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Fujinaga H, Sakai Y, Yamashita T, Arai K, Terashima T, Komura T, Seki A, Kawaguchi K, Nasti A, Yoshida K, Wada T, Yamamoto K, Kume K, Hasegawa T, Takata T, Honda M, Kaneko S. Biological characteristics of gene expression features in pancreatic cancer cells induced by proton and X-ray irradiation. Int J Radiat Biol 2019; 95:571-579. [PMID: 30557072 DOI: 10.1080/09553002.2019.1558297] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
BACKGROUND Radiation therapy is an important alternative treatment for advanced cancer. The aim of the current study was to disclose distinct alterations of the biological characteristics of gene expression features in pancreatic cancer cells, MIAPaCa-2, following proton and X-ray irradiation. MATERIALS AND METHODS Using cDNA microarray, we examined the gene expression alterations of MIAPaCa-2 cells following proton or X-ray irradiation. We also isolated the surviving MIAPaCa-2 cells after irradiation and analyzed their gene expression profiles. RESULTS Although the cytocidal effects of both types of irradiation were similar at sufficient doses in vitro and in vivo, the affected gene expression profile alterations of MIAPaCa-2 cells irradiated with protons were distinct from those irradiated with X-ray. Interestingly, clustering analysis of gene expression of the surviving MIAPaCa-2 cells was also completely discernible between the two types of irradiation. However, a similar cytocidal effect was still observed in the proton- and X-ray-irradiated surviving cells after re-irradiation, commonly showing biological effects related to apoptosis and cell cycle processes. CONCLUSIONS Proton irradiation treatment for pancreatic cancer provides the distinct biological effect of steady gene expression alterations compared to X-ray irradiation; however, surviving cells from both types of irradiation were still susceptible to the cytocidal effects induced by proton re-irradiation treatment.
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Affiliation(s)
- Haruo Fujinaga
- a Disease control and homeostasis , Kanazawa University , Kanazawa , Japan
| | - Yoshio Sakai
- b Department of Gastroenterology , Kanazawa University Hospital , Kanazawa , Japan
| | - Tatsuya Yamashita
- b Department of Gastroenterology , Kanazawa University Hospital , Kanazawa , Japan
| | - Kuniaki Arai
- b Department of Gastroenterology , Kanazawa University Hospital , Kanazawa , Japan
| | - Takeshi Terashima
- b Department of Gastroenterology , Kanazawa University Hospital , Kanazawa , Japan
| | - Takuya Komura
- c System Biology , Kanazawa University , Kanazawa , Japan
| | - Akihiro Seki
- c System Biology , Kanazawa University , Kanazawa , Japan
| | - Kazunori Kawaguchi
- b Department of Gastroenterology , Kanazawa University Hospital , Kanazawa , Japan
| | - Alessandro Nasti
- a Disease control and homeostasis , Kanazawa University , Kanazawa , Japan
| | - Keiko Yoshida
- a Disease control and homeostasis , Kanazawa University , Kanazawa , Japan
| | - Takashi Wada
- d Department of Nephrology , Kanazawa University Hospital , Kanazawa , Japan
| | | | - Kyo Kume
- e The Wakasa Wan Energy Research Center , Tsuruga , Japan
| | | | - Takushi Takata
- e The Wakasa Wan Energy Research Center , Tsuruga , Japan
| | - Masao Honda
- b Department of Gastroenterology , Kanazawa University Hospital , Kanazawa , Japan
| | - Shuichi Kaneko
- a Disease control and homeostasis , Kanazawa University , Kanazawa , Japan.,b Department of Gastroenterology , Kanazawa University Hospital , Kanazawa , Japan.,c System Biology , Kanazawa University , Kanazawa , Japan
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Li YF, Yuan YY, Zhang YM, Zhao N, Zhang Q, Meng FX, Gao RP, Yu BF, Zhang YH, Guo R, Wang HL, Xie J, Xu J, Qin Q, Dong XS. HSVtk/GCV system on hepatoma carcinoma cells: Construction of the plasmid pcDNA3.1‑pAFP-TK and targeted killing effect. Mol Med Rep 2017; 16:764-772. [PMID: 28560395 PMCID: PMC5482189 DOI: 10.3892/mmr.2017.6657] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2016] [Accepted: 03/17/2017] [Indexed: 12/17/2022] Open
Abstract
Previous studies demonstrated that herpes simplex virus thymidine kinase (HSVtk) could phosphorylate non-toxic gancyclovir (GCV) efficiently to produce phosphorylated products that result in cell apoptosis, to kill tumor cells. The present study aimed to construct a plasmid vector, pcDNA3.1-pAFP-TK, carrying the suicide gene driven by the alpha-fetoprotein (AFP) promoter, to investigate the cytotoxicity of HSVtk/GCV suicide gene system on hepatoma carcinoma cells. Reverse transcription-polymerase chain reaction and western blotting results demonstrated that the HSVtk gene was effectively expressed in HepG2 hepatoma carcinoma cells transfected with pcDNA3.1-pAFP-TK plasmid, whereas HSVtk gene expression was not detected in normal HL-7702 liver cells. In addition, MTT assays indicated that cell viability of HepG2 cells with the plasmid pcDNA3.1-pAFP-TK decreased in a dose-dependent manner following treatment with GCV for 48 h. Flow cytometry also revealed that the cell apoptosis rate and mitochondrial membrane potential reduction rate in the HepG2 cells treated with HSVtk/GCV suicide gene system were significantly higher than in the control group. Apoptosis rates in the control group and the pcDNA3.1-pAFP-TK group were (1.00±0.62%) and (38.70±6.03%), respectively. Mitochondrial membrane potential reduction rates in the control group and the pcDNA3.1-pAFP-TK group were (0.57±0.11%) and (22.84±5.79%), respectively. Caspase-3 staining demonstrated that activated caspase-3 increased significantly in the HepG2 cells treated with HSVtk/GCV suicide gene system, whereas in the control group activated caspase-3 increase was not observed. The results of the present study, therefore, indicated that HSVtk suicide gene was obviously expressed in the HepG2 cells and that the HSVtk/GCV system was effective at killing HepG2 hepatoma carcinoma cells.
