-
Yasumoto Yamasaki,
Hiroshi Tazawa,
Yuuri Hashimoto, Toru Kojima,
Shinji Kuroda,
Shuya Yano,
Ryosuke Yoshida,
Futoshi Uno,
Hiroyuki Mizuguchi,
Akira Ohtsuru,
Yasuo Urata,
Shunsuke Kagawa,
Toshiyoshi Fujiwara
[show abstract]
[hide abstract]
ABSTRACT: Oncolytic viruses engineered to replicate in tumour cells but not in normal cells could be used as tumour-specific vectors carrying the therapeutic genes. We previously developed a telomerase-specific oncolytic adenovirus, OBP-301, that causes cell death in human cancer cells with telomerase activities. Here, we further modified OBP-301 to express the wild-type p53 tumour suppressor gene (OBP-702), and investigated whether OBP-702 induces stronger antitumour activity than OBP-301. The antitumour effect of OBP-702 was compared to that of OBP-301 on OBP-301-sensitive (H358 and H460) and OBP-301-resistant (T.Tn and HSC4) human cancer cells. OBP-702 suppressed the viability of both OBP-301-sensitive and OBP-301-resistant cancer cells more efficiently than OBP-301. OBP-702 caused increased apoptosis compared to OBP-301 or a replication-deficient adenovirus expressing the p53 gene (Ad-p53) in H358 and T.Tn cells. Adenovirus E1A-mediated p21 and MDM2 downregulation was involved in the apoptosis caused by OBP-702. Moreover, OBP-702 significantly suppressed tumour growth in subcutaneous tumour xenograft models compared to monotherapy with OBP-301 or Ad-p53. Our data demonstrated that OBP-702 infection expressed adenovirus E1A and then inhibited p21 and MDM2 expression, which in turn efficiently induced apoptotic cell death. This novel apoptotic mechanism suggests that the p53-expressing OBP-702 is a promising antitumour reagent for human cancer and could improve the clinical outcome.
European journal of cancer (Oxford, England: 1990) 01/2012; 48(14):2282-91. · 4.12 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hypoxia is a microenvironmental factor that contributes to the invasion, progression and metastasis of tumor cells. Hypoxic tumor cells often show more resistance to conventional chemoradiotherapy than normoxic tumor cells, suggesting the requirement of novel antitumor therapies to efficiently eliminate the hypoxic tumor cells. We previously generated a tumor-specific replication-competent oncolytic adenovirus (OBP-301: Telomelysin), in which the human telomerase reverse transcriptase (hTERT) promoter drives viral E1 expression. Since the promoter activity of the hTERT gene has been shown to be upregulated by hypoxia, we hypothesized that, under hypoxic conditions, the antitumor effect of OBP-301 with the hTERT promoter would be more efficient than that of the wild-type adenovirus 5 (Ad5). In this study, we investigated the antitumor effects of OBP-301 and Ad5 against human cancer cells under a normoxic (20% oxygen) or a hypoxic (1% oxygen) condition. Hypoxic condition induced nuclear accumulation of the hypoxia-inducible factor-1α and upregulation of hTERT promoter activity in human cancer cells. The cytopathic activity of OBP-301 was significantly higher than that of Ad5 under hypoxic condition. Consistent with their cytopathic activity, the replication of OBP-301 was significantly higher than that of Ad5 under the hypoxic condition. OBP-301-mediated E1A was expressed within hypoxic areas of human xenograft tumors in mice. These results suggest that the cytopathic activity of OBP-301 against hypoxic tumor cells is mediated through hypoxia-mediated activation of the hTERT promoter. Regulation of oncolytic adenoviruses by the hTERT promoter is a promising antitumor strategy, not only for induction of tumor-specific oncolysis, but also for efficient elimination of hypoxic tumor cells.
PLoS ONE 01/2012; 7(6):e39292. · 4.09 Impact Factor
-
Toru Kojima,
Yuichi Watanabe,
Yuuri Hashimoto,
Shinji Kuroda,
Yasumoto Yamasaki,
Shuya Yano,
Masaaki Ouchi,
Hiroshi Tazawa,
Futoshi Uno,
Shunsuke Kagawa,
Satoru Kyo,
Hiroyuki Mizuguchi,
Yasuo Urata,
Noriaki Tanaka,
Toshiyoshi Fujiwara
[show abstract]
[hide abstract]
ABSTRACT: The aim of this study was to develop a less invasive way of targeting lymph node metastasis for the treatment of human gastrointestinal cancer. Lymphatic invasion is a major route for cancer cell dissemination, and adequate treatment of locoregional lymph nodes is required for curative treatment in patients with malignancies.
