ケモカインMCP-1が誘導する肝癌自殺遺伝子治療の再発予防効果の検討

金沢大学附属病院これまでの研究により、自殺遺伝子(HSV-tk)と単球走化因子(MCP-1)を発現するアデノウイルスベクターを作製し、MCP-1産生量によって抗腫瘍効果が顕著に変化することを報告した(Cancer Gene Ther. 2003, J. Immunol. 2007)。MCP-1は抗腫瘍効果と腫瘍増殖の相反する作用を示すことが報告されており、臨床応用をめざした至適投与量に関する詳細な検討はなされていない。そこで今年度は、肝癌自殺遺伝子治療に併用するMCP-1の投与量とin vitro, in vivoで抗腫瘍免疫の賦活作用について検討した。具体的にはHSV-tk、MCP-1を発現するAd-tk、Ad-MCP1を用いて、Ad-tk濃度を固定した条件下でAd-MCP1の濃度(1,1/10,1/100 dose of Ad-tk)を変化させ、以下の検討を行った。(1)MCP-1産生量の測定。(2)皮下肝癌モデルでの抗腫瘍効果。(3)腫瘍、脾臓でのサイトカインの発現量の検討。(4)活性化マクロファージのサイトカイン産生能の検討。(5)腫瘍再移植に対する抑制効果。結果を以下に示す。Ad-tkとの併用下で、Ad-MCP1の濃度に相関してMCP-1産生量は増加した。Ad-MCP1投与量が1/10以上あれば、高い腫瘍増殖抑制効果を認めた。一方、肝癌再移植に対しては、Ad-MCP1投与量が1/10治療群で高い再発抑制効果を認めた。方法(3),(4)の検討から、これらの治療効果はTh1(IL-12, IL-18)/Th2(IL-10)サイトカインの産生量の差と相関していた。また脾臓において、Ad-MCP1投与量が1/100群<1群<1/10群の順に、NK、T細胞の数が増加していた。至適量のMCP-1の併用によって、マクロファージの活性化を介して高い腫瘍縮小と再発抑制効果が得られたことより、MCP-1が肝癌に対する免疫遺伝子治療の開発に寄与する可能性が示唆された。なお、現在論文(Gene Therapy)投稿中である。研究課題/領域番号:18790450, 研究期間(年度):2006 – 2007出典：「ケモカインMCP-1が誘導する肝癌自殺遺伝子治療の再発予防効果の検討」研究成果報告書　課題番号18790450（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-18790450/)を加工して作

自殺遺伝子と単球走化因子を用いたbicistronicアデノウイルスベクターによる肝癌に対する抗腫瘍効果の増強

取得学位 : 博士（医学）, 学位授与番号 : 医博甲第1581号, 学位授与年月日 : 平成15年6月30日, 学位授与大学 : 金沢大

Optimal amount of monocyte chemoattractant protein-1 enhances antitumor effects of suicide gene therapy against hepatocellular carcinoma by M1 macrophage activation

[email protected] 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. © 2008 Japanese Cancer Association

Prevention of intrahepatic metastasis of liver cancer by suicide gene therapy and chemokine ligand 2/monocyte chemoattractant protein-1 delivery in mice

金沢大学医薬保健研究域医学系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/MLow), 3/3 (T/MHigh) and Ad-tk/Ad-LacZ = 3/3 (T/L) were injected into BALB/c mice.Results: Intrahepatic tumor growth was significantly lower in T/MLow mice. By contrast, no tumor suppression was observed in T/MHigh mice. The tumor-specific cytolytic activities of splenocytes from T/MLow and T/MHigh 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/MLow mice. In addition, interleukin-12 production was elevated in these tissues. Vascular endothelial growth factor-A expression and CD31+ microvessels were increased in T/MHigh 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. © 2010 John Wiley & Sons, Ltd

Diagnostic utility of aberrant methylation of tissue factor pathway inhibitor 2 in pure pancreatic juice for pancreatic carcinoma

医薬保健研究域医学系The tissue factor pathway inhibitor 2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor. Recently, the aberrant methylation of TFPI-2 was detected frequently in pancreatic carcinoma (PCa) tissues but not in normal pancreatic tissues. We analyzed the aberrant methylation of TFPI-2 in the pure pancreatic juice (PPJ) aspirated endoscopically from patients with various pancreatic diseases. Using the highly sensitive methylation-specific polymerase chain reaction (MSP) and quantitative MSP (Q-MSP) assay, we investigated the aberrant methylation of TFPI-2 in nine human PCa cell lines and in the PPJ from patients with PCa, intraductal papillary mucinous neoplasms (IPMN) and chronic pancreatitis (CP). The incidence of aberrant TFPI-2 methylation was seven (77.8%) of nine PCa cell lines by Q-MSP. In cell lines, the expression of TFPI-2 mRNA by quantitative reverse transcription-polymerase chain reaction showed an inverse correlation to the aberrant methylation of TFPI-2. The incidence of aberrant TFPI-2 methylation in the PPJ was 21 (58.3%) of 36 PCa patients, three (17.6%) of 17 IPMN and one (4.8%) of 21 CP by MSP assay. Using a suitable cut-off value of 2.5 according to the receiver operating characteristic curve, the incidence of aberrant TFPI-2 methylation in the PPJ by real-time MSP was 18 (62.1%) of 29 PCa patients, one (5.1%) of 17 IPMN and three (14.3%) of 21 CP, respectively. The incidence of quantitative TFPI-2 hypermethylation in the PPJ with PCa was significantly higher than that with IPMN (P < 0.001) or CP (P < 0.001). Moreover, the aberrant methylation rate of TFPI-2 in the PPJ was 100%, as observed (6/6) in the PCa patients with liver metastasis, and 86.7% (26/30) in stages IVa + IVb of PCa by Q-MSP assay. These results suggest that promoter methylation of TFPI-2 in the PPJ may be a useful marker in the diagnosis and progression of PCa using an endoscopically feasible approach. © 2006 Japanese Cancer Association

