吴茱萸碱联合吉西他滨对舌鳞癌荷瘤裸鼠模型的抑制作用及其机制研究

研究中药吴茱萸碱联合西药吉西他滨对舌鳞癌裸鼠皮下移植瘤的抑制作用并探讨其作用机制。建立舌鳞癌荷瘤裸鼠模型,药物干预后观察裸鼠瘤体积、进食量、进水量及对外界刺激的应激反应变化,HE染色观察移植瘤及各脏器组织病理结构,透射电镜观察肿瘤组织超微结构,Western blot及免疫组化检测NF-κB及Bcl-2家族基因蛋白表达。结果显示,较模型对照组及单药组,联合组移植瘤体积显著减小,荷瘤裸鼠的生活质量提高,肿瘤细胞病理性核分裂像数目减少,凋亡率增加,Bax、Ik-βα蛋白表达明显增加,Bcl-2、Bcl-xl、P65表达水平明显减少。吴茱萸碱联合吉西他滨抗肿瘤作用机制可能与调控NF-κB及Bcl-2家族蛋白有关。     

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。     

固本抑瘤Ⅲ号方联合吉西他滨对人胰腺癌裸鼠异位移植瘤的抑瘤作用

目的探讨固本抑瘤Ⅲ号方以及与吉西他滨联合治疗人胰腺癌裸鼠异位移植瘤的抑瘤作用。方法将40只荷瘤裸鼠随机分为对照组、吉西他滨组、固本抑瘤Ⅲ号联合吉西他滨组、固本抑瘤Ⅲ号组,每组10只。于接种后第8天开始给药,观察指标为瘤重、裸鼠体重、移植瘤体积。结果吉西他滨组、固本抑瘤Ⅲ号联合吉西他滨组、固本抑瘤Ⅲ号组的抑瘤率分别为49.2%、68.9%和28.0%。固本抑瘤Ⅲ号联合吉西他滨组较吉西他滨组抑瘤作用更强(P0.05)。吉西他滨组、固本抑瘤Ⅲ号联合吉西他滨组、固本抑瘤Ⅲ号组移植瘤体积均较对照组明显降低,差异有显著性。固本抑瘤Ⅲ号联合吉西他滨组体重下降明显,体重较对照组、吉西他滨组及固本抑瘤Ⅲ号组明显下降,差异有显著性。结论固本抑瘤Ⅲ号方具有增加吉西他滨化疗治疗人胰腺癌裸鼠腋下移植瘤疗效的作用。     

斑蝥素酸镁对人肝癌细胞SMMC-7721裸鼠皮下移植瘤凋亡的影响

建立人肝癌细胞SMMC-7721裸鼠皮下移植瘤模型,实验组每只裸鼠瘤周注射斑蝥素酸镁6.26×10-5mmol,对照组给予相同容积的无菌生理盐水瘤周注射。给药22 d后,观察斑蝥素酸镁对皮下移植瘤增殖的影响,并在此基础上,利用HE染色观察药物对人肝癌裸鼠皮下移植瘤组织形态学特征的影响。实验发现斑蝥素酸镁组移植瘤细胞体积变小、胞浆固缩、嗜酸性变,细胞核固缩、碎裂。透射电镜观察人肝癌裸鼠皮下移植瘤组织超微结构的改变,镜下见斑蝥素酸镁组移植瘤细胞核膜基本消失、核染色质聚集成团等改变。免疫组化二步法检测人肝癌裸鼠皮下移植瘤组织中bcl-2、bax的表达水平,结果显示斑蝥素酸镁组瘤组织中bcl-2的表达低于生理盐水组,而bax的表达高于生理盐水组(P0.05)。本实验提示斑蝥素酸镁能明显抑制人肝癌裸鼠皮下移植瘤的增殖,并能诱导移植瘤细胞发生凋亡,其机制可能与上调bax和下调bcl-2表达有关。     

miR-451a促进胰腺癌细胞对吉西他滨的敏感性

目的:探究miR-451a在胰腺癌吉西他滨耐药中的功能。方法:通过低浓度梯度递增法建立胰腺癌吉西他滨耐药细胞株,microRNA(miRNA)测序筛选耐药相关miRNA;细胞存活曲线、克隆形成实验及流式凋亡实验分析miR-451a对胰腺癌细胞耐药的影响;裸鼠成瘤实验检测在动物体内模型中miR-451a对胰腺癌吉西他滨耐药的调控作用;调取TCGA数据分析miR-451a表达水平与胰腺癌病人预后的相关性。结果:胰腺癌吉西他滨耐药细胞株中miR-451a表达水平明显下调;miR-451a过表达增加胰腺癌细胞对吉西他滨的敏感性,增强了吉西他滨抑制细胞增殖和诱导细胞凋亡的作用;miR-451a诱导了裸鼠皮下肿瘤对吉西他滨敏感;miR-451a低表达与胰腺癌患者不良预后相关。结论:miR-451a增强了胰腺癌细胞对吉西他滨的敏感性。     