Human telomerase reverse transcription (hTERT) is the catalytic subunit of telomerase, which is highly active in cancer cells but quiescent in most normal somatic cells. OBP-301 (Telomelysin) is an attenuated adenovirus with oncolytic potency that contains the hTERT promoter element to regulate viral replication. We examined whether OBP-301 injected into the primary tumor might be useful for purging micrometastasis from regional lymph nodes in an orthotopic colorectal cancer model.
OBP-301 was intratumorally injected into HT29 tumors orthotopically implanted into the rectum in BALB/c nu/nu mice. By using a highly sensitive quantitative PCR analysis that targets the human-specific Alu sequence, we showed that OBP-301 caused viral spread into the regional lymphatic area and selectively replicated in neoplastic lesions, resulting in tumor-cell-specific death in metastatic lymph nodes. Moreover, although the surgical removal of primary tumors increased the tendency of lymph node metastasis, preoperative intratumoral injection of virus significantly reduced lymph node metastasis.
Our results indicate that intratumoral injection of OBP-301 mediates effective in vivo purging of metastatic tumor cells from regional lymph nodes, which may help optimize treatment of human cancer, especially gastrointestinal malignancies.
Annals of surgery 06/2010; 251(6):1079-86. · 7.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Multimodal approaches combining drugs that differentially function is the most popular regimen for treating human cancer. Understanding the molecular mechanisms underlying the synergistic, potentiative, and antagonistic effects of drug combinations could facilitate the discovery of novel efficacious combinations. We previously showed that telomerase-specific replication-competent adenovirus (Telomelysin, OBP-301), in which the human telomerase reverse transcriptase promoter controls the adenoviral E1 gene expression, induces a selective antitumor effect in human cancer cells. Here, using E1-deleted replication-deficient adenovirus expressing the p53 tumor suppressor gene (Advexin, Ad-p53) and OBP-301, we investigate how these adenoviruses that kill tumor cells with different mechanisms could work in combination on human cancer. We found that E1-deficient Ad-p53 could kill cancer cells more efficiently in the presence of OBP-301 than Ad-p53 alone or OBP-301 alone, because Ad-p53 could become replication-competent by being supplied adenoviral E1 from coinfected OBP-301 in trans. Ad-p53 plus OBP-301 induced high levels of p53 protein expression without p21 induction, resulting in apoptotic cell death documented by active caspase-3 expression with a cytometric bead array and an increased subdiploid apoptotic fraction of the cell cycle. For in vivo evaluation, nude mice xenografted with human lung tumors received intratumoral injection of OBP-301 and/or Ad-p53. Analysis of the growth of implanted tumors showed an enhanced antitumor effect in combination therapy. Our data show that Ad-p53 in combination with OBP-301 induces not only oncolytic but also apoptotic cancer cell death and enhances antitumor activity in vitro and in vivo, providing potential merits as a multimodal treatment for human cancer.
Molecular Cancer Therapeutics 06/2010; 9(6):1884-93. · 5.23 Impact Factor
-
Toru Kojima,
Yuuri Hashimoto,
Yuichi Watanabe,
Shunsuke Kagawa,
Futoshi Uno,
Shinji Kuroda,
Hiroshi Tazawa,
Satoru Kyo,
Hiroyuki Mizuguchi,
Yasuo Urata,
Noriaki Tanaka,
Toshiyoshi Fujiwara
[show abstract]
[hide abstract]
ABSTRACT: The presence of circulating tumor cells (CTCs) in the peripheral blood is associated with short survival, making the detection of CTCs clinically useful as a prognostic factor of disease outcome and/or a surrogate marker of treatment response. Recent technical advances in immunocytometric analysis and quantitative real-time PCR have made it possible to detect a few CTCs in the blood; however, there is no sensitive assay to specifically detect viable CTCs. Here, we report what we believe to be a new approach to visually detect live human CTCs among millions of peripheral blood leukocytes, using a telomerase-specific replication-selective adenovirus expressing GFP. First, we constructed a GFP-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication (OBP-401; TelomeScan). We then used OBP-401 to establish a simple ex vivo method that was able to detect viable human CTCs in the peripheral blood. The detection method involved a 3-step procedure, including the lysis of rbc, the subsequent addition of OBP-401 to the cell pellets, and an automated scan using fluorescence microscopy. OBP-401 infection increased the signal-to-background ratio as a tumor-specific probe, because the fluorescent signal was amplified only in viable, infected human tumor cells, by viral replication. This GFP-expressing virus-based method is remarkably simple and allows precise enumeration of CTCs.
