Enhanced antitumor effect of EGF R-targeted p21WAF-1 and GM-CSF gene transfer in the established murine hepatoma by peritumoral injection

One of the major obstacles in current cancer gene therapy is the lack of a gene delivery system with high efficiency and targetability. In this paper, a nonviral gene delivery system GE7, which was designed to target EGF receptor (EGF R) overexpressed on the surface of cancer cel Is through an EGF R-binding oligopeptide (GE7), was used for in vivo gene therapy in a murine subcutaneous hepatoma model. It was demonstrated that the GE7 system could target the reporter gene beta-gal to EGF R-expressing hepatoma cells with high efficiency after in vitro transfection and in vivo peritumoral injection. To improve the therapeutic effect elicited by single gene transfer, human cyclin-dependent kinase inhibitor gene p21WAF-1 and murine cytokine gene GM-CSFwere used simultaneously in peritumoral injection of the GE7/DNA polyplex. The results showed that combined gene transfer of p21WAF-1 and GM-CSF could inhibit the growth of pre-established tumor more effectively and prolong the survival time of hepatoma-bearing mice more significantly than the transfer of a single gene. Apoptosis in the tumor tissues were found when injected with the p21WAF1-DNA polyplex. Prominent inflammatory infiltration was observed in the tumor tissue transfected with the GM-CSF DNA polyplex. Our data demonstrate that the GE7 system-mediated, EGF R-targeted cotransfer of p21WAF-1 and GM-CSF genes exhibit more potent antitumor effect by inducing tumor cell apoptosis and inflammatory responses.     

小鼠甲胎蛋白基因顺式调控元件的选择性克隆重组及功能研究

目的构建并选择肝癌特异性甲胎蛋白(AFP)基因启动元件。方法采用PCR方法从小鼠肝脏克隆AFP基因增强子I、抑制子和启动子,经过不同组合构建了两个真核表达载体:pEGFP-1-EP(含调控序列I:增强子I 启动子)和pEGFP-1-ERP(含调控序列II:增强子I 抑制子 启动子)。通过瞬时转染,采用荧光显微镜观察和流式细胞仪检测所构建调控序列分别在小鼠肝癌细胞株Hepa1-6、黑色素瘤细胞株B16和成纤维细胞株NIH3T3的活性。结果所得AFP基因增强子I、抑制子和启动子序列与Genebank中公布的相关序列一致;荧光显微镜下:pEGFP-1-EP和pEGFP-1-ERP转染Hepa1-6细胞呈强阳性反应,pEGFP-1(空载体)转染Hepa1-6和三种质粒转染的B16、NIH3T3细胞均呈阴性反应;流式细胞仪检测结果显示:pEGFP-1-EP转染Hepa1-6阳性细胞率和平均荧光强度高于pEGFP-1-ERP转染Hepa1-6细胞,两者阳性细胞率和平均荧光强度均高于pEGFP-1转染Hepa1-6细胞;pEGFP-1转染Hepa1-6和三种质粒转染B16、NIH3T3阳性细胞率和平均荧光强度均较低。结论我们所构建小鼠AFP基因调控序列I和调控序列II的启动活性均具有肝癌特异性,且前者高于后者,并选择调控序列I在下一步肝癌特异性基因治疗中使用。     

转4-1BBL 基因的小鼠肝癌细胞瘤苗刺激脾细胞产生细胞因子的研究

 目的 研究转4-1BBL基因的小鼠肝癌细胞瘤苗体外刺激同系小鼠脾细胞产生细胞因子（IL-2、TNF-α和GM-CSF）的能力。方法 以丝裂霉素C（MMC）处理高表达转m4-1 BBL基因的小鼠Hepa1-6肝癌细胞，制成肿瘤细胞瘤苗（TCV），体外与同系小鼠脾淋巴细胞共同培养后，观察其对脾细胞产生细胞因子（IL-2、TNF-α和GM—CSF）的影响。结果 TCV-4-1 BBL刺激后，脾细胞体外分泌细胞因子IL-2、TNF-α和GM-CSF的水平明显增高。结论 转4-1BBL基因的小鼠肝癌细胞瘤苗能刺激脾细胞产生细胞因子IL-2、TNF-α和GM-CSF。     

