Interleukin-10 gene transfer to peritoneal mesothelial cells suppresses peritoneal dissemination of gastric cancer cells due to a persistently high concentration in the peritoneal cavity

Interleukin (IL)-10 has potent biological properties including an inhibitory action on the proliferation and metastasis of various cancer cells. However, it is difficult to maintain a high concentration of this cytokine as it has a short half life. In this study, we evaluated whether peritoneal mesothelial cells (PMCs) could be suitable for maintaining a high concentration of IL-10 using adenoviral gene transfer. We also evaluated the therapeutic effects of an intraperitoneal injection with adenoviral vector containing mouse IL-10 gene (Ad-mIL-10) using a mouse peritoneal dissemination model of MKN45 gastric cancer cells. We demonstrated that in vitro transfection efficiency of a recombinant adenovirus containing the bacterial beta-galactosidase gene (Ad-LacZ) was approximately 10-fold higher for primarily isolated PMCs than MKN45. The entire peritoneum was transfected until 3 weeks after an intraperitoneal Ad-LacZ injection. Ad-mIL-10 treatment increased intraperitoneal IL-10 levels until 3 weeks after treatment, and then significantly inhibited peritoneal cancer growth by inhibiting angiogenesis. This treatment also improved cachexia and prolonged mice survival. We thus concluded that IL-10 gene transfer in PMCs could be a new strategy for the prevention of peritoneal dissemination of gastric cancer due to the resulting persistently high IL-10 concentration in the peritoneal cavity.     

ICAM-2 gene therapy for peritoneal dissemination of scirrhous gastric carcinoma.

PURPOSE: Human scirrhous gastric carcinoma develops peritoneal dissemination with high frequency, and the prognosis of patients with peritoneal metastasis is poor. There have been few reports of an immunogene therapy for peritoneal dissemination. Intercellular adhesion molecule (ICAM)-2 is a second ligand of leukocyte function-associated antigen-1, which functions as a costimulatory molecule for effector cells. In the present study, we examined whether ICAM-2 transfection using adenovirus vector is effective gene therapy for peritoneal metastasis of gastric cancer. EXPERIMENTAL DESIGN: We constructed an adenovirus vector, AdICAM-2, that encodes the full-length human ICAM-2 gene under control of the cytomegalovirus promoter. This vector expresses high levels of ICAM-2 on the human gastric cancer cell line OCUM-2MD3, which has high peritoneal metastatic ability in nude mice. We investigated the antitumor effects of gene transfer of ICAM-2 using the adenovirus vector AdICAM-2 in vitro and in vivo. RESULTS: ICAM-2 expressed on OCUM-2MD3 cells by AdICAM-2 demonstrated significantly high adhesiveness to and cytotoxicity against peripheral blood mononuclear cells in vitro compared with the control adenovirus vector AdlacZ. Intratumoral injection of AdICAM-2 significantly inhibited the growth of s.c. tumor. Mice with peritoneal metastasis survived for a significantly longer time after AdICAM-2 injection, compared with injection of AdlacZ. Histopathological findings revealed that many natural killer cells infiltrated the peritoneal metastatic lesions after AdICAM-2 injection. CONCLUSIONS: These findings suggest that transduction of ICAM-2 into cancer cells enhances the adhesion and activation of natural killer cells, resulting in a reduction of peritoneal metastasis. ICAM-2 transfection using adenovirus vector might be an effective form of gene therapy for peritoneal metastasis of gastric cancer.     

Suppression of peritoneal implantation of gastric cancer cells by adenovirus vector-mediated NK4 expression

Peritoneal dissemination is the most common mode of metastasis in gastric cancer. We previously reported the importance of milky spots (MS), peritoneal lymphoid tissues, as selective sites of cancer implantation in peritoneal dissemination. In the present study, we first demonstrated that intraperitoneal injection of adenovirus vector encoding the GFP gene into tumor-free nude mice resulted in GFP expression at omental and mesenteric MS; MS macrophages were target cells for adenovirus infection. We confirmed that intraperitoneal injection of adenovirus vector encoding the NK4 gene (AdNK4) resulted in NK4 production localized to the peritoneal cavity, especially the omentum. Adenovirus vector-mediated MS-selective transgene expression was markedly impaired in tumor-bearing mice whose MS had already been replaced by infiltrating cancer cells. However, prior injection of AdNK4 successfully inhibited MS-selective cancer cell implantation, resulting in suppression of peritoneal dissemination and prolongation of survival. Adenovirus vector-mediated MS-selective delivery of a therapeutic gene may prevent peritoneal dissemination of gastric cancer.     

