Down-regulation of lung resistance related protein by RNA interference targeting survivin induces the reversal of chemoresistances in hepatocellular carcinoma

Background Both survivin and lung resistance related protein （LRP） are hepatocellular carcinoma （HCC）. But the relationship between survivin and LRP is investigate the effects of down-regulation of survivin on LRP expressions and the both in vitro and in vivo. related to the chemoresistances in ndefinite. The aim of this study was to reversal of chemoresistances in HCC Methods The expressions of survivin were detected by RT-PCR and Western blotting in HCC cell line SMMC-7721 and SMMC-7721/ADM. The sensitivities of these two cell lines to ADM were evaluated by MTT assays. SiRNA which targeted survivin was transfected into SMMC-7721/ADM cells, then the sensitivity of SMMC-7721/ADM cells to ADM and the expressions of survivin and LRP were detected respectively. SMMC-7721/ADM cells were transplanted subcutaneously into nude mice to establish xenograft tumors. Antitumor activities of RNA interference （RNAi） targeting survivin, various doses of ADM and combination therapies were observed respectively. Possible toxicities were evaluated. LRP expression changes were tested. Student＇s ttest was used for evaluating statistical significance. Results The expressions of survivin in SMMC-7721/ADM cell line showed significant elevation compared to those in SMMC-7721 cell line （P 〈0.05）. Positive siRNA down-regulated the expressions of survivin significantly （P 〈0.05）. SiRNA targeting survivin could sensitize SMMC-7721/ADM cells to ADM and down-regulate the expressions of LRP significantly （P 〈0.05）. Growths of the tumors were significantly inhibited in positive siRNA group as compared with those in the control group from the 8th day （P 〈0.05）. Combination therapies caused significant tumor inhibitions compared with tumors of nude mice in the other three groups respectively （P 〈0.05）. No toxicities were found in nude mice treated by siRNA and combination therapies. The expressions of LRP were markedly reduced in tumors treated with siRNA targeting survivin （P 〈0.05）. Conclusions Down regulation of survivin gene by RNAi can increase chemosensitivity of HCC both in vitro and in vivo. The reversal of drug resistance may be reduced through the inhibitions of LRP.     

免疫毫微粒的内化作用及其逆转人肝癌细胞多药耐药性的研究

目的：观察免疫毫微粒能否内化进入人肝癌细胞及其多药耐药性(MDR)细胞，研究免疫毫微粒能否逆转MDR。方法：将抗人肝癌阿霉素白蛋白免疫毫微粒(HAbl8-ADR-HSA-NP)与人肝癌株SMMC-7721细胞及其MDR细胞(SMMC-7721／MDR^ )共同孵育，采用扫描电镜，激光共聚焦显微镜，透射电镜技术观察免疫毫微粒与靶细胞的结合及免疫毫微粒的内化过程；采用MTT比色分析法测定免疫毫微粒对靶细胞的杀伤率。计算免疫毫微粒对靶细胞的IC50和MDR细胞的耐药倍数(RF)。结果：激光共聚焦显微镜观察，SMMC-7721细胞表面和胞浆中均有许多荧光毫微粒；透射电镜观察。免疫毫微粒与SMMC-7721细胞及其MDR细胞在37℃孵育后，胞浆中可见有免疫毫微粒，随时间的延长细胞变性损伤越严重；当SMMC-7721及其MDR细胞预先经HAbl8抗体处理，再与HAbl8-ADR-HSA-NP孵育，则胞浆中未见免疫毫微粒；扫描电镜显示，多个HAbl8-ADR-HSA-NP紧密结合在SMMC-7721／MDR^ 表面。MDR细胞对于免疫毫微粒的耐药倍数(2.1)较其对于游离ADR(4．4)明显降低。结论：人肝癌特异性阿霉素白蛋白免疫毫微粒通过抗体介导能特异性内化进入人肝癌SMMC-7721细胞及其MDR细胞；该免疫毫微粒能增强MDR细胞对阿霉素杀伤的敏感性。有一定程度逆转MDR作用。     