脂质体介导mIL-12基因与MHCⅠ基因联合对小鼠实验性肝癌的治疗作用

背景与目的：白细胞介素-12及MHCⅠ基因均已单独用于肿瘤基因治疗,为探索两者的抗肿瘤协同效应,本研究探讨小鼠白细胞介素-12（mIL-12)基因与同种异型的MHCⅠ（小鼠为H-2K）基因联合治疗Balb/C小鼠实验性肝癌。方法：分别应用含mIL-12基因,C57BL/6小鼠H-2K^bcDNA及绿色荧光蛋白（GFP）报告基因的真核表达质粒载体pcDNA3。体外经新型脂质体LipofectAMINE2000(LF2000)介导转染小鼠肝癌细胞株MM45T.Li,检测转染细胞外源基因的表达,将经LF2000介导pcDNA3/mIL-12与pcDNA3/H-2K^b转染的MM45T.Li细胞,注射于小鼠皮下,观察致瘤性的变化,在荷瘤Balb/C小鼠瘤内注射上述脂质体-DNA复合物,观察瘤体生长情况及小鼠生存期的改变。结果：LF2000介导pcDNA3/GFP转染MM45T.Li细胞的最佳条件为脂质体与DNA为3：1（μg：μg）,转染效率达30%,经LF2000介导pcDNA3/mIL-12与pcDNA3/H-2K^b转染的MM45T.Li细胞,RT-PCR检测有mIL-12和H-2K^bcDNA的特异扩增片段,WesternBlot检测显著有57kDaH-2K^b蛋白表达,ELISA检测mIL-12分泌量达48ng/ml/10^6细胞,经LF2000介导pcDNA3/mIL-12与pcDNA3/H-2K^b转染的MM45T.Li细胞,其致瘤性下降;荷瘤Balb/C小鼠瘤内注射该脂质体-DNA复合物,FACS检测显示小鼠脾脏淋巴细胞中CD3^ ,CD4^ 和CD8^ 的数量治疗组较对照组高,肿瘤生长相对缓慢,且pcDNA3/mILp-12与pcDNA3/H-2K^b联合治疗具有一定的正协同效应。结论：mIL-12基因与MHCⅠ基因联合治疗小鼠肝癌具有正协同效应,增强了抗肿瘤效果。     

Growth suppression and immunogenicity enhancement of Hep-2 or primary laryngeal cancer cells by adenovirus-mediated co-transfer of human wild-type p53, granulocyte-macrophage colony-stimulating factor and B7-1 genes

Co-transfer of immunomodulatory and antiproliferative genes may be the basis for new strategies to potentiate tumor regression. In this study, we evaluated the in vitro effect of the introduction of human wild-type p53, granulocyte-macrophage colony-stimulating factor (GM-CSF), and B7-1 genes via recombinant adenovirus on the growth and immunogenicity of Hep-2 or primary laryngeal cancer cells. By the introduction of wild-type p53 gene, the growth of Hep-2 cells was inhibited via enhanced apoptosis. By the introduction of GM-CSF and B7-1 genes, the immunogenicity of cancer cells was enhanced. Significant proliferation of tumor infiltrating lymphocytes (TILs) and tumor-specific cytotoxicity of cytotoxic T lymphocytes (CTLs) were induced in vitro. Furthermore, the combinative effect of GM-CSF and B7-1 was even more evident than that of any one of them singly. These results suggest that the co-transfer of human wild-type p53, GM-CSF and B7-1 genes into tumor cells via recombinant adenovirus may be further developed into a potential combination gene therapy strategy for cancer.     

EGF对肝癌细胞及正常肝细胞内ras基因表达的影响

目的 揭示正常肝细胞和肝癌细胞对表皮生长因子（ｅｐｉｄｅｒｍａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ＥＧＦ）不同的应答能力,以及ＥＧＦ对两种不同细胞株内ｒａｓ基因表达的影响。方法 运用Ｗｅｓｔｅｒｎ免疫印迹技术对正常肝细胞株Ｌ－０２和肝部细胞株Ｈｅｐ－Ｇ２ｋ ｐ２１＾ｒａｓ的表达进行比较分析。结果 在两种细胞株Ｌ－０２和Ｈｅｐ－Ｇ２中以ｐ２１＾ｒａｓ的表达。Ｈｅｐ－Ｇ２细胞的ｐ２１＾ｒａｓ表达量在ＥＧＦ     