Adenovirus-mediated transfection of caspase-8 augments anoikis and inhibits peritoneal dissemination of human gastric carcinoma cells

Caspase-8 is a member of the cysteine protease family that modulates apoptosis induced by a variety of cell death signals and has recently been found to be activated during the process of anoikis, which is a form of apoptosis caused by loss of anchorage in epithelial cells. We previously demonstrated that the inhibition of anoikis promotes peritoneal dissemination of human gastric carcinoma MKN45 cells, which are anchorage dependent. This suggests that augmentation of anoikis may suppress dissemination of carcinoma cells. To determine whether extrinsic overexpression of caspase-8 can augment anoikis in MKN45 cells, we transfected them with the caspase-8 gene using an adenoviral (Adv) vector (Adv-caspase-8). Here we demonstrate that Adv-caspase-8 infection, at 15 multiplicity of infection (MOI), can augment anoikis in MKN45 cells and suppresses MKN45 peritoneal dissemination in SCID mice. The inhibitory effect on peritoneal dissemination resulted in a prolonged survival compared with that in control mice. In contrast, the Adv-caspase-8 (15 MOI) had no distinct effect on cell viability or growth either of attached MKN45 cells or of s.c. tumor growth in SCID mice. Thus, Adv-mediated overexpression of caspase-8 suppressed peritoneal dissemination mainly through augmentation of anoikis. In addition, Adv-caspase-8-mediated augmentation of anoikis was similarly observed in another gastric carcinoma MKN74 cell line. In contrast, Adv-p53 could not augment anoikis in MKN45 cells. These results imply that Adv-mediated gene transfer of caspase-8 can selectively induce apoptosis in detached carcinoma cells and, thus, shows potential as a novel cancer therapy against dissemination of gastric and probably other carcinoma cells originating from epithelial tissues.     

Intraperitoneal injection of adenovirus expressing antisense K-ras RNA suppresses peritoneal dissemination of hamster syngeneic pancreatic cancer without systemic toxicity

We examined the antitumor effect and safety of the adenovirus-mediated expression of antisense K-ras RNA in two peritoneal dissemination models of pancreatic cancer. First, we found that the infection of an adenovirus vector expressing antisense human K-ras RNA (AxCA-AS) induced significant apoptosis in vitro in human pancreatic cancer cells with K-ras mutation. Second, the intraperitoneal (ip) injection of AxCA-AS effectively suppressed the growth of human pancreatic cancer cells in the peritoneal cavity of nude mice. Third, in the hamster syngeneic peritoneal dissemination model, the ip injection of an adenovirus expressing antisense hamster K-ras RNA significantly suppressed the peritoneal growth of hamster pancreatic cancer cells, and no significant systemic toxicity was observed in the treated hamsters. This study suggests a feasibility of the development of a therapeutic strategy against pancreatic cancer based on the adenovirus-mediated transduction of an antisense K-ras construct.     

Efficacy of oncolytic reovirus against human gastric cancer with peritoneal metastasis in experimental animal model

The prognosis of gastric cancer patients with peritoneal dissemination is extremely poor, and the development of an effective treatment is necessary. The aim of this study was to investigate the efficacy of oncolytic reovirus against peritoneal metastasis in human gastric cancer using an experimental animal model. Four human gastric cancer cell lines, including MKN45p, NUGC4, MKN7 and KatoIII, a normal NIH3T3 cell line as a control, and reovirus serotype 3, were used in this study. We evaluated the cytopathic effect of reovirus and the Ras activity in each gastric cancer cell line in vitro. To evaluate oncolytic efficacy in vivo, reovirus (1x10(8) PFU) was administered into the peritoneal cavity of nude mice on days 7, 8 and 9 after inoculation with MKN45p cells. Mean volume of ascites and the total number and weight of the peritoneal tumors were measured after sacrifice. After reovirus infection, cytopathic effect was observed in all four gastric cancer cell lines, but not in the control cells. Ras activation assay showed that Ras activity in all four gastric cancer cell lines increased to a higher level than that in the control cells. In the animal model experiments, mean volume of ascites and the total number and weight of the peritoneal tumors in the reovirus treatment group were significantly lower than those in the control group. In conclusions, intraperitoneal administration of reovirus could be useful as a new modality against peritoneal metastasis in gastric cancer.     