Anti-tumor effect of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice

Objective: To investigate anti-tumor effect of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice. Methods: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors in nude mice by subcutaneous injection. Then the subcutaneous tumors were implanted into the liver of nude mice, and the orthotopic transplantation tumor models of human hepatocellular carcinoma were established. Seventy-five models were randomized into 5 groups ( n = 15) . Bufalin was injected intraperitoneally into the 3 groups at dose of 1.5,1 and 0.5 mg/kg for day 15 - 24, respectively. NS group were injected equal volume saline as above and adriamycin were injected intraperitoneally into ADM group at dose of 8.0 mg/kg for day 15. Ten mice in each group were killed at day 25 and detected on morphological and ultrastructural changes in myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscope. The survival time in each group     

Potent antitumor efficacy of an E1B 55kDa-deficient adenovirus carrying murine endostatin in hepatocellular carcinoma

Data from clinical trails have shown that the antitumoral effect of ONYX-015, an E1B 55kDa-deficient adenovirus, as monotherapy is insufficient. To enhance its efficiency, CNHK200-mE, another E1B 55kDa-deficient adenovirus armed with a mouse endostatin gene was constructed and its antitumoral activities against hepatocellular carcinoma （HCC） in vitro and in vivo were investigated. The selective replication and cytotoxicity of CNHK200-mE in Hep3B and HepG Ⅱ cells independent of p53 status were confirmed via TCID50 and 3-（4,5dimetylthiazol）-2,5-diphenyltetrazolium bromide （MTT） assays. Potent tumor growth suppression on SMMC-7721 xenografts in nude mice was observed and a synergistic effect of the carrier virus and the therapeutic gene was suggested. Moreover, in comparison with. the nonreplicative adenovirus carrying the same therapeutic gene, amplified transgene expression of mouse endostatin in vitro and in vivo were confirmed by Western blotting and ELISA assay. The effective angiogenesis inhibition and replication of CNHK200-mE in nude mice xenografts were demonstrated by immunohistochemistry. In conclusion, the recombinant adenovirus CNHK200-mE is a replication-competent oncolytic virus mediating high expression of therapeutic gene. Because CNHK200-mE is capable of replicating in and lysing HCC cells selectively with effective tumor growth suppression and antiangiogenic activity on HCC xenografts in nude mice, it holds good potential for the treatment of HCC.     

抗人肝癌免疫毫微球的制备及其抗癌效果观察

目的 制备抗人肝癌免疫毫微球,观察其活性及抗癌效果.方法 抗人肝癌单克隆抗体HAb18通过异型双功能交联剂SPDP与载阿霉素(ADR)人血清白蛋白毫微球[HSA(ADR)-NS]偶联,制成抗人肝癌免疫毫微球HAb18-HSA(ADR)-NS,使用凝集试验及免疫荧光检测其活性,光镜和电镜下观察其与人肝癌细胞株SMMC-7721特异性结合.MTT法检测该免疫毫微球的体外杀伤性.于人肝癌裸鼠模型上分别使用HAbl8-HSA(ADM)-NS、HSA(ADM)-NS及ADM,检测3者的肿瘤抑制率.结果 HAb18-HSA(ADM)-NS具有单抗活性,能与肝癌细胞特异结合;其体外杀伤SMMC-7721细胞IC50值为44.6μg/ml,与HSA(ADM)-NS(345.5μg/ml)及ADM(365.5μg/ml)相比,明显降低;体内肿瘤抑制率比HSA(ADM)-NS及ADM明显增强(P＜0.001).结论 HAb18-HSA(ADM)-NS具有免疫活性,对肝癌细胞有主动靶向性,体内外均具有比HSA(ADM)-NS及ADM更强的抗癌效果.     