Herpes simplex virus thymidine kinase and granulocyte macrophage colony-stimulating factor combination gene therapy in a murine CT26 cell colon cancer model

We evaluated the antitumor effects of combination gene therapy on CT26 mouse colon cancer cells, using the genes for herpes simplex virus thymidine kinase gene HSV-TK combined with granulocyte macrophage colony-stimulating factor (GM-CSF) compared with HSV-TK alone. Cells, unmodified or retrovirally transduced with HSV-TK or GM-CSF, were inoculated subcutaneously into syngeneic BALB/c mice in various combinations. HSV-TK and GM-CSF were also delivered using different routes (in separate cells vs doubly transfected single cells). Both HSV-TK (with i.p. ganciclovir - GCV - treatment) and GM-CSF genes had independent antitumor effects, and given together they caused significant reduction in tumor volumes compared with the HSV-TK gene alone (P < 0.001). Following GCV treatment, however, the treated/control ratios for tumor volumes were not different between tumors containing either HSV-TK alone or both genes (0.27 vs 0.25, respectively). Thus, the presence of GM-CSF did not increase the bystander effect of HSV-TK. Tumors receiving genes transferred in separate cells tended to be more consistently suppressed after GCV treatment than when both genes were transferred in the same cells, although this was not statistically significant. Thus, combination GM-CSF and HSV-TK gene therapy produced greater therapeutic efficacy than HSV-TK alone, but the bystander effect was not enhanced by GM-CSF.     

Adenovirus-mediated combined P16 gene and GM-CSF gene therapy for the treatment of established tumor and induction of antitumor immunity

Antitumor effects of combined transfer of P16 and cytokine genes were investigated in this study. The adenovirus harboring the P16 gene (AdP16) and murine granulocyte-macrophage colony-stimulating factor gene (AdGM-CSF) were utilized for the treatment of established tumors. The mice were inoculated subcutaneously with Renca cells and, 6 days later, received an intratumoral injection of AdP16 in the presence or absence of AdGM-CSF. The results demonstrated that tumor-bearing mice treated with AdP16 in combination with AdGM-CSF showed more potent inhibition of tumor growth and survived much longer than did mice treated with AdP16, AdGM-CSF, adenovirus expressing beta-galactosidase, or phosphate-buffered saline alone (P<.01). The tumor mass showed obvious necrosis and inflammatory cell infiltration, and more CD(4)(+) and CD(8)(+) T cells infiltrating the tumor after combined therapy. After combined therapy, the expression of MHC-1 (H-2K(d)) and Fas molecules on freshly isolated tumor cells increased greatly. The activity of specific cytotoxic T lymphocytes was also found to be induced more significantly after the combined therapy (P<.01). Our results demonstrated that combined therapy with P16 and GM-CSF genes can inhibit the growth of established tumors in mice significantly and induce antitumor immunity of the host efficiently.     

Experimental therapy of hepatoma with artemisinin and its derivatives: in vitro and in vivo activity, chemosensitization, and mechanisms of action

PURPOSE: ART and its derivatives, clinically used antimalarial agents, have recently shown antitumor activities. However, the mechanisms underlying these activities remain unclear. This study was designed to determine their antitumor efficacy and underlying mechanisms of action in human hepatoma cells. EXPERIMENTAL DESIGN: The in vitro cytotoxicities of ART, DHA, artemether, and artesunate were compared in human hepatoma cells, HepG2 (p53 wild-type), Huh-7 and BEL-7404 (p53 mutant), and Hep3B (p53 null), and a normal human liver cell line, 7702. Based on their activity and specificity, ART and DHA were further investigated for their in vitro and in vivo antitumor effects and their effects on the protein expression of genes associated with cell proliferation and apoptosis. RESULTS: ART and DHA exerted the greatest cytotoxicity to hepatoma cells but significantly lower cytotoxicity to normal liver cells. The compounds inhibited cell proliferation, induced G(1)-phase arrest, decreased the levels of cyclin D1, cyclin E, cyclin-dependent kinase 2, cyclin-dependent kinase 4, and E2F1, and increased the levels of Cip1/p21 and Kip1/p27. They induced apoptosis, activated caspase-3, increased the Bax/Bcl-2 ratio and poly(ADP-ribose) polymerase, and down-regulated MDM2. In mice bearing HepG2 and Hep3B xenograft tumors, ART and DHA inhibited tumor growth and modulated tumor gene expression consistent with in vitro observations. DHA increased the efficacy of the chemotherapeutic agent gemcitabine. CONCLUSIONS: ART and DHA have significant anticancer effects against human hepatoma cells, regardless of p53 status, with minimal effects on normal cells, indicating that they are promising therapeutics for human hepatoma used alone or in combination with other therapies.     