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Affiliation(s)
- Yong-Fang Li
- Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Yang-Yang Yuan
- Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Ying-Min Zhang
- Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Na Zhao
- Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Qi Zhang
- Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Fan-Xiu Meng
- Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Ran-Peng Gao
- Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Bao-Feng Yu
- Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Yue-Hong Zhang
- Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Rui Guo
- Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Hai-Long Wang
- Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Jun Xie
- Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
| | - Jun Xu
- Department of General Surgery, Affiliated Tumor Hospital of Shanxi Medical University, Taiyuan, Shanxi 030013, P.R. China
| | - Qin Qin
- Central Laboratory, Shanxi Provincial People's Hospital, Shanxi Medical University, Taiyuan, Shanxi 030012, P.R. China
| | - Xiu-Shan Dong
- Department of General Surgery, Shanxi Dayi Hospital, Taiyuan, Shanxi 030032, P.R. China
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Miyazaki H, Takabe K, Yeudall WA. Chemokines, chemokine receptors and the gastrointestinal system. World J Gastroenterol 2013; 19:2847-2863. [PMID: 23704819 PMCID: PMC3660811 DOI: 10.3748/wjg.v19.i19.2847] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/23/2012] [Revised: 11/13/2012] [Accepted: 04/27/2013] [Indexed: 02/06/2023] Open
Abstract
The biological properties of tumor cells are known to be regulated by a multitude of cytokines and growth factors, which include epidermal growth factor receptor agonists and members of the transforming growth factor β family. Furthermore, the recent explosion of research in the field of chemokine function as mediators of tumor progression has led to the possibility that these small, immunomodulatory proteins also play key roles in carcinogenesis and may, therefore, be potential targets for novel therapeutic approaches. In this review, we will summarize recently reported findings in chemokine biology with a focus on the gastrointestinal tract.
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Marukawa Y, Nakamoto Y, Kakinoki K, Tsuchiyama T, Iida N, Kagaya T, Sakai Y, Naito M, Mukaida N, Kaneko S. Membrane-bound form of monocyte chemoattractant protein-1 enhances antitumor effects of suicide gene therapy in a model of hepatocellular carcinoma. Cancer Gene Ther 2012; 19:312-319. [PMID: 22402625 DOI: 10.1038/cgt.2012.3] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2011] [Revised: 12/05/2011] [Accepted: 01/26/2012] [Indexed: 12/16/2022]
Abstract
Suicide gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system combined with monocyte chemoattractant protein-1 (MCP-1) provides significant antitumor efficacy. The current study was designed to evaluate the antitumor immunity of a newly developed membrane-bound form of MCP-1 (mMCP-1) in an immunocompetent mouse model of hepatocellular carcinoma (HCC). A recombinant adenovirus vector (rAd) harboring the human MCP-1 gene and the membrane-spanning domain of the CX3CL1 gene was used. Large amounts of MCP-1 protein were expressed and accumulated on the tumor cell surface. The growth of subcutaneous tumors was markedly suppressed when tumors were treated with mMCP-1, as compared with soluble MCP-1, in combination with the HSV-tk/GCV system (P<0.01). The numbers of Mac-1-, CD4- and CD8a-positive cells were significantly higher in tumor tissues (P<0.05), and tumor necrosis factor (TNF) mRNA expression levels with mMCP-1 were almost five-fold higher than those with soluble MCP-1. These results indicate that the delivery of the mMCP-1 gene greatly enhanced antitumor effects following the apoptotic stimuli by promoting the recruitment and activation of macrophages and T lymphocytes, suggesting a novel strategy of immune-based gene therapy in the treatment of patients with HCC.