Synergistic effect of hyperthermia and neferine on reverse multidrug resistance in adriamycin-resistant SGC7901/ADM gastric cancer cells

Multidrug resistance(MDR) plays a major obstacle to successful gastric cancer chemotherapy.The purpose of this study was to investigate the MDR reversal effect and mechanisms of hyperthermia in combination with neferine(Nef) in adriamycin(ADM) resistant human SGC7901/ADM gastric cancer cells.The MDR cells were heated at 42℃ and 45℃ for 30 min alone or combined with 10 μg/mL Nef.The cytotoxic effect of ADM was evaluated by MTT assay.Cellular plasma membrane lipid fluidity was detected by fluorescence polarization technique.Intracellular accumulation of ADM was monitored with high performance liquid chromatography.Mdr-1 mRNA,P-glycoprotein(P-gp),γH2AX expression and γH2AX foci formation were determined by real-time PCR,Western blot and immunocytochemical staining respectively.It was found that different heating methods induced different cytotoxic effects.Water submerged hyperthermia had the strongest cytotoxicity of ADM and Nef combined with hyperthermia had a synergistic cytotoxicity of ADM in the MDR cells.The water submerged hyperthermia increased the cell membrane fluidity.Both water submerged hyperthermia and Nef increased the intracellular accumulation of ADM.The water submerged hyperthermia and Nef down-regulated the expression of mdr-1 mRNA and P-gp.The water submerged hyperthermia could damage DNA and increase the γH2AX expression of SGC7901/ADM cells.The higher temperature was,the worse effect was.Our results show that combined treatment of hyperthermia with Nef can synergistically reverse MDR in human SGC7901/ADM gastric cancer cells.     

Morphological Study on the Mechanism of Tumor-selective Cytocidal Action of Tumor Necrosis Factor

Using light microscopy and electron microscopy, we observed the morphological changes inheuman hepatocellular carcinoma cell line (SMMC-7721) treated with tumor necrosis tumor necrosis factor (TNF)and the cytocidal effect of TNF on the heterotransplanted human hepatocellular carcinoma. It wasfound that the changes of the injury occurred earlier in the cell membranes than in the nuclei duringthe course of TNF killing of SMMC-7721 cells and there were similar lesions around the necroticarea in the heterotransplanted human hepatocellular carcinoma in the nude mice as compared withthose produced in SMMC-7721 cells. In addition, the determination of the DNA content in TNF-treated SMMC-7721 cells and controls revealed no significant difference between them. On the basisof these results and Darzynkiewicz's proposals, it is suggested that TNF exerts its tumor-selectivekilling effect by binding to a specific to a specific plasma membrane receptor to disturb synthesis or assembly ofcell membrane components, thus causing the plasma membrane injury and finally cell lysis.     

THE INFLUENCE OF HUMAN SINGLE CHAIN INTELEUKIN-12 GENE TRANSDUCTION ON THE BIOLOGICAL BEHAVIOR OF HEPATOMA 7721 CELLS

Objective. To investigate the anti-tumor effects of human single chain interleukin-12 (hscIL-12). Method. pcDNA/hscIL-12 recombinant was transfected into human hepatic carcinoma cells (7721 cells) by lipofectin method. The 7721/hscIL-12 cells which secrete hscIL-12 stably, were obtained via G418 selection, and in vitro the influence of hscIL-12 gene transduction on the growth of tumor cells was evaluated by cellcycle analysis. In vivo, genetically engineered 7721 cells (7721/hscIL-12, 7721/pcDNA) and parental cells were implanted into BALB/c nude mice, respectively. 7721/pcDNA and 7721/hscIL-12 groups were divided into two sub-groups on day 8: one was administered with hPBL twice, 6 days at interval; the other was given equalvolume of PBS. Mice were sacrificed on day 26, and spleens and tumors were taken out for histologic assay. Results. hscIL-12 produced stably by 7721/hscIL-12 cells had bioactivity, and it was proved by Western blot, immunocytochemistry, and in situ hybridization. In vitro, compared with 7721 and 7721/pcDNA, the7721/hscIL-12 grew much more slowly. FACS assay showed apparent G1 arrest of 7721/hscIL-12 cells. In ani-mal experiment, on day 8 after inoculation, the tumors of 7721 and 7721/pcDNA group were up to 5 -7mm,while those of 7721/hscIL-12 group were 2 -4mm. When treated with hPBL, the tumor of 7721/hscIL-12 groupdisappeared completely. Histologically, the tumors from 7721/hscIL-12 without hPBL treatment had numerouslymphocyte infiltration, the tumor cells displayed depression looking, atrophy, focal necrosis and apoptosis, whereas the tumors of 7721 and 772l/pcDNA groups grew thrivingly.Conclusion. hsclL-12 transduced 7721 cells could induced significant antitumor immune response which resulted in tumor regression totally when the hPBL was inoculated, and also hscIL-12 has certain effects on mice immune system. These findings suggest that hscIL-12 and hscIL-12 gene therapy might have promising prospects in clinical application.     