Adenovirus-mediated calponin h1 gene therapy directed against peritoneal dissemination of ovarian cancer: bifunctional therapeutic effects on peritoneal cell layer and cancer cells.

PURPOSE: Calponin h1 (CNh1), one of the family of actin-binding proteins, stabilizes the filaments of actin and modulates various cellular biological phenotypes. Recent studies revealed the close correlation between the invasive tumor spread and the reduced expression of CNh1 and alpha-smooth muscle actin in the surrounding stromal cells. The purpose of this study is to evaluate the efficacy of i.p. CNh1 gene therapy against peritoneal dissemination of ovarian cancer. EXPERIMENTAL DESIGN: We used an adenoviral vector to induce the CNh1 gene into peritoneal cells and ovarian cancer cells as a means of enhancing or inducing the expression of alpha-smooth muscle actin as well as CNh1. The efficacy of gene transfer was examined by in vitro cell culture and in vivo animal experiments. RESULTS: The formation of longer and thicker actin fibers was observed in each transfected cell line, and the localization of these fibers coincided with that of externally transducted CNh1. With respect to changes in cell behavior, the CNh1-transfected peritoneal cells acquired an ability to resist ovarian cancer-induced shrinkage in cell shape; thus, cancer cell invasion through the monolayer of peritoneal cells was inhibited. In addition, CNh1-transfected ovarian cancer cells showed suppressed anchorage-independent growth and invasiveness, the latter of which accompanied impaired cell motility. The concomitant CNh1 transfection into both peritoneal cells and ovarian cancer cells produced an additive inhibitory effect with respect to cancer cell invasion through the peritoneal cell monolayer. By in vivo experiments designed to treat nude mice that had been i.p. inoculated with ovarian cancer cells, we found that the i.p. injected CNh1 adenovirus successfully blocked cancer-induced morphologic changes in peritoneal cell surface and significantly prolonged the survival time of tumor-bearing mice. Moreover, CNh1 adenovirus could successfully enhance the therapeutic effect of an anticancer drug without increase in side effects. CONCLUSIONS: Thus, CNh1 gene therapy against peritoneal dissemination of ovarian cancer is bifunctionally effective (i.e., through inhibitory effects on the infected peritoneal cell layers that suppress cancer invasion and through direct antitumor effects against invasion and growth properties of cancer cells).     

Carcinoembryonic antigen-targeted selective gene therapy for gastric cancer through FZ33 fiber-modified adenovirus vectors.

PURPOSE: A major problem when using the adenoviral vectors for gene therapy applications is thought to be related to low transduction efficiency in cancer cells or to side effects in normal cells. There is an urgent requirement to improve the specificity of gene delivery in the context of cancer gene therapy. EXPERIMENTAL DESIGN: We constructed a genetically modified adenovirus incorporating an IgG Fc-binding motif from the Staphylococcus protein A, Z33, within the HI loop (Adv-FZ33). A remarkable degree of targeted gene delivery to gastric cancer cells was obtained with Adv-FZ33 with the fully human anti-carcinoembryonic antigen (CEA) monoclonal antibody, C2-45. RESULTS: In vitro LacZ or EGFP gene expression after Adv-FZ33 infection via C2-45 was 20 times higher than control monoclonal antibody in MKN-45 at 1,000 viral particles/cell. We generated Ax3CAUP-FZ33 (UP-FZ33), which is an Adv-FZ33 derivative vector expressing a therapeutic gene (i.e., Escherichia coli uracil phosphoribosyltransferase), which converts 5-fluorouracil (5-FU) directly to 5-fluoro-UMP. UP-FZ33 with C2-45 enhanced the cytotoxicity of 5-FU by 10.5-fold in terms of IC(50) against MKN-45 compared with control IgG4. In a nude mouse peritoneal dissemination model, tumor growth in mice treated with UP-FZ33/C2-45/5-FU was significantly suppressed, and tumor volumes were less than one-fourth of those of the control IgG4 group (P < 0.05). The median survival time of the UP-FZ33/C2-45/5-FU group was significantly longer than those treated with PBS or 5-FU only (P < 0.01). CONCLUSIONS: These data suggest that CEA-targeted FZ33 mutant adenovirus-mediated gene delivery offers a strong and selective therapeutic modality against CEA-producing cancers.     