抗肝癌hdsFv-hEDN重组免疫毒素对荷人肝癌裸鼠皮下移植瘤生长的抑制作用

目的:研究抗肝癌hdsFv-hEDN重组免疫毒素对荷人肝癌裸鼠皮下移植瘤生长的抑制作用,评价其作用为导向治疗药物的临床应用价值.方法:将体外培养的人肝癌细胞系SMMC-7721细胞接种于裸鼠皮下,建立荷人肝癌裸鼠皮下移植瘤动物模型,随机分为hdsFv-hEDN治疗组和对照组,分别给予尾静脉注射hdsFv-hEDN和生理盐水,1次/日,共2周.比较各组裸鼠皮下移植瘤生长速度、肿瘤体积和重量,并计算肿瘤的抑制率.取各组裸鼠肿瘤组织,心,肺,肝,肾组织HE染色,光学显微镜下观察.结果:抗肝癌hdsFv-hEDN治疗组裸鼠皮下移植瘤生长抑制作用显著,肿瘤生长速度减慢,肿瘤体积从42.62±0.57 mm3增加到74.28±2.59 mm3、瘤重为155.82±14.43 mg,而对照组肿瘤体积从41.94±0.91 mm3增加到127.42±4.81 mm3、瘤重为283.28±15.21 mg,两组比较差异非常显著.肿瘤体积抑瘤率和瘤重抑瘤率分别达到41.59±0.02%和45.51±0.09%.组织学观察抗肝癌hdsFv-hEDN组肿瘤组织出现大片坏死,凋亡明显增加,心、肝、肺、肾等重要器官未见明显异常.结论:抗肝癌hdsFv-hEDN重组免疫毒素对荷人肝癌裸鼠皮下移植瘤生长具有良好的抑制作用.     

血管形成在双歧杆菌预防大肠癌生长中的作用

目的 从血管形成角度探索青春型双歧杆菌预防大肠癌生长的途径。方法 建立大肠癌裸鼠移植瘤模型,以免疫组化法检测大肠癌组织血管内皮生长因子（VEGF）的蛋白表达水平及其微血管密度（MVD）。结果 双歧杆菌预防组大肠癌VEGF的阳性细胞密度及MVD的数量均明显低于肿瘤对照组（P〈0．01）。结论 青春型双歧杆菌能下调大肠癌VEGF的表达,进而抑制其血管形成,这可能是它预防大肠癌生长的途径之一。     

抗VEGFR-2嵌合Fab抗体对裸鼠肝癌原位移植瘤新生血管生成的影响

目的:检测抗VEGFR-2嵌合Fab抗体对裸鼠肝癌原位移植瘤血管生成的影响.方法:建立裸鼠肝癌H22细胞原位移植瘤模型,随机分成生理盐水组(n=12)和抗体组(n=12).采用免疫组织化学SP染色法对两组肝脏移植瘤进行血管染色,观察其微血管密度(MVD)情况.结果:成功建立裸鼠H22肝癌原位移植瘤模型,HE染色显示肝脏移植瘤为肝细胞肝癌,免疫组化结果显示肝脏实体瘤内微血管密度抗体组较生理盐水组显著性减少(25.64± 1.53 vs 8.65± 1.79,P＜0.05).结论:抗VEGFR-2嵌合Fab抗体能够抑制裸鼠肝癌原位移植瘤的血管生成.     