GM-CSF gene or B7-1 gene modified murine EL-4 cells vaccine

Since 1990, gene transduced tumor vaccine has been studied. Many articles reported that tumor cells transduced with some cytokine or costimulatory molecule could induce system antitumor immunity[1,2] In this study, EL-4 lymphoma was transduced with recombinant retrovirus containing the murine GM-CSF gene and B7-1 gene, respectively. The effect of gene transduction on antitumor immunity was investigated.MATERIALS AND METHODSMice and Cell Lines Female C57BL/6 mice were bought from …     

Resveratrol suppresses growth of Ishikawa cells through down-regulation of EGF

Resveratrol and other polyphenols have anti-carcinogenic and anti-tumorigenic activities in various carcinomas. However, such studies are limited in endometrial cancer. We hypothesize that resveratrol suppresses cancer growth through modulation of cell cycle and cell growth regulatory genes. To test this hypothesis, we treated endometrial cancer cells (Ishikawa cell line) with resveratrol (1, 10, 50 and 100 micro M) for 1, 3, 5 and 7 days, and analyzed for growth signal genes (EGF and VEGF), cell cycle regulatory genes (p53 and p21), and apoptosis-related genes (bcl-2 and bax). Results of these experiments demonstrate that after resveratrol treatment, the growth of Ishikawa cells was inhibited in a dose dependent manner. The gene and protein expression data suggest that resveratrol treatment significantly decreased EGF, whereas VEGF was up-regulated in Ishiwaka cell lines. Interestingly, protein expressions of p21 and Bax were decreased, even though their mRNA expressions did not show significant changes. The present study suggests that resveratrol can suppress proliferation of Ishikawa cells through down-regulation of EGF.     

Cancer gene therapy of adenovirus-mediated anti-4-1BB scFv in immunocompetent mice

The use of immunostimulatory molecule genes aiming at enhancing anti-tumor immunity has emerged as a new approach to treat cancers. 4-1BB signaling, an important costimulatory pathway delivering a signal for T cell activation, survival and growth, has become one of the most promising targets for cancer immunotherapy. In this work, a recombinant nonreplicative adenovirus (Ad.4-1BB scFv) carrying a single-chain Fv fragments (scFv) specific for 4-1BB gene (anti-4-1BB scFv) was generated, haracterized and explored for its stimulation of anti-tumor immunity in immunocompetent C57BL/6 mice. Ad.4-1BB scFv could efficiently infect murine hepatoma Hepa 1-6 cells and induce anti-4-1BB scFv expression on the cell surface. Moreover, Ad.4-1BB scFv did not cause obvious cytotoxicity effect on human and murine tumor cell lines (A549, PLC/PRF/5, Hepa 1-6 and TC-1) even at a high MOI, which suggested Ad.4-1BB scFv had no direct effect on tumor cells. Intratumoral injection of Ad.4-1BB scFv to established Hepa 1-6 tumors significantly suppressed the tumor growth in C57BL/6 mice. The anti-tumor effect might be mainly attributed to the anti-4-1BB scFv-mediated immune activity, as evidenced by enhanced interferon-gamma-producing splenic cells and increased lymphocytes infiltration in the tumor microenvironment. These results indicated that nonreplicative adenovirus carrying the anti-4-1BB scFv gene possessed powerful in vivo anti-tumor efficacy and might be a valuable tool for cancer immunotherapy.     