生存素反义核酸抑制肝癌细胞增殖及对5-FU的增敏作用

目的 探讨生存素反义核酸抑制肝癌细胞增殖及对5-FU的增敏作用.方法 WST法,台盼蓝拒染法、克隆形成抑制实验检测聚乙烯亚胺携带生存素反义核酸(PEI-ASODN)对SMMC-7721细胞的增殖抑制作用.建立小鼠肝癌腋下移植瘤和腹水瘤模型,检测PEI-ASODN联合5-FU对肝癌移植瘤小鼠瘤重、瘤体积的改变,对肝癌腹水瘤小鼠平均存活天数的影响.结果 不同浓度的PEI-ASODN作用于SMMC-7721细胞48 h后,0.75 μmol/L的PEI-ASODN分别作用细胞24、48、72和96 h后,均能对细胞产生明显的抑制作用;PEI-ASODN对细胞的克隆形成也有显著的抑制作用,与细胞对照组相比有显著的统计学差异(P<0.01 ).肝癌腋下移植瘤小鼠,各治疗组的瘤重、瘤体积均得到不同程度的抑制.其中以联合用药组(5-FU+PEI-ASODN)最明显,瘤重抑瘤率高达56.91%,瘤体积抑制率高达57.83%;肝癌腹水瘤小鼠模型,联合用药组生命延长率为94.09%,与生理盐水组相比,具有显著性差异(P<0.01).结论 聚乙烯亚胺携带生存素反义核酸(PEI-ASODN)可显著抑制肝癌细胞生长,并可提高荷肝癌小鼠对5-FU的敏感性.

Abstract：

Objective To investigate the inhibitory effect of survivin antisense oligodeoxynuleotides (ASODN) mediated by polyethylenimine (PEI) on the proliferation of hepatocelluar carcinoma SMMC-7721 cells, and assess its detect on the chemosensitivity of the cells to 5-FU. Methods The inhibitory effect of PE1-ASODN on SMMC-7721 cell proliferation was assessed using WST-8 test, trypan blue staining, and cell clone formation test. In mice bearing transplanted hepatocarcinoma and ascites tumor derived from H22 cells, 5-FU combined with PEI-ASODN was administered, and the weight and volume of the subcutaneous tumors were measured to calculate the tumor inhibition rate, and the average survival time of the mice was calculated. Results Incubation of the cells with different concentrations of PEI-ASODN for 48 h significantly inhibited the cell proliferation as compared with the control group, but PEI or ASODN alone produced no significant inhibitory effects. At 24, 48, 72, 96 h of incubation of the SMMC-7721 cells with 0.75 μmol/L PEI-ASODN, the cell proliferation was suppressed significantly, and incubation with PEI-ASODN at 0.25-0.75 μmol/L for 7 days resulted in significantly inhibited cell clone formation. No significant inhibition was detected in ASODN and PEI group. The tumor weight and volume were reduced in all the treated groups. The tumor inhibition rate was 56.91% and volume inhibition rate was 57.83% in 5-FU+PEI-ASODN group, significantly different from those in the normal saline group (P<0.01). In mice bearing ascites tumor, the average survival time was 22.0 days in saline group, and 42.7 days 5-FU+PEI-ASODN group. The life-prolongation rate of 5-FU+PEI-ASODN was 94.09% when compared with the survival time in saline group. A cooperative effect was detected between 5-FU and PEI-ASODN. Conclusion PEI-ASODN complex can significantly inhibit the proliferation of hepatocarcinoma SMMC-7721 cells and enhance the chemosensitivity of the tumor cells to 5-FU.     