The Journal of clinical investigation 10/2009; 119(10):3172-81. · 15.39 Impact Factor
-
Yuji Kurihara,
Yuichi Watanabe,
Hideki Onimatsu, Toru Kojima,
Tatsuo Shirota,
Masashi Hatori,
Dong Liu,
Satoru Kyo,
Hiroyuki Mizuguchi,
Yasuo Urata,
Satoru Shintani,
Toshiyoshi Fujiwara
[show abstract]
[hide abstract]
ABSTRACT: Long-term outcomes of patients with squamous cell carcinoma of the head and neck (SCCHN) remain unsatisfactory despite advances in combination of treatment modalities. SCCHN is characterized by locoregional spread and it is clinically accessible, making it an attractive target for intratumoral biological therapies.
OBP-301 is a type 5 adenovirus that contains the replication cassette in which the human telomerase reverse transcriptase promoter drives expression of the E1 genes. OBP-401 contained the replication cassette and the green fluorescent protein (GFP) gene. The antitumor effects of OBP-301 were evaluated in vitro by the sodium 30-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate assay and in vivo in an orthotopic xenograft model. Virus spread into the lymphatics was also orthotopically assessed by using OBP-401.
Intratumoral injection of OBP-301 resulted in the shrinkage of human SCCHN tumors orthotopically implanted into the tongues of BALB/c nu/nu mice and significantly recovered weight loss by enabling oral ingestion. The levels of GFP expression following ex vivo infection of OBP-401 may be of value as a positive predictive marker for the outcome of telomerase-specific virotherapy. Moreover, whole-body fluorescent imaging revealed that intratumorally injected OBP-401 could visualize the metastatic lymph nodes, indicating the ability of the virus to traffic to the regional lymphatic area and to selectively replicate in neoplastic lesions, resulting in GFP expression and cell death in metastatic lymph nodes.
These results illustrate the potential of telomerase-specific oncolytic viruses for a novel therapeutic and diagnostic approach, termed theranostics, for human SCCHN.
Clinical Cancer Research 05/2009; 15(7):2335-43. · 7.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A phase I dose-escalation study of telomerase-specific oncolytic adenovirus, OBP-301 (Telomelysin), is now under way in the United States to assess feasibility and to characterize its pharmacokinetics in patients with advanced solid tumors. The present preclinical study investigates whether OBP-301 and a chemotherapeutic agent that is commonly used for lung cancer treatment, gemcitabine, are able to enhance antitumor effects in vitro and in vivo. The antitumor effects of OBP-301 infection and gemcitabine were evaluated by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt assay. In vivo antitumor effects of intratumoral injection of OBP-301 in combination with systemic administration of gemcitabine were assessed on nu/nu mice s.c. xenografted with human lung tumors. OBP-301 infection combined with gemcitabine resulted in very potent synergistic cytotoxicity in human lung cancer cells. The three human lung cancer cell lines treated with OBP-301 for 24 hours tended to accumulate in S phase compared with controls. The proportion of cells in S phase increased from 43.85% to 56.41% in H460 cells, from 46.72% to 67.09% in H322 cells, and from 38.22% to 57.67% in H358 cells. Intratumoral injection of OBP-301 combined with systemic administration of gemcitabine showed therapeutic synergism in human lung tumor xenografts. Our data suggest that the combination of OBP-301 and gemcitabine enhances the antitumor effects against human lung cancer. We also found that the synergistic mechanism may be due to OBP-301-mediated cell cycle accumulation in S phase. These results have important implications for the treatment of human lung cancer.