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Affiliation(s)
- Y Marukawa
- Department of Disease Control and Homeostasis, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan
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5
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Sunagozaka H, Honda M, Yamashita T, Nishino R, Takatori H, Arai K, Yamashita T, Sakai Y, Kaneko S. Identification of a secretory protein c19orf10 activated in hepatocellular carcinoma. Int J Cancer 2011; 129:1576-85. [PMID: 21128247 DOI: 10.1002/ijc.25830] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2010] [Accepted: 11/15/2010] [Indexed: 11/07/2022]
Abstract
The identification of genes involved in tumor growth is crucial for the development of inventive anticancer treatments. Here, we have cloned a 17-kDa secretory protein encoded by c19orf10 from hepatocellular carcinoma (HCC) serial analysis of gene expression libraries. Gene expression analysis indicated that c19orf10 was overexpressed in approximately two-thirds of HCC tissues compared to the adjacent noncancerous liver tissues, and its expression was significantly positively correlated with that of alpha-fetoprotein (AFP). Overexpression of c19orf10 enhanced cell proliferation of AFP-negative HLE cells, whereas knockdown of c19orf10 inhibited cell proliferation of AFP-positive Hep3B and HuH7 cells along with G1 cell cycle arrest. Supplementation of recombinant c19orf10 protein in culture media enhanced cell proliferation in HLE cells, and this effect was abolished by the addition of antibodies developed against c19orf10. Intriguingly, c19orf10 could regulate cell proliferation through the activation of Akt/mitogen-activated protein kinase pathways. Taken together, these data suggest that c19orf10 might be one of the growth factors and potential molecular targets activated in HCC.
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Affiliation(s)
- Hajime Sunagozaka
- Department of Gastroenterology, Kanazawa University Hospital, Kanazawa, Ishikawa, Japan
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6
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Kakinoki K, Nakamoto Y, Kagaya T, Tsuchiyama T, Sakai Y, Nakahama T, Mukaida N, Kaneko S. Prevention of intrahepatic metastasis of liver cancer by suicide gene therapy and chemokine ligand 2/monocyte chemoattractant protein-1 delivery in mice. J Gene Med 2010; 12:1002-1013. [PMID: 21157824 DOI: 10.1002/jgm.1528] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
BACKGROUND The prognosis of patients with hepatocellular carcinoma (HCC) remains poor, largely as a result of intrahepatic metastasis. Using a mouse model of intrahepatic metastasis, we investigated whether chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) could potentiate the antitumor effects of the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system. METHODS Mouse hepatoma cells infected with recombinant adenovirus vectors expressing HSV-tk, CCL2/MCP-1 and LacZ at multiplicities of infection of Ad-tk/Ad-MCP1 = 3/0.03 (T/M(Low)), 3/3 (T/M(High)) and Ad-tk/Ad-LacZ = 3/3 (T/L) were injected into BALB/c mice. RESULTS Intrahepatic tumor growth was significantly lower in T/M(Low) mice. By contrast, no tumor suppression was observed in T/M(High) mice. The tumor-specific cytolytic activities of splenocytes from T/M(Low) and T/M(High) mice were comparable. Immunohistochemical analysis of liver tissues showed similar infiltration by Mac-1(+) and T cells in these animals, whereas the proportions of classical activated (M1) monocytes/macrophages were significantly higher in T/M(Low) mice. In addition, interleukin-12 production was elevated in these tissues. Vascular endothelial growth factor-A expression and CD31(+) microvessels were increased in T/M(High) mice. CONCLUSIONS Collectively, these results demonstrate that an adequate amount of CCL2/MCP-1, together with the HSV-tk/GCV system, may induce T helper 1-polarized antitumor effects without inducing tumor angiogenesis in the microenvironment of intrahepatic HCC progression.
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Affiliation(s)
- Kaheita Kakinoki
- Disease Control and Homeostasis, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan
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7
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Tsuchiyama T, Nakamoto Y, Sakai Y, Mukaida N, Kaneko S. Optimal amount of monocyte chemoattractant protein-1 enhances antitumor effects of suicide gene therapy against hepatocellular carcinoma by M1 macrophage activation. Cancer Sci 2008; 99:2075-2082. [PMID: 19016769 PMCID: PMC11158831 DOI: 10.1111/j.1349-7006.2008.00951.x] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2008] [Revised: 06/18/2008] [Accepted: 06/27/2008] [Indexed: 12/01/2022] Open
Abstract
Suicide gene therapy combined with chemokines provides significant antitumor efficacy. Coexpression of suicide gene and monocyte chemoattractant protein-1 (MCP-1) increases antitumor effects in murine models of hepatocellular carcinoma (HCC) and colon cancer. However, it is unclear whether the doses administered achieved the maximum antitumor effects. We evaluated antitumor effects of various amounts of recombinant adenovirus vector (rAd) expressing MCP-1 in the presence of a suicide gene in a murine model of HCC. HCC cells were transplanted subcutaneously into BALB/c nude mice, and transduced with a fixed amount of Ad-tk harboring the suicide gene, HSV-tk, and various doses of Ad-MCP1 harboring MCP-1 (ratios of 1:1, 0.1:1, and 0.01:1 relative to Ad-tk). Growth of primary tumors was suppressed when treated with Ad-tk plus Ad-MCP1 (1:1 and 1:0.1) as compared with Ad-tk alone. The antitumor effects against tumor rechallenge tended to be high in the Ad-tk plus Ad-MCP1 group (1:0.1). The effects were dependent on production of Th1 type-cytokines. Delivery of an optimal amount of rAd expressing MCP-1 enhanced the antitumor effects of suicide gene therapy against HCC by M1 macrophage activation, suggesting that this is a plausible form of cancer gene therapy to prevent HCC progression and recurrence.