应用荷人卵巢癌腹水瘤模型研究免疫脂质体的导向治疗

用卵巢癌单抗脂质体阿霉素交联物对16只荷人卵巢癌裸鼠腹水瘤模型进行导向治疗。腹水瘤模型更接近实际,符合临床。结果表明,导向治疗组的疗效明显高于对照组、阿霉素组及正常IgG脂质体组。     

以脂质体为载体的卵巢癌单克隆抗体导向治疗实验研究

应用卵巢癌单克隆抗体脂质体阿霉素的交联物（ＭＬＡ），对卵巢细胞系ＳＫＯＶ３进行生长抑制实验。对２４只荷ＳＫＯＶ３的裸鼠皮下移植瘤和１６只荷ＳＫＯＶ３的裸鼠腹水瘤模型进行导向治疗实验。结果表明，ＭＬＡ对细胞的杀伤和对裸鼠体内导治疗效果明显于共他治疗组。要癌治疗有临床意义。     

TNP-470与阿霉素联合使用治疗小鼠膀胱癌

目的观察血管形成抑制剂TNF-470与化疗药物阿霉素联合应用抑制小鼠膀胱癌的协同作用.方法分别予TNP-470、阿霉素、TNP-470 阿霉素治疗TN39膀胱癌荷瘤小鼠,观察肿瘤生长及血管形成、细胞凋亡情况.结果治疗后12 d,TNP-470、阿霉素及TNP-470 阿霉素治疗组抑瘤率分别为46.3%、21.4%及56.5%,TNP-470组微血管密度明显减少、凋亡指数增多,阿霉素组凋亡指数增多,TNP-470 阿霉素组凋亡指数进一步增加.结论TNP-470与阿霉素治疗小鼠膀胱肿瘤,有明显的抗肿瘤协同作用,其机理可能是通过抑制血管形成及化疗药物细胞毒性作用而促使肿瘤细胞凋亡进一步增加.     

The Inhibition of Apoptosis of Hepatoma Cells Induced by HBx Is Mediated by Up-regulation of Survivin Expression

Abnormalityofproliferationandapoptosisinma lignanttumorsisubiquitousandinvolvedmanygenes.Studiesshowedthathepatocellularcarcinoma(HCC) ,oneofthemostcommonmalignancesinChi na ,hasmanyabnormalexpressionofgenesrelatedtoproliferationandapoptosis .Recentstudiesindicatedthatsurvivin ,whichbelongstothefamilyofapopto sisinhibitionprotein ,hadahighexpressionlevelinHCCandwasrelatedtothemechanismsofabnormalapoptosisofHCCcells[1] .PatientswithHCCinChi nashowedahighinfectiousrateofover 80 %ofhep atit…     

绿色荧光裸鼠的建立和验证

目的建立系统性表达绿色荧光蛋白的裸鼠,接种人源肺癌细胞验证该模型是否具有免疫缺陷性,并观察双色荧光的成像效果。方法利用系统性表达绿色荧光蛋白的C57BL/6J小鼠与BALB/C裸小鼠多代杂交和互交,建立稳定表达绿色荧光蛋白的裸鼠。大体解剖观察胸腺生长情况,整体和器官荧光成像验证绿色荧光蛋白的表达情况。以2×106/只的剂量对其皮下腋下接种表达红色荧光蛋白的人类A549肺癌细胞(RFP-A549),通过观测肿瘤生长来验证模型的免疫缺陷性。同时,利用红色荧光标记的肿瘤和绿色宿主鼠,对双色的整体成像效果进行观测。结果构建出系统性表达绿色荧光蛋白的裸鼠,大体解剖可见胸腺缺失。在激发光的激发下,绿色荧光裸鼠全身发出清晰的绿色荧光,脑、心脏、肺脏、肝脏、肾脏,肠胃及胰腺等主要器官可见明显绿色荧光。接种RFP-A549细胞后,成瘤率达到100%,整体动物荧光成像表现出清晰的双色。结论本研究构建出的绿色荧光裸鼠,动物整体可以清晰地表达绿色荧光并具有免疫缺陷性     