重组人内皮抑素联合TP方案对MCF-7裸鼠移植瘤的影响

目的:探讨重组人内皮抑素联合TP方案对MCF-7裸鼠移植瘤模型血管生成及肿瘤生长的作用.方法:将36只荷瘤鼠随机分为A、B两大组,A、B两大组内均设有单一化疗组、联合用药组及对照组;单一化疗组给予紫杉醇 顺铂(TP方案).联合用药组给予重组人内皮抑素 TP,对照组等体积生理盐水;比较A大组内各组裸鼠的抑瘤率,肿瘤生长曲线.ELISA方法检测血清血管内皮生长因子(VEGF),取肿瘤组织病理切片观察微血管密度(MVD)、凋亡指数,并观察B组所有动物生存期.结果:联合用药组VEGF,MVD,较单一化疗组及对照组低,凋亡指数比单一化疗组高,有统计学差异(P＜0.05),联合用药组与化疗组生存时间的差异无统计学意义(P＞0.05),与对照组比较有统计学差异,生存时间较对照组长(P＜0.05).结论:重组人内皮抑素联合TP方案对MCF-7裸鼠移植瘤对肿瘤生长及肿瘤血管抑制优于单一化疗,但1个周期联合化疗用药不能延长生存期.     

姜黄素对晚期宫颈癌患者Bcl-xL、Bcl-2、EphA2及EphrinA1表达的影响

目的:探究姜黄素对宫颈癌晚期放疗患者Bcl-x L、Bcl-2、Eph A2与Ephrin A1表达的影响。方法:收集160例晚期宫颈癌放疗患者的宫颈癌石蜡标本,分为姜黄素组与对照组,均给予放射治疗,姜黄素组放疗期间加服姜黄素片剂,采用免疫组化等实验方法,观察两组病例标本Bcl-x L、Bcl-2、Eph A2与Ephrin A1的表达情况,比较两组病理标本化疗前后的凋亡细胞指数(AI)与微血管密度(MVD)。结果:姜黄素组的Bcl-x L、Bcl-2、Eph A2与Ephrin A1的表达阳性率分别为8.3%、43.33%、46.67%、15.0%,均显著低于对照组(P0.05);放疗后姜黄素组的AI显著高于对照组(P0.05),MVD显著低于对照组(P0.05)。结论:姜黄素可抑制Bcl-x L、Bcl-2的表达以促进肿瘤细胞凋亡,同时降低Eph A2与Ephrin A1的表达以减少肿瘤微血管形成,有显著的抗肿瘤与放疗增敏作用。     

Combination Therapy with Gossypol Reveals Synergism against Gemcitabine Resistance in Cancer Cells with High BCL-2 Expression

Although gemcitabine is highly active in several cancer types, intrinsic and acquired drug resistance remains a major challenge. Overexpression of Bcl-2 has been associated with gemcitabine resistance. The aim of this study is to determine whether gossypol can overcome gemcitabine resistance in cell lines with high level of Bcl-2 expression in combination drug therapy. Our study demonstrated that in 10 cell lines derived from different cancers, high Bcl-2 baseline expression was observed in cell lines that were resistant to gemcitabine (GEM-R). Furthermore, synergistic effect of combination therapy was observed in gemcitabine-resistant (GEM-R) cell lines with high Bcl-2 expression, but not in a gemcitabine-sensitive (GEM-S) cell lines regardless of Bcl-2 expression. Gossypol treatment resulted in the decrease of anti-apoptotic genes such as Bcl-2 and Bcl-xl and an upregulation of the pro-apoptotic gene, Noxa. Furthermore, the addition of gossypol to gemcitabine resulted in lower expressions of anti-apoptotic genes compared to gemcitabine alone. Gene expression profiling in GEM-R and GEM-S cell lines suggest that anti-apoptotic genes such as pAkt and PI3KR2 may play important role in gemcitabine resistance, while pro-apoptotic Bcl-2 related genes (Bad, Caspase-6 and Calpain-1) may regulate synergistic interaction in combination therapy.     

维甲酸对肝癌细胞HepG2糖基化磷脂酰肌醇特异性磷酯酶D表达及细胞生物学特性的影响

应用MTT、流式细胞仪、免疫印迹法检测全反式维甲酸(ATRA)单独或联合糖基化磷脂酰肌醇特异性磷酯酶D(GPI-PLD)特异性抑制剂1,10-二氮杂菲对肝癌细胞HepG2生物学特性的改变.ATRA使肝癌细胞HepG2 GPI-PLD基因表达及酶活性上调,并呈现剂量和时间依赖性.ATRA可抑制肝癌细胞HepG2增殖,使肝癌细胞Caspase-3表达水平显著增加,Bcl-2表达水平下调,促进肝癌细胞凋亡(P<0.05).ATRA联合1,10-二氮杂菲诱导组细胞,Bcl-2、细胞增殖活性较ATRA单独诱导组显著增强,Caspase-3、凋亡率显著下降.维甲酸可促进肝癌细胞HepG2 GPI-PLD基因表达上调,高活性的GPI-PLD有助于维甲酸抑制肝癌细胞增殖,促进肝癌细胞凋亡.     