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MESH Headings
- Adenoviridae/genetics
- Animals
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/therapy
- Cell Line, Tumor
- Chemokine CCL2/genetics
- Disease Models, Animal
- Enzyme-Linked Immunosorbent Assay
- Genes, Transgenic, Suicide/genetics
- Genetic Therapy/methods
- Genetic Vectors
- Humans
- Immunohistochemistry
- Liver Neoplasms, Experimental/genetics
- Liver Neoplasms, Experimental/therapy
- Macrophage Activation/genetics
- Macrophages, Peritoneal/immunology
- Male
- Mice
- Mice, Nude
- Xenograft Model Antitumor Assays
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Affiliation(s)
- Tomoya Tsuchiyama
- Disease Control and Homeostasis, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan
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8
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Tsuchiyama T, Nakamoto Y, Sakai Y, Marukawa Y, Kitahara M, Mukaida N, Kaneko S. Prolonged, NK cell-mediated antitumor effects of suicide gene therapy combined with monocyte chemoattractant protein-1 against hepatocellular carcinoma. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2007; 178:574-583. [PMID: 17182598 DOI: 10.4049/jimmunol.178.1.574] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Tumor recurrence rates remain high after curative treatments for hepatocellular carcinoma (HCC). Immunomodulatory agents, including chemokines, are believed to enhance the antitumor effects of tumor cell apoptosis induced by suicide gene therapy. We therefore evaluated the immunomodulatory effects of a bicistronic recombinant adenovirus vector (rAd) expressing both HSV thymidine kinase and MCP-1 on HCC cells. Using an athymic nude mouse model (BALB/c-nu/nu), primary s.c. tumors (HuH7; human HCC cells) were completely eradicated by rAd followed by treatment with ganciclovir. The same animals were subsequently rechallenged with HCC cells, tumor development was monitored, and the recruitment or activation of NK cells was analyzed immunohistochemically or by measuring IFN-gamma mRNA expression. Tumor growth was markedly suppressed as compared with that in mice treated with a rAd expressing the HSV thymidine kinase gene alone (p < 0.001). Suppression of tumor growth was associated with the elevation of serum IL-12 and IL-18. During suppression, NK cells were recruited exclusively, and Th1 cytokine gene expression was enhanced in tumor tissues. The antitumor activity, however, was abolished either when the NK cells were inactivated with anti-asialo GM1 Ab or when anti-IL-12 and anti-IL-18 Abs were administered. These results indicate that suicide gene therapy, together with delivery of MCP-1, eradicates HCC cells and exerts prolonged NK cell-mediated antitumor effects in a model of HCC, suggesting a plausible strategy to prevent tumor recurrence.
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Affiliation(s)
- Tomoya Tsuchiyama
- Department of Gastroenterology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan
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9
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Kagaya T, Nakamoto Y, Sakai Y, Tsuchiyama T, Yagita H, Mukaida N, Kaneko S. Monocyte chemoattractant protein-1 gene delivery enhances antitumor effects of herpes simplex virus thymidine kinase/ganciclovir system in a model of colon cancer. Cancer Gene Ther 2006; 13:357-366. [PMID: 16224495 DOI: 10.1038/sj.cgt.7700908] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2005] [Revised: 08/12/2005] [Accepted: 08/12/2005] [Indexed: 01/10/2023]
Abstract
Suicide gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system is a well-characterized tool for cancer gene therapy; however, it does not yet exhibit sufficient efficacy to cure patients of malignancies. We have reported that adenovirally delivered monocyte chemoattractant protein (MCP)-1 augmented the antitumor effects of the HSV-tk/GCV system in an athymic nude mouse model. The current study, which uses an immunocompetent mouse model of colon cancer, was designed to evaluate the antitumor effects of MCP-1 gene delivery in conjunction with this suicide gene therapy system. Subcutaneous tumor foci were directly transduced with both recombinant adenoviruses (rAds) expressing an HSV-tk gene and either of the MCP-1, CD80 and LacZ genes, followed by GCV administration. The growth of tumors was markedly suppressed by codelivery of HSV-tk and MCP-1 genes, which was exclusively associated with the recruitment of monocytes/macrophages, T helper 1 (Th1) cytokine gene expression and cytotoxic activity of the splenocytes. Furthermore, the antitumor effects were more efficient than that obtained by the combination of HSV-tk and CD80 genes. These results suggest an immunomodulatory effect of MCP-1 in the context of suicide gene therapy of colon cancer via orchestration of innate and acquired immune responses.