NF-κB decoy寡核苷酸联合阿霉素诱导裸鼠肝癌细胞HepG_2凋亡

目的:探讨NF-κB decoy寡核苷酸协同阿霉素对裸鼠肝癌细胞HepG2的作用及机制。方法:接种传代培养的人肝癌细胞HepG2于裸鼠皮下,局部注射NF-κB decoy寡核苷酸和/或阿霉素进行治疗。观测裸鼠瘤体大小变化,计算抑瘤率;HE染色观察肝癌组织结构和细胞形态改变;TUNEL法检测细胞凋亡。结果:NF-κB decoy寡核苷酸联合阿霉素治疗组裸鼠肝癌细胞的生长受到明显抑制,抑瘤率为80.52%;肿瘤重量、体积明显小于单用NF-κBdecoy寡核苷酸、阿霉素治疗组及对照组;联合组癌组织凋亡增加,凋亡指数为(39.8±1.6)%,与其余3组比较差异均有统计学意义(P<0.05)。结论:NF-κB decoy寡核苷酸协同阿霉素能抑制NF-κB活性,促进肝癌细胞凋亡,增加其对化疗药物的敏感性。     

人肝癌裸鼠移植模型阿霉素诱导法建立肝癌多药耐药细胞系

目的:建立人肝癌多药耐药细胞系研究其耐药特性及机制.方法:给予移植人肝癌细胞的裸小鼠腹腔注射阿霉素(ADM)长期诱导,获得多药耐药细胞系BEL-7402/ADM.相差显微镜和HE染色观察细胞,MTT法检测耐药细胞的多药耐药性,流式细胞术检测耐药细胞表面多药耐药基因(mdr)的表达产物P糖蛋白(P-gp)、多药耐药相关蛋白(MRP)及谷胱甘肽硫转移系统(GSH/GST)的表达.结果:BEL-7402/ADM对多种抗癌药物产生耐药,对阿霉素的耐药性提高了20.33倍.BEL-7402/ADM细胞表面P-gp和MRP表达阳性,与对照细胞BEL-7402表达比较,差异十分显著(P<0.01).GSH/GST表达无明显变化.结论:BEL-7402/ADM具有明确的多药耐药性,该模型的建立对研究肝癌多药耐药的产生及逆转具有重要价值.     

原位小鼠乳腺癌肝转移模型

目的 探讨利用HER-2阳性细胞系SKBr-3建立自发性乳腺癌肝转移模型，为乳腺癌肝转移的机制及治疗学研究提供有价值的模型。方法 利用SKBr-3乳腺癌细胞株接种至16只裸鼠第二对乳腺脂肪垫，建立裸鼠第一代原位乳腺癌模型，再利用建立成功的原位肿瘤组织在16只裸鼠原位进行瘤组织块第二代原位种植，最后利用第二代组织块种植成功的瘤组织对16只裸鼠再次进行原位肿瘤种植，分别观察肿瘤肝转移情况。结果 在第一代肿瘤细胞原位种植的肿瘤中，没有发生肝转移现象。而在第二代组织原位种植瘤中，只有1只发生肝转移，而在第三代的组织原位肿瘤种植模型中出现了接近81．25％（13／16）的肝转移。无一只出现肉眼肺转移。结论 乳腺癌裸鼠体内反复传代可以建立高效、相对特异性的自发性乳腺癌肝转移模型，为肝转移机制及治疗研究提供很好的模型。     

人胃癌细胞悬液裸小鼠原位移植模型建立及其部分生物学特性

目的 建立人胃癌裸小鼠原位移植模型,观察其部分生物学特性。方法 用人胃低分化腺癌细胞（ＳＧＣ－７９０１）悬液种植于小裸小鼠胃壁,观察原位移植瘤的生长情况、移植成功率和自发转移的发生率。结果 原位移植成功率（成瘤率）为６７％。移植瘤的局部淋巴结转移率８５％,远处淋巴结转移率５０％,肝转移发生率３０％。荷瘤鼠的中位生存期为１６周,晚期出现消瘦和全身衰竭。结论 该裸小鼠原位移植模型具有人胃癌自然生长过程