应用细胞因子缓释微球的肿瘤疫苗治疗肝癌的研究

目的 探讨采用细胞因子缓释微球的肿瘤疫苗预防和治疗肝癌的疗效及抗癌机制。方法 我们研制开发了一种肿瘤疫苗,其组成是固定的肿瘤细胞或组织碎片、细胞因子缓释微球和免疫辅助药。采用多聚甲醛固定的小鼠Hepal-6细胞或肿瘤碎片、微球包装的GM—CSF和／IL—2和合成TiterMax Gold等不同成份的瘤苗皮内接种C57BL／6J小鼠,随后肝内接种活体Hepal-6细胞。结果 对照组15只小鼠全部发展成肝肿瘤;含有固定Hepal-6细胞和IL-2及GM—CSF微球的肿瘤疫苗,80％小鼠获得保护。再加入免疫辅助剂TiterMax Gold的肿瘤疫苗,则87％小鼠获得保护。将Hepal-6细胞接种于左躯干皮下。肿瘤长至直径5mm时,皮内接种肿瘤疫苗2次。结果显示,对照组肿瘤继续生长。疫苗组在第2次接种后7—10天,10只小鼠中9只肿瘤生长受到抑制,随后明显缩小。60％小鼠的肿瘤完全消散。细胞毒性实验结果显示,未接种疫苗的小鼠脾细胞不能杀灭Hepal-6细胞和其他肿瘤细胞;而接种疫苗的小鼠脾细胞对Hepal-6细胞杀瘤活性达41％,但对B16—Fl,Lewis肺癌细胞(LLC),肾癌细胞(Renca),膀胱癌细胞(MBT-2)则无效。疫苗的Ⅰ期临床实验结果显示肝癌疫苗能有效地预防肝癌术后复发,诱导DTH反应。结论 肝癌疫苗能有效预防和治疗原发性肝癌,其抗瘤机制是诱导内源性抗原特异性CTL反应,其杀瘤特性是由典型的：MHC—Ⅰ限制的CD8^ T细胞所介导的。     

Combined IL-12 and GM-CSF gene therapy for murine hepatocellular carcinoma.

Among various immunotherapeutic approaches, interleukin-12 (IL-12) is particularly appealing because of its superior antitumor effects, which have been demonstrated in preclinical as well as clinical studies. However, IL-12 therapy was often accompanied by severe side effects due mainly to the supranormal induction of interferon-gamma. To optimize the therapeutic efficacy and lower the side effects of IL-12, we have investigated the antitumor activity of combined IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene therapy in a highly malignant and poorly immunogenic murine hepatocellular carcinoma model. Using a versatile hydrodynamics-based DNA delivery method, we showed that the combined gene delivery of IL-12 and GM-CSF induced very strong antitumor cellular immunity and achieved significant therapeutic efficacy, whereas each cytokine gene alone yielded appreciable but less effects. We also observed that the combined therapy induced lower levels of interferon-gamma than did IL-12 alone. These results suggest that combined IL-12 and GM-CSF therapy can render a stronger antitumor effect as well as lowering potential side effects.     

The immune anti-tumor effects of GM-CSF and B7-1 gene transfection are enhanced by surgical debulking of tumor

Malignant mesothelioma (MM) is a solid tumor largely unresponsive to conventional therapies. Immunological gene therapy shows promise in murine models and human clinical trials; however, the role of surgery in combination with gene therapy has not been widely studied. The aim of this study was to determine if debulking surgery improved the effectiveness of gene therapy in a murine MM model. Mice were subcutaneously inoculated with the MM cell line, AC29, at two different sites, 4 days apart, to allow a surgical and distal site tumor to develop. Once tumors were established, the surgical site tumor was debulked and vaccination of syngeneic tumor transfectants encoding genes for IL-4, IL-2, GM-CSF, B7-1 or allogeneic MHC molecules commenced at a site away from both tumors, and tumor growth was measured. Neither debulking surgery nor gene therapy alone delayed tumor growth. However, there was a clear delay of tumor growth when debulking surgery was combined with vaccination of tumor transfectants expressing B7-1 or high levels of GM-CSF. Combinations of these two transfectants did not lead to a synergistic effect. This study demonstrates that debulking surgery can augment the immunostimulatory effects of immunological gene therapy and can delay tumor growth. This has implications for the future design of human gene therapy trials for solid tumors such as MM.     