Intracellular targeting therapy of cisplatin-encapsulated transferrin-polyethylene glycol liposome on peritoneal dissemination of gastric cancer

Peritoneal dissemination in gastric cancer is a common fatal clinical condition with few effective therapies available. We studied the therapeutic effect of a tumor-targeting drug delivery system that uses cisplatin-encapsulated and Tf-conjugated PEG liposomes (Tf-PEG liposomes) in nude mice with peritoneal dissemination of human gastric cancer cells. Small unilamellar Tf-PEG, PEG or DSPC/CH liposomes (bare liposomes) encapsulating cisplatin were prepared by reverse-phase evaporation followed by extrusion. Electron microscopic studies revealed that Tf-PEG liposomes were internalized into tumor cells by receptor-mediated endocytosis. To examine the biodistribution of each liposome and cisplatin level, nude mice were inoculated i.p. with 10(7) MKN45P human gastric tumor cells. On the fourth day after tumor inoculation, (3)H-CHE-labeled and cisplatin-encapsulated Tf-PEG, PEG or bare liposome were inoculated i.p. The Tf-PEG liposome-administered group maintained high liposome and cisplatin levels in ascites and showed a prolonged residence time in the peripheral circulation. Uptake of Tf-PEG liposomes into the liver and spleen was significantly lower than that of bare liposomes. Uptake of Tf-PEG liposomes in disseminated tumor cells of ascites and the greater omentum was significantly higher than that of PEG or bare liposomes and a significant increase in cisplatin levels was observed in these tumor cells. Mice receiving Tf-PEG liposomes 1 and 4 days after the day of tumor inoculation showed significantly higher survival rates compared with those receiving PEG liposomes without Tf, bare liposomes or free cisplatin solution. These results suggest that cisplatin-encapsulated Tf-PEG liposomes may be useful as a new intracellular targeting carrier for treatment of gastric cancer with peritoneal dissemination.     

Prevention of peritoneal metastasis of human gastric cancer cells in nude mice by S-1, a novel oral derivative of 5-Fluorouracil

OBJECTIVE: Recent clinical trials have suggested that oral administration of a new anti-cancer agent, S-1, seems a promising therapy for advanced gastric cancer. In this study, we assessed the efficacy of S-1 against peritoneal dissemination of gastric cancer in a newly developed animal model and investigated the efficacy of S-1 from a pharmacokinetic angle. METHODS: Human gastric cancer cells (MKN-45) were injected into the peritoneal cavity of nude mice. The cancer cells were transduced using an enhanced green fluorescent protein (EGFP)-expressing plasmid vector, enabling micrometastatic foci to be accurately assessed with a high level of detection sensitivity. To investigate pharmacokinetics, the concentration of 5-FU was determined in tumor, peritoneum and plasma. RESULTS: Fourteen and 21 days after intraperitoneal injection, a significant difference in the number of fluorescent foci was observed between the control group and the S-1 group (p = 0.02 and p = 0.0024, respectively. The therapeutic effect of S-1 was significantly greater than that of 5-FU. Furthermore, S-1 treatment greatly improved the survival time and cachexia. The area under the curve of 5-FU in tumor was higher than in the peritoneum and plasma. CONCLUSION: Oral S-1 is a promising chemotherapy for peritoneal dissemination of gastric cancer.     