Molecular Cancer Therapeutics 05/2009; 8(4):980-7. · 5.23 Impact Factor
-
Yoshihiro Ikeda, Toru Kojima,
Shinji Kuroda,
Yoshikatsu Endo,
Ryo Sakai,
Masayoshi Hioki,
Hiroyuki Kishimoto,
Futoshi Uno,
Shunsuke Kagawa,
Yuichi Watanabe,
Yuuri Hashimoto,
Yasuo Urata,
Noriaki Tanaka,
Toshiyoshi Fujiwara
[show abstract]
[hide abstract]
ABSTRACT: Soluble factors in the tumor microenvironment may influence the process of angiogenesis; a process essential for the growth and progression of malignant tumors. In this study, we describe a novel antiangiogenic effect of conditional replication-selective adenovirus through the stimulation of host immune reaction. An attenuated adenovirus (OBP-301, Telomelysin), in which the human telomerase reverse transcriptase promoter element drives expression of E1 genes, could replicate in and cause selective lysis of cancer cells. Mixed lymphocyte-tumor cell culture demonstrated that OBP-301-infected cancer cells stimulated PBMC to produce IFN-gamma into the supernatants. When the supernatants were subjected to the assay of in vitro angiogenesis, the tube formation of HUVECs was inhibited more efficiently than recombinant IFN-gamma. Moreover, in vivo angiogenic assay using a membrane-diffusion chamber system s.c. transplanted in nu/nu mice showed that tumor cell-induced neovascularization was markedly reduced when the chambers contained the mixed lymphocyte-tumor cell culture supernatants. The growth of s.c. murine colon tumors in syngenic mice was significantly inhibited due to the reduced vascularity by intratumoral injection of OBP-301. The antitumor as well as antiangiogenic effects, however, were less apparent in SCID mice due to the lack of host immune responses. Our data suggest that OBP-301 seems to have antiangiogenic properties through the stimulation of host immune cells to produce endogenous antiangiogenic factors such as IFN-gamma.
The Journal of Immunology 03/2009; 182(3):1763-9. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cancer gene therapy and oncolytic virotherapy have been studied extensively. However, their clinical application is hampered by their weak anticancer activity. We previously constructed a replicating adenovirus (OBP-301, Telomelysin), in which the human telomerase reverse transcriptase (hTERT) promoter drives expression of the adenoviral E1 genes, and causes selective lysis of human cancer cells. We hypothesized that combination adenoviral therapy containing OBP-301 and a nonreplicating adenovirus expressing the proapoptotic Bax gene could overcome the weakness and augment the anticancer efficacy of each modality. Combination treatment resulted in marked Bax protein expression and enhanced efficacy in in vitro cell viability assay, when compared with either single treatment. However, combination treatment was not as effective in suppressing both subcutaneous and pleural disseminated tumors compared with OBP-301 treatment alone. Further investigation revealed that combination treatment resulted in suppressed E1A protein expression associated with reduced viral replication. Our results suggest that Bax gene therapy in combination with oncolytic adenovirotherapy potentially augments their antitumor activity, but further improvements may be required to maximize the combinatorial effect in vivo, for the Bax gene expression to avoid interference with production of the oncolytic virus.
International Journal of Cancer 07/2008; 122(11):2628-33. · 5.44 Impact Factor
-
Hiroyuki Kishimoto, Toru Kojima,
Yuichi Watanabe,
Shunsuke Kagawa,
Toshiya Fujiwara,
Futoshi Uno,
Fuminori Teraishi,
Satoru Kyo,
Hiroyuki Mizuguchi,
Yuuri Hashimoto,
Yasuo Urata,
Noriaki Tanaka,
Toshiyoshi Fujiwara
[show abstract]
[hide abstract]
ABSTRACT: Currently available methods for detection of tumors in vivo such as computed tomography and magnetic resonance imaging are not specific for tumors. Here we describe a new approach for visualizing tumors whose fluorescence can be detected using telomerase-specific replication-competent adenovirus expressing green fluorescent protein (GFP) (OBP-401). OBP-401 contains the replication cassette, in which the human telomerase reverse transcriptase (hTERT) promoter drives expression of E1 genes, and the GFP gene for monitoring viral replication. When OBP-401 was intratumorally injected into HT29 tumors orthotopically implanted into the rectum in BALB/c nu/nu mice, para-aortic lymph node metastasis could be visualized at laparotomy under a three-chip color cooled charged-coupled device camera. Our results indicate that OBP-401 causes viral spread into the regional lymphatic area and selectively replicates in neoplastic lesions, resulting in GFP expression in metastatic lymph nodes. This technology is adaptable to detect lymph node metastasis in vivo as a preclinical model of surgical navigation.
Nature Medicine 11/2006; 12(10):1213-9. · 22.46 Impact Factor
-
