protein of Bcl-2. BA could increase the production of reactive oxygen species and reduce mitochondrial membrane potential of cancer cell, suggesting that BA induced cancer cells apoptosis by mitochondrial mediated pathways. Furthermore, BA significantly inhibited the migration and invasion of colorectal cancer cells, reduced the expression of matrix metalloproteinase (MMPs) and increased the expression of MMPs inhibitor (TIMP-2). In addition, the growth of tumor was significantly suppressed by intraperitoneal administration of 20 mg/kg/day of BA in a xenograft tumor mouse model of HCT-116. Histopathological and immunohistochemical analysis showed that MMP-2+ cells and Ki-67+ cells were reduced and cleaved caspase-3+ cells were increased in tumor tissues of mice after BA administration. The results showed that BA not only promoted the apoptosis of colorectal cancer cells, but also inhibited the metastasis of cancer cells. Our results suggest that BA can be a potential natural drug to inhibit the growth and metastasis of colorectal cancer.     

The bisphosphonate zoledronic acid inhibits the growth of HCT-116 colon carcinoma cells and induces tumor cell apoptosis

Besides its preventive action on bone resorption the third generation bisphosphonate zoledronic acid (ZOL) has been shown to display potent inhibitory action on the formation of bone metastases of various human cancers. Recent research also indicates an antitumoral effect on primary tumors and visceral metastases. Here we investigate for the first time the effect of ZOL on the human colon carcinoma cell line HCT-116. ZOL strongly inhibited the proliferation and soft agar colony formation of HCT-116 cells and caused a G1 cell cycle arrest in a population of ZOL treated cells. This cell cycle arrest was accompanied by an induction of apoptosis via a caspase dependent mechanism. Activation of Caspases 3, 7, 8 and 9, cleavage of PARP as well as the release of cytochrome C into the cytosol were detected in HCT-116 cells treated with low micromolar concentrations of ZOL. The induction of the mitochondrial pathway of apoptosis was accompanied by a translocation of Bax into the mitochondria, Bid activation and a decrease of overall Bcl-2 expression. We also detected a cytosolic increase of apoptosis inducing factor (AIF), a trigger of caspase-independent apoptosis. Taken together, our data indicate a potent antitumoral and apoptosis inducing effect of ZOL on HCT-116 colon carcinoma cells. Lilian Sewing and Florian Steinberg contributed equally to this work.     

Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth

We isolated and identified an endogenous 24-kDa human basement membrane-derived inhibitor of angiogenesis and tumor growth, termed canstatin. Canstatin, a fragment of the alpha2 chain of type IV collagen, was produced as a recombinant molecule in Escherichia coli and 293 embryonic kidneys cells. Canstatin significantly inhibited human endothelial cell migration and murine endothelial cell tube formation. Additionally, canstatin potently inhibited 10% fetal bovine serum-stimulated endothelial cell proliferation and induced apoptosis, with no inhibition of proliferation or apoptosis observed on non-endothelial cells. Inhibition of endothelial proliferation was not concomitant with a change in extracellular signal-regulated kinase activation. We demonstrate that apoptosis induced by canstatin was associated with a down-regulation of the anti-apoptotic protein, FLIP. Canstatin also suppressed in vivo growth of large and small size tumors in two human xenograft mouse models with histology revealing decreased CD31-positive vasculature. Collectively, these results suggest that canstatin is a powerful therapeutic molecule for suppressing angiogenesis.