Spatial distribution of intraperitoneally administrated paclitaxel nanoparticles solubilized with poly (2-methacryloxyethyl phosphorylcholine-co n-butyl methacrylate) in peritoneal metastatic nodules

Intraperitoneal (i.p.) administration of paclitaxel nanoparticles (PTX-30W) prepared by solubulization with the amphiphilic copolymer of 2-methacryloxyethyl phosphorylcholine and n-butyl methacrylate can efficiently suppress the growth of peritoneal metastasis. In this study, we characterized the drug distribution of i.p. injected PTX-30W in peritoneal tumor and liver in a mouse model using MKN45, human gastric cancer cells. Oregon green-conjugated PTX-30W showed perivascular accumulation in MKN45 tumor in the peritoneum at 24 h after intravenous (i.v.) injection; however, the amount of PTX in tumor was markedly less than that in liver. In contrast, a larger amount of PTX accumulated in the peripheral area of disseminated nodules at 1 h after i.p. injection and the area gradually enlarged. The depth of PTX infiltration reached 1 mm from the tumor surface at 48 h after i.p. injection, and the fluorescence intensity was markedly greater than that in liver. Interestingly, i.p. injected PTX preferentially accumulated in relatively hypovascular areas, and many tumor cells in the vicinity of PTX accumulation showed apoptosis. This unique accumulation pattern and lesser washout in hypovascular areas are thought to be attributable to the superior penetrating activity of PTX-30W, and thus, PTX-30W is considered to be highly suitable for i.p. chemotherapy for peritoneal dissemination.     

Efficacy of intra-peritoneal and intra-venous injection of monoclonal antibody A7-NCS conjugates against peritoneal dissemination of the gastric cancer

The monoclonal antibody A7 (Mab A7) against human colonic cancer also reacts with human gastric cancer at a high rate. We produced a conjugate of neocarzinostatin (NCS) with Mab A7 (A7-NCS). The in vitro anticancer effect of A7-NCS on the antigen-positive human gastric cancer cell line MKN45 was stronger than that of free NCS. Nude mice models of peritoneal dissemination were established by the intra-peritoneal inoculation of MKN45. These models were divided into three groups. The anticancer effect observed in the group that received the intra peritoneal injection of A7-NCS was superior to that observed in the group that received the intra-venous injection and the group that received no treatment. In conclusion, the intra-peritoneal injection of A7-NCS was a useful treatment method for the peritoneal dissemination of gastric cancer.     

胃癌细胞uPA表达与腹膜种植潜能的相关性研究

目的探讨不同胃癌细胞系尿激酶型血浆纤溶酶原激活因子(uPA)的表达及其与腹膜种植能力的关系。方法采用半定量RT-PCR、Western blot和ELISA方法,比较不同胃癌细胞系(AGS、SGC7901、MKN45和MKN28)uPA表达和uPA活性的差异。将胃癌细胞直接注入裸鼠腹腔,建立腹膜种植模型,通过观察裸鼠生存时间等方法,比较不同胃癌细胞系的腹膜种植潜能。结果uPA表达以SGC7901细胞最高,uPA活性以MKN45细胞最高,AGS细胞uPA表达和活性均最低。AGS未发生腹膜种植肿瘤;SGC7901和MKN45均出现大小不等的腹膜种植肿瘤;MKN28出现伴有血性腹水且大小相似的腹膜种植肿瘤;但MKN45最早形成腹膜种植肿瘤,并且该组裸鼠的生存时间最短。结论各株胃癌细胞的uPA表达在转录和翻译水平相一致,但uPA的表达量和uPA活性大小有明显差异,并且这二者与腹膜种植能力呈正相关。     

Intraperitoneal administration of an adenovirus vector carrying REIC/Dkk-3 suppresses peritoneal dissemination of scirrhous gastric carcinoma

Expression levels of the novel tumor suppressor gene REIC/Dkk-3 are reduced in many human cancers. We have previously showed that an adenovirus vector carrying REIC/Dkk-3 (Ad-REIC) induced apoptosis of cancer cells selectively and exerted bystander antitumor effects via ER stress. We examined possible effects of Ad-REIC in a peritoneal dissemination model of scirrhous gastric carcinoma (SGC). Among various types of gastric cancer, SGC continues to be associated with the worst prognosis due to a high incidence of metastases in the peritoneal cavity. We found that a single intraperitoneal injection of Ad-REIC suppressed tumor dissemination and disease progression. Immunomodulation by Ad-REIC led to recruitment of natural killer cells inside tumor nodules. We conclude that Ad-REIC gene therapy may be a potential tool in combinatorial approaches to achieve curative effects in SGC.     