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Affiliation(s)
- T Kagaya
- Department of Gastroenterology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan
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Chiu CC, Li CH, Fuh TS, Chen WL, Huang CS, Chen LJ, Ung WH, Fang K. The suppressed proliferation and premature senescence by ganciclovir in p53-mutated human non-small-lung cancer cells acquiring herpes simplex virus-thymidine kinase cDNA. ACTA ACUST UNITED AC 2005; 29:286-93. [PMID: 15916863 DOI: 10.1016/j.cdp.2005.02.001] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2005] [Accepted: 02/08/2005] [Indexed: 11/28/2022]
Abstract
The concerted actions of molecular networks determine how cells undergo proliferation, death or aging. Here we show that the highly invasive, tumorigenic human non-small-cell-lung cancer (NSCLC) cells carrying mutated p53 alleles were transfected with herpes simplex virus-thymidine kinase (HSV-tk) cDNA and the selected clone was susceptible to exogenous ganciclovir (GCV). The work further indicated that, in the stable HSV-tk transfectants, GCV suppressed cell proliferation by inducing G(2)/M cell cycle arrest and premature senescence and the potency can be amplified through bystander effect. The growth suppression of the established tumor xenografts in nude mice can be successfully targeted by GCV. These data showed that the GCV-suppressed tumor cell proliferation can be coordinated by cell cycle arrest and cellular senescence in HSV-tk transfectant lacking wild-type p53.
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Affiliation(s)
- C-C Chiu
- Department of Biological Science, National Taiwan Normal University, Taipei, Taiwan, Republic of China
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Iyoda T, Nagata K, Akashi M, Kobayashi Y. Neutrophils Accelerate Macrophage-Mediated Digestion of Apoptotic Cells In Vivo as Well as In Vitro. THE JOURNAL OF IMMUNOLOGY 2005; 175:3475-83. [PMID: 16148089 DOI: 10.4049/jimmunol.175.6.3475] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
It is generally believed that the clearance of apoptotic cells does not lead to inflammation. In contrast, we previously found that injection of apoptotic cells into the peritoneal cavity induced the expression of an inflammatory chemokine, MIP-2, and infiltration of neutrophils, and that anti-MIP-2 Abs suppressed the infiltration significantly. Because our previous study showed that whole-body x-irradiation caused neutrophil infiltration into the thymus along with T cell apoptosis, we examined the role of neutrophils in apoptotic cell clearance. Neutrophil infiltration reached a peak 12 h after irradiation with 1 Gy of x-rays. Immunohistological analysis revealed that apoptotic cells disappeared dramatically from 10.5 to 12 h after x-irradiation. As neutrophils moved from an inner area of the cortex to the periphery, apoptotic cells disappeared concomitantly. Either anti-MIP-2 or anti-CXCR2 Abs suppressed neutrophil infiltration significantly, and the suppression of neutrophil infiltration by anti-MIP-2 Abs delayed the disappearance of apoptotic cells. Moreover, macrophage-mediated digestion of apoptotic thymocytes was accelerated in vitro on coculturing with neutrophils, even if neutrophils were separated from macrophages. These results suggest that neutrophils are recruited to the thymus mainly by MIP-2 after whole-body x-irradiation and that such neutrophils may not induce inflammation but rather accelerate complete digestion of apoptotic cells by macrophages.
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Affiliation(s)
- Takuya Iyoda
- Division of Molecular Medicine, Department of Biomolecular Science, Faculty of Science, Toho University, Funabashi, Japan
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12
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Parker JN, Meleth S, Hughes KB, Gillespie GY, Whitley RJ, Markert JM. Enhanced inhibition of syngeneic murine tumors by combinatorial therapy with genetically engineered HSV-1 expressing CCL2 and IL-12. Cancer Gene Ther 2005; 12:359-68. [PMID: 15678154 DOI: 10.1038/sj.cgt.7700784] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Herpes simplex viruses type 1 (HSV-1) that lack the gamma(1)34.5 gene are unable to replicate in the central nervous system (CNS), but maintain replication competence in actively dividing tumors. To determine if antitumor therapy by M002, a gamma(1)34.5(-) HSV that expresses interleukin-12 (IL-12), could be augmented by combinatorial therapy with another gamma(1)34.5-deleted HSV-1 engineered to express the chemokine CCL2, Neuro-2a tumors were established subcutaneously in the syngeneic A/J mouse strain. Tumors received multiple injections intratumorally either of saline, the parent, non-cytokine-expressing virus R3659, M002, M010 (gamma(1)34.5(-) HSV expressing CCL2), or a combination of M002 and M010. Efficacies were evaluated by monitoring inhibition of tumor growth over time. Results demonstrated the following: (1) inhibition of tumor growth was most pronounced in tumors treated with a combination of M002 and M010; (2) enhanced tumor growth inhibition for the combinatorial treatment group was statistically significant compared to either M002 or M010 alone; and (3) the variability between slopes of the tumor growth rates within an individual treatment group appeared to be virus-dependent, and was reproducible between experiments. Our results demonstrate that combinatorial cytokine/chemokine gamma(1)34.5(-) HSV therapies can provide superior antitumor effects in experimental tumors as a model for malignancies arising in the brain.