Membrane-bound form of monocyte chemoattractant protein-1 enhances antitumor effects of suicide gene therapy in a model of hepatocellular carcinoma

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.Cancer Gene Therapy advance online publication, 9 March 2012; doi:10.1038/cgt.2012.3

Advanced hepatocellular carcinoma treated effectively with irinotecan via hepatic arterial infusion followed by proton beam therapy

金沢大学医学部附属病院がん高度先進治療センターWe report a 48-year-old man with hepatocellular carcinoma (HCC) treated with hepatic arterial infusion (HAI) chemotherapy followed by proton beam therapy. The HCC lesion in this patient was 88 mm in diameter, with portal vein tumor thrombosis in the right lobe of the liver. He was first treated with 5-fluorouracil, cisplatin, and isovorin, administered by HAI, combined with interferon-α, and he was subsequently treated with epirubicin and mitomycin-C administered by HAI. However, no definite efficacy of either of these treatments was observed. Then, after 3 weeks\u27 continuous administration of irinotecan by HAI, the tumor size decreased to 68 mm in diameter. However, 3 months after reduction of the tumor, the tumor had become enlarged to 100 mm in diameter and intrahepatic metastases were prominent. Angiographic findings indicated that the HCC was fed not only from the right hepatic artery but also from the left gastric and right and left subphrenic arteries. After rearrangement of the arteries, and 3 months\u27 continuous HAI chemotherapy with irinotecan, plus hyperthermia, the tumor size had decreased to 50 mm in diameter. The reduction rate of the main tumor according to the Response Evaluation Criteria in Solid Tumors was 43%; therefore, the efficacy of this treatment was judged as a partial response. Two months after reduction of the tumor, the patient\u27s serum alpha-fetoprotein (AFP) level was elevated, and so docetaxel was administered by HAI instead of irinotecan. The liver tumors showed gradual enlargement during the administration of docetaxel, although the AFP level was suppressed. Proton beam therapy was instituted and the liver tumors showed necrosis after this therapy. The patient died of hepatic failure and distant metastases 6 years after the onset of HCC. As far as we know, this is the first case report of HCC treated effectively with irinotecan administered by HAI followed by proton beam therapy in which tumor suppression and the long-term survival of the patient were observed. © 2009 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases

In-situ water-immersion experiments on amorphous silicates in the MgO-SiO2 system : implications for the onset of aqueous alteration in primitive meteorites

Amorphous silicates, abundant in primitive carbonaceous chondrites, are among the most primitive materials from the early Solar System. They show evidence of some aqueous alteration in the meteorite parent bodies, but it is not clear how this highly reactive material changed at an early stage after contact with water. Herein, we report in-situ experiments on the aqueous alteration of amorphous silicate nanoparticles (typically 70 nm in diameter); we used two different compositions that are similar to forsterite (MgO/SiO2 = 2.02) and enstatite (MgO/SiO2 = 1.15) in the simple MgO-SiO2 system to under-stand basic reaction principles at the onset of the aqueous alteration. The experiments were performed in pure water at room temperature using X-ray diffraction (XRD), transmission electron microscopy (TEM), and pH measurements. The in-situ TEM images of the nanoparticles in particular those with the forsterite composition gradually became difficult to recog- nize in water. The pH value of the water also increased with time, suggesting that preferential Mg2+ dissolution occurred from the amorphous silicates right after mixing with water. The in-situ XRD patterns showed that magnesium silicate hydrate (M-S-H), which is a poorly crystalline phase like a phyllosilicate, newly appeared. The M-S-H seems to have been formed via a dissolution-precipitation process. Its formation rate from amorphous silicates was considerably higher than from crystalline silicates, because amorphous silicates are highly metastable and have high solubility in water. M-S-H formation from the for-sterite composition, which has a highly unstable amorphous structure, is ten times faster than from the enstatite composition. The M-S-Hs show string-like or tiny fragmental textures in the final dried products that are very similar to those observed in the matrices of some primitive carbonaceous chondrites. M-S-H would have been the initial product formed in the aqueous alteration of amorphous silicates in the meteorites; thus, it is an important tracer of early aqueous activity at low temperatures in the early Solar System. By comparing the in-situ observations with those obtained after drying the experimental samples, we found two types of M-S-Hs: epigenetic M-S-Hs which have a slightly Si-rich composition formed during drying, and syn- genetic M-S-Hs formed by in-situ alteration. Carbonaceous chondrites may also contain these two types of hydrous silicates, and this should be investigated to understand the conditions for aqueous alteration in the early Solar System in more detail. The present study clearly showed the importance of Mg/Si ratio in the precursor materials, although the actual chondrites are in more complicated multi-component system. Future experiments based on the present results can extend the investigation to the system containing Fe, S, and other components as in carbonaceous chondrites. (C) 2020 Elsevier Ltd. All rights reserved