Functional analysis of peritoneal lymphoid tissues by GFP expression in mice--possible application for targeting gene therapy against peritoneal dissemination

It is important to develop an efficient adjuvant therapy for the prevention of the postoperative peritoneal recurrence of gastrointestinal cancers such as gastric cancer or pancreatic cancer. Milky sports (MS) are peritoneal lymphoid tissues broadly distributed on the peritoneal tissues such as the omentum, mesentery, or Douglas pouch, and are the selective implantation sites of disseminated cancer cells. In this study, we introduced GFP gene into various cancer cell lines and host-derived cells such as peritoneal macrophages and dendritic cells by adenovirus vetor and injected them i.p. into mice. We then investigated the sites of GFP expression on the peritoneum by fluorescence microscopy. The results showed that green fluorescence was detected specifically for the MS sites that stained black with activated carbon particles (CH40), despite the differences in the cell type injected. These findings indicate that 1) the physioanatomical characteristics of MS may play an essential role in the formation of the initial disseminated lesions at MS, and 2) the host-derived cells accumulating near MS may be available as carriers of a therapeutic gene in intraperitoneal gene therapy against peritoneal dissemination.     

Significance of integrin alpha2/beta1 in peritoneal dissemination of a human gastric cancer xenograft model

In the present study, the role of integrin alpha2/beta1 in peritoneal dissemination of gastric cancer was investigated using an in vivo xenograft model for the highly metastatic MKN-45-P gastric cancer cells. Metastatic ability of MKN-45-P cells was significantly associated with the simultaneous expression of integrin alpha2 and alpha3 subunits. In an in vitro adhesion assay, neutralizing antibody for integrin alpha2 or beta1 subunit inhibited the adhesion of MKN-45-P cells to collagen type I and type IV. Moreover, the injection of anti-beta1 monoclonal antibody reduced the number of cancer cells on the peritoneum in nude mice that had been inoculated with MKN-45-P cells. These results suggest that integrin alpha2/beta1 represents a candidate target molecule available for the prevention of gastric cancer peritoneal dissemination.     

Preventive effect of matrix metalloproteinase inhibitor, R-94138, in combination with mitomycin C or cisplatin on peritoneal dissemination of human gastric cancer cell line TMK-1 in nude mice.

R-94138, a matrix metalloproteinase inhibitor, was examined for the ability to prevent peritoneal dissemination of a human gastric cancer xenograft, TMK-1. When the supernatant of a co-culture of TMK-1 cells and human normal fibroblast cells was subjected to gelatin zymography, it was clear that the protein expression of MMP-2 had been inhibited by R-94138. When TMK-1 was injected intraperitoneally (i.p.) into nude mice at 5 x 10(5) cells/body, the resulting peritoneal dissemination mimicked clinical carcinomatous peritonitis. When the maximum tolerated dose of mitomycin C (MMC) or cisplatin (DDP) was given 12 h after the tumor inoculation, peritoneal dissemination was completely inhibited, while the effect of R-94138 was limited when it was given i.p. at a dose of 20 mg/kg in a schedule of q.d. x 5 starting 12 h after tumor injection. MMC and DDP also suppressed peritoneal dissemination when they were administered 1 week after the tumor inoculation at a single dose of 2 and 3 mg/kg i.p., respectively. R-94138 inhibited peritoneal dissemination when it was administered i.p. at a dose of 30 mg/kg in a schedule of q.d. x 5 starting from 1 week after tumor injection. The combination of MMC and R-94138 increased the preventive effect on peritoneal dissemination. R-94138 seems to be a promising candidate to prevent peritoneal dissemination of gastric cancer.     