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Affiliation(s)
- Jacqueline N Parker
- Department of Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35294-3410, USA
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13
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Djeha HA, Todryk SM, Pelech S, Wrighton CJ, Irvine AS, Mountain A, Lipinski KS. Antitumor immune responses mediated by adenoviral GDEPT using nitroreductase/CB1954 is enhanced by high-level coexpression of heat shock protein 70. Cancer Gene Ther 2005; 12:560-71. [PMID: 15665820 DOI: 10.1038/sj.cgt.7700807] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Gene-directed enzyme prodrug therapy (GDEPT) is a promising approach to local management of cancer through targeted chemotherapy. Killing localized tumors by GDEPT in a manner that induces strong antitumor cellular immune responses might improve local management and allow benefit in disseminated cancer. Here we evaluated the combination of nitroreductase (NTR)/CB1954 GDEPT with high-level expression of heat shock protein 70 (HSP70, a stress protein that can shuttle cytosolic peptides into antigen-presenting cells) for induction of antitumor immunity using adenovirus gene delivery in an aggressive and nonimmunogenic BALB/c syngeneic 4T1 breast cancer model. The mechanism of cell death and spectrum of stress proteins induced are likely to be important determinants of the resulting immune responses. We showed that NTR/CB1954 treatment of 4T1 cells gave both apoptotic and nonapoptotic killing. In vivo killing of 4T1 cells expressing NTR gave weak antitumor immunity and very limited induction of stress proteins including HSP70. High-level coexpression of HSP70 during NTR/CB1954-mediated killing of 4T1 cells in vivo gave much greater protection from tumor challenge (67% long-term survivors compared to 17%) and induced 4T1-specific cytotoxic T-cell responses. The enhancement of antitumor responses resulting from HSP70 coexpression was similar to that conferred by coexpression of GM-CSF.
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Affiliation(s)
- Hakim A Djeha
- ML Research, Keele University Science Park, Keele, Newcastle under Lyme, Staffordshire ST5 5SP, UK
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14
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Sakai Y, Morrison BJ, Burke JD, Park JM, Terabe M, Janik JE, Forni G, Berzofsky JA, Morris JC. Vaccination by genetically modified dendritic cells expressing a truncated neu oncogene prevents development of breast cancer in transgenic mice. Cancer Res 2004; 64:8022-8. [PMID: 15520211 DOI: 10.1158/0008-5472.can-03-3442] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Dendritic cells (DCs) are powerful antigen-presenting cells that process antigens and present peptide epitopes in the context of the major histocompatibility complex molecules to generate immune responses. DCs are being studied as potential anticancer vaccines because of their ability to present antigens to naive T cells and to stimulate the expansion of antigen-specific T-cell populations. We investigated an antitumor vaccination using DCs modified by transfer of a nonsignaling neu oncogene, a homologue of human HER-2/neu, in a transgenic model of breast cancer. BALB-neuT mice develop breast cancers as a consequence of mammary gland-specific expression of an activated neu oncogene. We vaccinated BALB-neuT mice with bone marrow-derived DCs transduced with Ad.Neu, a recombinant adenovirus expressing a truncated neu oncoprotein. The vaccine stimulated the production of specific anti-neu antibodies, enhanced interferon-gamma expression by T cells, and prevented or delayed the onset of mammary carcinomas in the mice. Over 65% of vaccinated mice remained tumor free at 28 weeks of age, whereas all of the mice in the control groups developed tumors. When challenged with a neu-expressing breast cancer cell line, vaccinated tumor-free animals had delayed tumor growth compared with controls. The antitumor effect of the vaccine was specific for expression of neu. Studies showed that CD4+ T cells were required in order to generate antitumor immunity. Importantly, the effectiveness of the vaccine was not diminished by preexisting immunity to adenovirus, whereas the protection afforded by vaccination that used direct injection of Ad.Neu was markedly reduced in mice with anti-adenovirus antibody titers. DCs modified by recombinant adenoviruses expressing tumor-associated antigens may provide an effective antitumor vaccination strategy.
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Affiliation(s)
- Yoshio Sakai
- Cancer Gene Therapy Section, Metabolism Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-1374, USA
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15
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Li PY, Lin JS, Feng ZH, He YF, Zhou HJ, Ma X, Cai XK, Tian DA. Combined gene therapy of endostatin and interleukin 12 with polyvinylpyrrolidone induces a potent antitumor effect on hepatoma. World J Gastroenterol 2004; 10:2195-200. [PMID: 15259064 PMCID: PMC4724992 DOI: 10.3748/wjg.v10.i15.2195] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To study the antitumor effect of combined gene therapy of endostatin and interleukin 12 (IL-12) with polyvinylpyrrolidone (PVP) on mouse transplanted hepatoma.
METHODS: Mouse endostatin eukaryotic plasmid (pSecES) with a mouse Igκ signal sequence inside and mouse IL-12 eukaryotic plasmid (pmIL-12) were transfected into BHK-21 cells respectively. Endostatin and IL-12 were assayed by ELISA from the supernant and used to culture endothelial cells and spleen lymphocytes individually. Proliferation of the latter was evaluated by MTT. H22 cells were inoculated into the leg muscle of mouse, which was injected intratumorally with pSecES/PVP, pmIL-12/PVP or pSecES + pmIL-12/PVP repeatedly. Tumor weight, serum endostatin and serum IL-12 were assayed. Tumor infiltrating lymphocytes, tumor microvessel density and apoptosis of tumor cells were also displayed by HE staining, CD31 staining and TUNEL.