Novel combination of Celecoxib and proteasome inhibitor MG132 provides synergistic antiproliferative and proapoptotic effects in human liver tumor cells

Molecular targeted therapy has shown promise as a treatment for advanced hepatocellular carcinoma (HCC). Celecoxib (Celebrex®) exhibits antitumor effects in human HCC cells, and its mechanism of action is mediated either by its ability to inhibit cyclooxygenase 2 (COX-2) or by a number of various other COX-2 independent effects. Proteasome inhibitors (PIs) can exert cell growth inhibitory and apoptotic effects in different tumor cell types, including HCC cells. The present study examined the interaction between celecoxib and the PI MG132 in two human liver tumor cell lines HepG2 and HA22T/VGH. Our data showed that each inhibitor reduced proliferation and induced apoptosis in a dose-dependent manner in both cell lines. Moreover, the combination of celecoxib with MG132 synergistically inhibited cell viability and increased apoptosis, as documented by caspase 3 and 7 activation, PARP cleavage, and down-regulation of Bcl-2. Celecoxib and MG132, both alone and synergistically in combination, induced expression of the endoplasmic reticulum (ER) stress genes ATF4, CHOP, TRB3 and promoted the splicing of XBP1 mRNA. Knockdown of TRB3 mRNA expression by small interference RNA significantly decreased combination-induced cell death in HA22T/VGH cells, whereas it increased combination-induced cell death in HepG2 cells, suggesting that activation of the ER stress response might have either a detrimental or a protective role in liver tumor cell survival. In conclusion, our data indicate that combination treatment with celecoxib and MG132 resulted in synergistic antiproliferative and proapoptotic effects against liver cancer cells, providing a rational basis for the clinical use of this combination in the treatment of liver cancer.     

Upregulated miR-182 increases drug resistance in cisplatin-treated HCC cell by regulating TP53INP1

Chemotherapy plays a crucial role in hepatocellular carcinoma (HCC) treatment especially for patients with advanced HCC. Cisplatin is one of the commonly used chemotherapeutic drugs for the treatment of HCC. However, acquisition of cisplatin resistance is common in patients with HCC, and the underlying mechanism of such resistance is not fully understood. In the study, we focused on identifying the role of miRNAs in chemotherapy resistance after cisplatin-based combination chemotherapy. We assayed the expression level of miR-182 after cisplatin-based chemotherapy in patients with advanced HCC, and defined the biological functions by real-time PCR analysis and CCK-8 assay. We found that miR-182 levels were significantly increased in HCC patients treated with cisplatin-based chemotherapy. miR-182 levels were also higher in cisplatin-resistant HepG2 (HepG2-R) cells than in HepG2 cells. Upregulated miR-182 significantly increased the cell viability, whereas miR-182 knockdown reduced the cell viability during cisplatin treatment. miR-182 inhibition also partially overcame cisplatin resistance in HepG2-R cell. Furthermore, we found that upregulated miR-182 inhibited the expression of tumor suppressor gene TP53INP1 (tumor protein 53-induced nuclear protein1) in vitro. In vivo, miR-182 and TP53INP1 expression was negatively correlated. We finally demonstrated that miR-182 increased cisplatin resistance of HCC cell, partly by targeting TP53INP1. These data suggest that miR-182/TP53INP1 signaling represents a novel pathway regulating chemoresistance, thus offering a new target for chemotherapy of HCC.     