Adenoviral-mediated gene transduction of the hepatocyte growth factor (HGF) antagonist, NK4, suppresses peritoneal metastases of gastric cancer in nude mice

The competitive inhibitory effects of NK4 (a specific hepatocyte growth factor (HGF)-antagonist) on the interaction between HGF and the c-Met/HGF receptor has been shown in HGF-mediated invasion of some distinct types of human cancer cells. Furthermore, NK4 has inhibitory effects on the angiogenic pathways driven by basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), as well as by HGF. In this study, to evaluate the therapeutic efficacy of adenoviral-mediated NK4 gene treatment, we employed animal models of peritoneal metastasis using two gastric cancer cell lines, the strongly c-Met expressing MKN45 cell line and the weakly c-Met-expressing cell line, TMK1. In both models, the total number and weight of peritoneal tumours per mouse and ascites treated early with AxCANK4 (administered 3 times 2, 7 and 12 days after the tumour inoculation) were significantly reduced compared with those treated with phosphate-buffered solution (PBS) and AxCALacZ (P < 0.05). In Factor-VIII-related-antigen-stained sections from peritoneal metastatic tumours, the inhibition of intratumour vessels was observed in tissues from tumours of MKN45 and TMK1 treated with AxCANK4. We also compared the therapeutic effect of early AxCANK4 treatment with that of late treatment (at 7, 12 and 17 days). Peritoneal metastases and ascites treated late with AxCANK4 showed less of an improvement than those treated early with AxCANK4 in both models. In addition, the inhibitory effect of cisplatin (CDDP) on peritoneal metastasis was significantly enhanced by AxCANK4, suggesting that the combination of intraperitoneal (i.p.) chemotherapy with NK4 gene therapy might be effective, even in cases of advanced peritoneal metastasis from gastric cancer. To conclude, these results show clearly that NK4 gene therapy inhibits peritoneal metastases from gastric cancer, regardless of the level of c-Met/HGF receptor expression in the tumour cells, and especially in the early stages of peritoneal metastasis.     

Intraperitoneal administration of paclitaxel solubilized with poly(2-methacryloxyethyl phosphorylcholine-co n-butyl methacrylate) for peritoneal dissemination of gastric cancer

Intraperitoneal (i.p.) administration of paclitaxel (PTX) is a hopeful therapeutic strategy for peritoneal malignancy. Intravenously (i.v.) injected nanoparticle anticancer drugs are known to be retained in the blood stream for a long time and favorably extravasated from vessels into the interstitium of tumor tissue. In this study, we evaluated the effect of i.p. injection of PTX (PTX-30W), which was prepared by solubulization with water-soluble amphiphilic polymer composed of PMB-30W, a co-polymer of 2-methacryloxyethyl phosphorylcholineand n -butyl methacrylate, for peritoneal dissemination of gastric cancer. In a peritoneal metastasis model with transfer of MKN45P in nude mice, the effct of i.p. administration of PTX-30W was compared with conventional PTX dissolved in Cremophor EL (PTX-Cre). The drug accumulation in peritoneal nodules was evaluated with intratumor PTX concentration and fluorescence microscopic observation. PTX-30W reduced the number of metastatic nodules and tumor volume significantly more than did conventional PTX dissolved in Cremophor EL (PTX-Cre), and prolonged the survival time ( P  < 0.05). PTX concentration in disseminated tumors measured by HPLC was higher in the PTX-30W than in the PTX-Cre group up to 24 h after i.p. injection. Oregon green–conjugated PTX-30W, i.p. administered, preferentially accumulated in relatively hypovascular areas in the peripheral part of disseminated nodules, which was significantly greater than the accumulation of PTX-Cre. I.p. administration of PTX-30W may be a promising strategy for peritoneal dissemination, due to its superior characteristics to accumulate in peritoneal lesions. ( Cancer Sci  2009; 100: 1979–1985)     

Establishment of a visible peritoneal micrometastatic model from a gastric adenocarcinoma cell line by green fluorescent protein

To establish a visible peritoneal micrometastatic model, an enhanced green fluorescent protein (EGFP) expressing plasmid vector was transfected into the gastric cancer cell line, MKN-45. Highly expressing EGFP cells were injected into the peritoneal cavity of nude mice. Then, the peritoneum and abdominal organs were harvested and observed on days 1, 4 and 7. Fluorescence stereomicroscopy identified peritoneal micrometastases that had been undetected by stereomicroscopy. Micrometastases as small as a single cell are detectable in this model. This peritoneal micrometastatic model should be a useful tool for research on metastasis of gastric cancer.