RESULTS: Endostatin and IL-12 were secreted after transfection, which could inhibit the proliferation of endothelial cells or promote the proliferation of spleen lymphocytes. Tumor growth was highly inhibited by 91.8% after injection of pSecES + pmIL-12/PVP accompanied by higher serum endostatin and IL-12, more infiltrating lymphocytes, fewer tumor vessels and more apoptosis cells compared with injection of pSecES/PVP, pmIL-12/PVP or vector/PVP.
CONCLUSION: Mouse endostatin gene and IL-12 gene can be expressed after intratumoral injection with PVP. Angiogenesis of hepatoma can be inhibited synergisticly, lymphocytes can be activated to infiltrate, and tumor cells are induced to apoptosis. Hepatoma can be highly inhibited or eradiated.
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Affiliation(s)
- Pei-Yuan Li
- Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
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16
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Nakamura M, Kyo S, Kanaya T, Yatabe N, Maida Y, Tanaka M, Ishida Y, Fujii C, Kondo T, Inoue M, Mukaida N. hTERT-promoter-based tumor-specific expression of MCP-1 effectively sensitizes cervical cancer cells to a low dose of cisplatin. Cancer Gene Ther 2004; 11:1-7. [PMID: 14681721 DOI: 10.1038/sj.cgt.7700650] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2003] [Indexed: 11/08/2022]
Abstract
Cervical cancers at advanced stages or with recurrent status are mainly treated by platinum-based chemotherapy, such as cisplatin. However, a novel strategy to reduce the minimally effective dose is required to prevent severe adverse effects that limit the effectiveness of the treatment. Monocyte chemoattractant protein-1 (MCP-1) is a subtype of chemokines that can promote monocyte/macrophage infiltration and enhance their phagocytosis at not only sites of inflammatory lesions but also of tumors. The present study applies MCP-1-based gene therapy to treat cervical cancers. To achieve tumor-specific expression of MCP-1, retroviral expression vector was constructed using the human telomerase reverse transcriptase gene (hTERT) promoter. Retroviral expression of MCP-1 into cervical cancer ME180 cells did not affect their proliferation either in vitro or in vivo. However, when combined with a suboptimal low dose of cisplatin, tumor formation was obviously reduced in clones transduced with MCP-1, but not in control clones. Histological examination revealed that a substantial number of macrophages infiltrated the tumor sites of MCP-1-transduced cells, but not of controls. These findings suggest that MCP-1 expression sensitizes cervical cancer cells to an otherwise ineffective low dose of cisplatin, possibly by inducing the migration of macrophages to eradicate tumor cells. This system may be a novel strategy for chemotherapy combined with immunogene therapy against otherwise intractable cervical cancers.
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Affiliation(s)
- Mitsuhiro Nakamura
- Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, 13-1, Takaramachi, Kanazawa, Ishikawa 920-8641, Japan
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17
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Mukaida N. CANCER GENE THERAPY USING CYTOKINE AND CHEMOKINE GENES. ANNALS OF CANCER RESEARCH AND THERAPY 2004; 12:33-51. [DOI: 10.4993/acrt.12.33] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/04/2025]
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18
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Gérolami R, Uch R, Bréchot C, Mannoni P, Bagnis C. Gene therapy of hepatocarcinoma: a long way from the concept to the therapeutical impact. Cancer Gene Ther 2003; 10:649-60. [PMID: 12944984 DOI: 10.1038/sj.cgt.7700610] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Hepatocellular carcinoma (HCC), the most prevalent histological form of primary liver cancer is one of the most frequent cancer worldwide. This pathology still requires the development of new therapeutical approaches. Gene therapy strategies focusing on the genetic manipulation of accessory cells involved in the immune reaction against cancer cells, or on the direct transduction of tumor cells with transgenes able to "suicide" cancer cells have been largely developed for more than ten years.
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Affiliation(s)
- René Gérolami
- Département de thérapie cellulaire et génique, EFS Alpes Méditerranée, Marseille, France
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Abstract
Chemokines play an important role in the generation of the immune system and in virtually every aspect of an immune response. The role of chemokines in antitumor immunity has been less straightforward to discern. A dichotomy exists in the field. One area of research has focused on the impact of tumor-derived chemokines, implicating them in everything from metastases to immune suppression. Another area of research has been dedicated to the introduction of chemokines into tumor cells in order to facilitate immune cell recruitment. In this review these two areas of investigation will be explored.
