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vector-mediated MS-selective transgene expression was markedly impaired in tumor-bearing mice whose MS had already been replaced by infiltrating cancer cells.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.

Gene therapy with secretory leukoprotease inhibitor promoter-controlled replication-competent adenovirus for non-small cell lung cancer.

Secretory leukoprotease inhibitor (SLPI) is highly expressed in almost all non-small cell lung cancers (NSCLCs), but not in the majority of other tumor types. In an attempt to create a specific gene therapy for NSCLC, we constructed AdSLPI.E1AdB, an adenovirus vector with a double expression cassette consisting of E1A driven by the SLPI promoter gene followed by E1B-19K under the control of the cytomegalovirus (CMV) promoter that can selectively replicate only in NSCLC cells. Infection with AdSLPI.E1AdB yielded E1A protein expression and adenovirus replication resulting in a >100-fold increase of the virus titers only in SLPI-producing NSCLC cells (A549, H358, and HS24 cells). In contrast, neither E1A protein nor replication was detected in non-SLPI-producing HepG2 cells. Treatment with AdSLPI.E1AdB significantly inhibited the proliferation of NSCLC cells in vitro in a dose-dependent manner, whereas the cell growth of HepG2 or normal human bronchial epithelial cells was not affected by AdSLPI.E1AdB infection. Direct injection of AdSLPI.E1AdB into A549 and H358 tumors in nude mice resulted in a marked reduction in tumor growth compared with controls (A549, 57%, P < 0.02; H358, 67%, P < 0.03). Histological examination revealed the replication of AdSLPI.E1AdB and strong induction of necrosis and apoptosis. In addition, we evaluated the combination of AdSLPI.E1AdB and AdCMV.NK4 encoding NK4 protein, which has strong antiangiogenic activity. E1A expressed by AdSLPI.E1AdB trans-acts on the replication of AdCMV.NK4 and thus increases the expression of NK4. Injection of these two vectors into H358 tumors resulted in a more striking reduction of tumor growth compared with single injection of each vector. These results suggest that AdSLPI.E1AdB could provide a selective therapeutic modality for NSCLC and that the combination of AdSLPI.E1AdB and AdCMV.NK4 may be a more effective gene therapy for NSCLC.

N-terminal deletion augments the cell-death-inducing activity of BAX in adenoviral gene delivery to nonsmall cell lung cancers.

Therapeutic modalities that overcome the antiapoptotic function of Bcl-2 that is often overexpressed in cancer cells are expected to be a novel strategy for cancer treatment. We previously reported that the leukemic cell death induced by an N-terminally truncated Bax (deltaN Bax: corresponding to amino acid 112-192 of full-length Bax) was not blocked by Bcl-2 or Bcl-x(L) owing to the lack of the BH3 domain needed to interact with the antiapoptotic Bcl-2 family molecules. In this study, we used the Cre-loxP system that allowed us to propagate adenoviruses expressing deltaN Bax, and investigated the effects of the deltaN Bax gene transfer into A549 and NCI-H1299 nonsmall cell lung cancer cell lines. deltaN Bax showed more cell-death-inducing activity in both cells than did the full-length Bax in vitro. It was found that the deltaN Bax-induced cell death was not inhibited by the pan-caspase inhibitor z-VAD-fmk, suggesting that deltaN Bax induces cell death through a caspase-independent mechanism. Intratumoral injection of adenoviruses expressing deltaN Bax into A549 tumors in Balb/c nude mice showed a significantly stronger suppression of tumor growth (74%) than full-length Bax (25%) compared to the control. Our results suggest that deltaN Bax may provide a better alternative than currently used cytotoxic genes in cancer gene therapy trials.

Intratracheal gene transfer of decorin reduces subpleural fibroproliferation induced by bleomycin.

Decorin, a small leucin-rich proteoglycan, is a negative regulator of transforming growth factor-beta, but the antifibrotic effect of decorin gene transfer has not been examined in a mouse model of usual interstitial pneumonia (UIP). We constructed a replication-defective recombinant adenovirus harboring human decorin gene (AdCMV.DC) and administered 1 x l0(9) plaque-forming units of AdCMV.DC intratracheally or intravenously to C57BL/6 mice with intraperitoneal injection of bleomycin, which induces a subpleural fibroproliferation, mimicking UIP, by day 28. Only intratracheal administration of AdCMV.DC increased decorin mRNA expression in the lung and decreased the hydroxyproline content augmented in bleomycin-induced pulmonary fibrosis (1.13 +/- 0.02 to 0.96 +/- 0.02, P = 0.006). In contrast, intravenous administration of AdCMV.DC increased the decorin expression only in the liver, but not in the lung, and without reducing lung fibrosis. These results indicate that adenoviral decorin gene transfer is effective only by direct administration to fibrosing lungs.