Enhanced sensitivity of human hepatoma cells to 5-fluorouracil by small interfering RNA targeting Bcl-2

This study was designed to reveal whether the apoptosis induced in human hepatocellular carcinoma (HCC) cell lines by 5-fluorouracil (5-FU) could be enhanced by transfecting Bcl-2 small interfering RNA (siRNA). Bcl-2 siRNA and control siRNA were transfected into cells following treatment with or without 5-FU. Suppression of Bcl-2 expression was confirmed by Western blotting; cell viability was evaluated by MTS assay, and the occurrence of apoptosis in cells was evaluated by apoptosis assay. Expression of Bcl-2 protein after transfection of 20 nM Bcl-2 siRNA was significantly lower than that of control. Incubation of all cell lines with Bcl-2 siRNA reduced cell viability 96 h after 5-FU treatment compared with all other controls: Huh-7 (P < 0.01), Huh-7 with hepatitis C replicon (P < 0.01), HepG2 (P < 0.01), HLE (P < 0.05). Moreover, the proportion of apoptosis in control siRNA, Bcl-2 siRNA, control siRNA prior to 5-FU treatment, and Bcl-2 siRNA prior to 5-FU treatment groups were (4.6 +/- 2.3)%, (7.5 +/- 0.5)%, (6.0 +/- 2.1)%, and (19.5 +/- 0.86)%, respectively. The Bcl-2 siRNA prior to 5-FU treatment group showed the strongest effect of inducing apoptosis. In conclusion, the combination Bcl-2 siRNA and 5-FU might represent a new therapeutic option for HCC.     

Sinomenine hydrochloride enhancement of the inhibitory effects of anti-transferrin receptor antibody-dependent on the COX-2 pathway in human hepatoma cells

Transferrin receptor (TfR) has been used as a target for the antibody-based therapy of cancer due to its higher expression in tumors relative to normal tissues. Great potential has been shown by anti-TfR antibodies combined with chemotherapeutic drugs as a possible cancer therapeutic strategy. In our study, we investigated the anti-tumor effects of anti-TfR monoclonal antibody (mAb) alone or in combination with sinomenine hydrochloride in vitro. Results suggested that anti-TfR mAb or sinomenine hydrochloride could induce apoptosis, inhibit proliferation, and affect the cell cycle. A synergistic effect was found in relation to tumor growth inhibition and the induction of apoptosis when anti-TfR mAb and sinomenine hydrochloride were used simultaneously. The expression of COX-2 and VEGF protein in HepG2 cells treated with anti-TfR mAb alone was increased in line with increasing dosage of the agent. In contrast, COX-2 expression was dramatically decreased in HepG2 cells treated with sinomenine hydrochloride alone. Furthermore, we demonstrated that the inhibitory effects of sinomenine hydrochloride and anti-TfR mAb administered in combination were more prominent than when the agents were administered singly. To sum up, these results showed that the combined use of sinomenine hydrochloride and anti-TfR mAb may exert synergistic inhibitory effects on human hepatoma HepG2 cells in a COX-2-dependent manner. This finding provides new insight into how tumor cells overcome the interference of iron intake to survive and forms the basis of a new therapeutic strategy involving the development of anti-TfR mAb combined with sinomenine hydrochloride for liver cancer.     

Anticancer drugs are synergistic with freezing in induction of apoptosis in HCC cells

Cryotherapy has been shown to be an important therapeutic alternative to surgery in the treatment of hepatocellular carcinoma (HCC). Here, the influence of cryo-chemotherapy on HCC was examined in vitro using the human HCC cell line Bel-7402, a drug-resistant HCC cell line originating from Bel-7402 cells (Bel-7402/R), as well as two control cell lines, the HCC cell line SMMC-7721 and a colorectal tumor cell line HIC-251. Cells were treated with either exposure to different freezing temperatures (ranging from −15 to −80 °C for 20 min), exposure to sub-lethal concentrations of anticancer chemotherapy drugs or a combination of cryotherapy and chemotherapy. Cell viability and apoptosis under each condition were investigated. We found that the combined treatment resulted in increases in both cell death and apoptosis compared to either treatment alone. The increased level of apoptosis observed in Bel-7402 cells after cryo-chemotherapy was inhibited in the presence of caspase inhibitors. Furthermore, Bax expression was increased 2- to 3-fold in cells exposed to the combination treatment compared with cells treated by freezing or drugs alone. In contrast, Bcl-2 levels remained constant. Although Bel-7402/R cells originated from the Bel-7402 cell line, they were more sensitive to the freezing procedure than the parental cell line. The level of Bax expression in Bel-7402/R cells was also higher than that observed in the parental cell line. In addition, we found that Bel-7402/R cells had lower levels of survivin mRNA than the parental Bel-7402 cells, in both untreated and treated cells. In conclusion, our data show that in HCC cells, apoptosis induced by cryotherapy can be synergistically enhanced using anticancer drugs.