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Affiliation(s)
- Mark S Brault
- Department of Biology, Lafayette College, Easton, Pennsylvania 18042, USA
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20
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Tsuchiyama T, Kaneko S, Nakamoto Y, Sakai Y, Honda M, Mukaida N, Kobayashi K. Enhanced antitumor effects of a bicistronic adenovirus vector expressing both herpes simplex virus thymidine kinase and monocyte chemoattractant protein-1 against hepatocellular carcinoma. Cancer Gene Ther 2003; 10:260-269. [PMID: 12679798 DOI: 10.1038/sj.cgt.7700571] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2002] [Indexed: 12/15/2022]
Abstract
The efficacy of the suicide gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system for the treatment of cancer is limited because of the insufficient gene transfer and the low killing activity. To enhance the antitumor activity, we determined whether recombinant adenovirus vector (rAd)s expressing both HSV-tk and monocyte chemoattractant protein-1 (MCP-1) genes could potentiate the destruction of hepatocellular carcinoma (HCC). The rAd Ad-tk-MCP1 harboring HSV-tk and MCP-1 genes in sequence under the universal CAG promoter was constructed with a bicistronic unit including the encephalomyocarditis virus-internal ribosomal entry site. The levels of HSV-tk expression and GCV-sensitive tumoricidal activity of Ad-tk-MCP1 were comparable to those of rAd expressing HSV-tk alone. The growth of subcutaneous tumors in athymic nude mice was markedly suppressed when tumors were treated with Ad-tk-MCP1 as opposed to another bicistronic vector Ad-MCP1-tk, rAd expressing either HSV-tk or MCP-1, or both of these vectors. The antitumor effects of Ad-tkMCP1 may be dependent on the activation of macrophages, since the recruitment of macrophages was observed tumor necrosis factor-alpha production was enhanced in the tumor tissue. Furthermore, the enhanced antitumor effect was abolished by inactivating macrophages with carrageenan treatment. These results demonstrated that a bicistronic rAd harboring both suicide and chemokine genes in sequence exerted the enhanced, macrophage-dependent, antitumor effects in a model of HCC and support the use of this strategy for the treatment of HCC.
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Affiliation(s)
- Tomoya Tsuchiyama
- Department of Gastroenterology, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8641, Japan
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Hu JY, Li GC, Wang WM, Zhu JG, Li YF, Zhou GH, Sun QB. Transfection of colorectal cancer cells with chemokine MCP-3 (monocyte chemotactic protein-3) gene retards tumor growth and inhibits tumor metastasis. World J Gastroenterol 2002; 8:1067-72. [PMID: 12439927 PMCID: PMC4656382 DOI: 10.3748/wjg.v8.i6.1067] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To evaluate the possibility of the induction of anti-tumor immune response by transfecting the colorectal cancer cells with chemokine MCP-3 gene.
METHODS: Mouse MCP-3 gene was transduced into mouse colorectal cancer cells CMT93 by using of Liposome. G418-resistant clones were selected and the MCP-3 mRNA expression was detected by RT-PCR. The chemotactic activity of MCP-3 in the cell culture supernatant was detected by Chemotaxis assay. The tumorigenicity of wild type CMT93 and CMT93 gene transfectants were detected by in vivo experiments. The immune cell infiltrations in tumor tissue and tumor metastasis were detected histopathologically.
RESULTS: MCP-3 mRNA expression was detected by RT-PCR in gene-transfected cells (CMT93/MCP-3), but not in control groups. And MCP-3 secreted in the cell culture supernatant possessed chemotatic activity. The results from in vivo experiments showed that the tumorigenicity of CMT93/MCP-3 had not decreased, but the tumors derived from CMT93/MCP-3 cells grew more slowly than those from CMT93 cells (1.021 ± 0.253) cm2vs (1.769 ± 0.371) cm2, P < 0.05) or CMT93/mock cells (1.021 ± 0.253) cm2vs (1.680 ± 0.643) cm2, P < 0.05). Histophathological results showed few immune cells infiltrating in the tumor tissue derived from the controls. In the tumor tissue derived from CMT93/MCP-3, infiltrating immune cells increased. In addition, no tumor metastasis was found in all mice inoculated with CMT93/ MCP-3 tumor cells. But all mice had tumor metastasis in CMT93 controls and 4 in 5 mice had tumor metastasis in CMT93/mock controls.
CONCLUSION: The results suggested that the transfection of chemokine MCP-3 gene could promote the induction of anti-colorectal cancer immunity, but the tumor growth could not be inhibited completely by merely MCP-3 gene transfection.
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Affiliation(s)
- Jin-Yue Hu
- Lab of Tumor Immunobiology, Cancer Research Institute, Xiangya Medical School, Central South University, Hunan Province, China
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22
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Abstract
Recombinant adenovirus (rAd) and recombinant adeno-associated virus (rAAV) are among the most extensively used vectors in gene therapy studies to date. These two vectors share some similar features such as a broad host range and ability to infect both proliferating and quiescent cells. However, they also possess their own unique set of properties that render them particularly attractive for gene therapy applications. rAd vectors can accommodate larger inserts, mediate transient but high levels of protein expression, and can be easily produced at high titers. Development of gutted rAd vectors has further increased the cloning capacity of these vectors. The gaining popularity of rAAV use in gene therapy can be attributed to its lack of pathogenicity and added safety due to its replication defectiveness, and its ability to mediate long-term expression in a variety of tissues. Site-specific integration, as occurs with wild-type AAV, will be a unique and valuable feature if incorporated into rAAV vectors, further improving their safety. This paper describes these properties of rAd and rAAV vectors, and discusses further development and vector improvements that continue to extend the utility of these vectors, such as cell retargeting by capsid modification, differential transduction by use of serotypes, and extension of the cloning capacity of rAAV vectors by dual vector heterodimerization.
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Affiliation(s)
- Chooi May Lai
- Centre for Ophthalmology and Visual Science, University of Western Australia, Nedlands, Western Australia
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