Quercetin regulates growth of Ishikawa cells through the suppression of EGF and cyclin D1

Quercetin and other polyphenols have anti-carcinogenic and anti-tumorigenic activity in various organs, however, studies of this activity are lacking in endometrial cancer. We hypothesize that quercetin has anti-proliferative activity and the mechanisms of quercetin action may be through modulation of cell cycle and cell growth regulatory genes. To test this hypothesis, we treated endometrial cancer cells (Ishikawa cell line) with quercetin, and cell proliferation, expression of growth signal genes (EGF, VEGF, and TGF-alpha), cell cycle genes (p53, p21, p73, and cyclin D1), and apoptosis-related genes (bcl-2 and bax) were analyzed. Results of these experiments demonstrate that after a 7-day exposure to 1, 10 and 100 micro M of quercetin, growth of Ishikawa cells was inhibited by 3, 51 and 87%, respectively. The gene and protein expression data suggest that quercetin treatment (100 micro M) significantly decreased EGF and cyclin D1, whereas VEGF was up-regulated in Ishiwaka cell lines. Other genes such as TGF-alpha, p53, p21, p73, bcl-2 and bax were not significantly changed with quercetin treatment in Ishiwaka cell lines. The present study suggests that quercetin can suppress proliferation of Ishikawa cells through down-regulation of EGF and cyclin D1.     

Intratumoural GM-CSF microspheres and CTLA-4 blockade enhance the antitumour immunity induced by thermal ablation in a subcutaneous murine hepatoma model

Purpose: We evaluated the effect of a new antitumour immunity regimen that included microwave ablation, intratumoural microspheres encapsulating granulocyte-macrophage colony stimulating factor (GM-CSF), and blockade of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4).

Materials and methods: C57BL6 mice with an established subcutaneous Hepa 1-6 hepatoma underwent microwave ablation, followed by intratumoural injection of GM-CSF microspheres, and intraperitoneal injection of anti-CTLA-4 antibodies. The therapeutic effects were evaluated by tumour growth, survival analysis, and cytotoxicity of T lymphocytes against Hepa 1–6.

Results: The co-administration of microwave thermal ablation, GM-CSF microspheres, and anti-CTLA-4 rejected tumour rechallenge in 90% of treated mice in a subcutaneous murine Hepa 1-6 model, and cured established distant tumour in 50% of the treated mice. This antitumour immune response was tumour-specific and mediated by natural killer (NK), CD4+, and CD8+ T cells.

Conclusions: Microwave ablation, followed by intratumoural GM-CSF microspheres, and anti-CTLA-4 antibodies results in the local eradication of tumours, rejection of tumours following rechallenge, and cures established distant tumours, suggesting that this is a promising regimen and one that is readily applicable in the clinic.     

Vaccination with dendritic cells pulsed with apoptotic cells elicits effective antitumor immunity in murine hepatoma models

Dendritic cell (DC)-based vaccine is a developing strategy to treat cancer including hepatoma. We evaluated the antitumor efficacy of vaccination with DCs pulsed with apoptotic cells, as compared to vaccination with DCs pulsed with cell lysates, in murine hepatoma models. Murine hepatoma cells, Hepa1-6, MH134 and BNL1ME.A.7R.1, and their syngeneic mice, C57BL/6, C3H/HeN and BALB/c, respectively, were used in the study. Protective and therapeutic antitumor effects of vaccination with bone marrow-derived DCs pulsed with irradiation or sulindac-induced apoptotic cells or cell lysates were analyzed. Immature DCs efficiently phagocytosed apoptotic cells and increased expression of CD86, a cell surface maturation marker. Vaccination with apoptotic cell-pulsed, but not cell lysate-pulsed, DCs promoted significant protective immunity against parental hepatoma in vivo. Spleen cells from mice vaccinated with apoptotic cell-pulsed DCs showed higher cytolytic activity and contained higher number of IFN-gamma producing cells against parental hepatoma cells than those from mice vaccinated with cell lysate-pulsed DCs in vitro. Polyriboinosinic polyribocytidylic acid [poly (I:C)], double strand RNA, further enhanced CD86 expression and the therapeutic efficacy of vaccination with DCs pulsed with apoptotic cells for pre-established hepatoma. These results suggest that vaccination with DCs pulsed with apoptotic cells and treated with poly (I:C) appears to be a promising approach as a new therapeutic means for hepatoma.