Gene transduction of NK4, HGF antagonist, inhibits in vitro invasion and in vivo growth of human pancreatic cancer.

In this study, we investigated the therapeutic effects of adenovirally-mediated transfer of the sequence of NK4, an antagonist for hepatocyte growth factor (HGF), against human pancreatic carcinoma. HGF has been implicated to play an important role in invasion and metastasis of various human cancers through tumor-stromal interactions. Although NK4 has been shown to block the metastatic behavior of cancer cells, problems with cellular delivery of NK4 must be addressed before it can be used for clinical trials. The effects of NK4 gene transduction mediated by recombinant adenovirus (Ad-NK4) were evaluated in a human pancreatic cancer cell line (SUIT-2) by in vitro scattering assays, invasion assays, and subcutaneous transplantation in nude mice. NK4 transduction markedly inhibited scattering and invasion of SUIT-2 cells stimulated by HGF without affecting cell proliferation in vitro. Furthermore, Ad-NK4 significantly inhibited the growth of tumors transplanted to nude mice. The tumor reduction induced by Ad-NK4 was associated with a decreased number of blood vessels surrounding the tumors. These findings suggest that Ad-NK4 gene therapy may be a unique and promising strategy for the treatment of pancreatic cancer.

High level of vascular endothelial growth factor in hemorrhagic pleural effusion of cancer.

Angiogenic cytokines, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and angiogenin, are candidates for the induction of pleural effusions because they have been implicated in the induction of neovascularization, vascular permeability, and hemorrhage both in the inflammatory process and in tumor progression. Thus, we hypothesized that these angiogenic factors in effusion might be involved in the clinical manifestation of malignant pleural disease. We measured the levels of VEGF, bFGF, and angiogenin in pleural effusions and sera from 40 patients. Pleural effusions due to malignancy (1,350 pg/ml) contained significantly higher levels of VEGF than effusions due to inflammatory diseases (102 pg/ml; p = 0.034). Furthermore, hemorrhagic effusions showed significantly higher VEGF levels (1,942 pg/ml) than non-hemorrhagic effusions (202 pg/ml; p = 0.016) in malignant patients. In contrast, neither bFGF nor angiogenin were correlated with any clinical manifestation of pleural effusion. Immunohistochemical study revealed that malignant cells in the pleura were stained with anti-VEGF antibody. Our data suggest that VEGF secreted from tumor cells may be involved in the accumulation of pleural effusion in malignancy, and that increased levels of VEGF may induce hemorrhagic effusion.

Targeting angiogenesis and HGF function using an adenoviral vector expressing the HGF antagonist NK4 for cancer therapy.

Hepatocyte growth factor (HGF) affects tumor growth/invasion and tumor neovascularization. A proposed HGF antagonist, NK4 (an amino-terminal kringle-domain peptide of HGF), inhibits tumor growth/invasion through the competition of HGF binding to its receptor, c-Met, and acts as an angiogenesis inhibitor. To investigate the in vivo effect of NK4 gene transfer, we constructed an adenovirus vector expressing human NK4 (AdCMV.NK4). Human lung cancer cell lines (A549 and H358) infected in vitro with AdCMV.NK4 yielded NK4 protein without a change in the cell growth rate. In contrast, direct injection of AdCMV.NK4 (1 x 10(9) pfu, twice) into established subcutaneous tumors in BALB/c nu/nu mice resulted in suppression of the tumors by 64% for A549 or by 91% for H358 compared with controls (P<0.02 or P<0.01, respectively). Counting of the tumor vessels revealed suppressed vascularity by 57% in H358 tumors when using AdCMV.NK4 (P<0.0001). Furthermore, systemic NK4 delivery by intraperitoneal injection of AdCMV.NK4 effectively suppressed both angiogenesis in the Matrigel assay (86% reduction, P<0.032), subcutaneous tumor growth in vivo (by 65% for H358, P<0.001), and hematogenous lung metastases without obvious side effects. These results indicate that NK4 elicits tumor-growth suppression in vivo through its anti-angiogenic activity and anti-HGF activity and that NK4 gene transfer can be an effective tool in the treatment of cancer.