Melanoma differentiation-associated gene 7/interleukin (IL)-24 is a novel ligand that regulates angiogenesis via the IL-22 receptor

The melanoma differentiation-associated gene 7 (mda-7), also called interleukin (IL)-24, suppresses the growth of some cancers in vitro and in vivo as a result of the ectopic expression of its protein. However, the function of the secreted form of the protein in cancer has not been previously studied. The purpose of this study was to determine the antiangiogenic function of a secreted form of the MDA-7/IL-24 protein (sMDA-7/IL-24). In vitro, sMDA-7/IL-24 inhibited both endothelial cell differentiation and migration of endothelial cells induced by vascular endothelial growth factor and basic fibroblast growth factor. The sMDA-7/IL-24-mediated inhibitory effect was 10-50 times more potent than endostatin, IFN-gamma, and IFN-inducible protein 10 in vitro. Furthermore, the inhibitory effect was not mediated by IFN or IFN-inducible protein 10. IL-22 receptor mediated the antiangiogenic activity of sMDA-7/IL-24. Administration of a blocking antibody to IL-22 receptor in conjunction with sMDA-7/IL-24 led to abrogation of inhibition of endothelial differentiation. sMDA-7/IL-24 inhibited vascular endothelial growth factor-induced angiogenesis as evidenced by reduced vascularization and hemoglobin content in in vivo Matrigel plug assays. In vivo, the growth of human lung tumor cells was significantly inhibited, and vascularization was reduced when the cells were mixed with 293 cells stably expressing sMDA-7/IL-24. Systemic administration of sMDA-7/IL-24 inhibited lung tumor growth in a mouse xenograft model. Associated with tumor growth inhibition was decreased tumor microvessel density and hemoglobin content, indicating the presence of antiangiogenic activity. These data demonstrate that sMDA-7/IL-24 is a novel and potent antiangiogenic effector and support the development of MDA-7/IL-24-based therapeutics.     

Measurement of fractional anisotropy using diffusion tensor MRI in supratentorial astrocytic tumors

In vivo, water diffusion displays directionality due to presence of complex microstructural barriers in tissue. The extent of directionality of water diffusion can be expressed as a fractional anisotropy (FA) value, using diffusion tensor MR imaging (DTI). The aim of this study was to determine whether FA values indicate microstructures in astrocytic tumors. We performed DTI in 31 patients with astrocytic tumor, and measured the FA values of tumor and normal brain regions prior to CT-guided stereotactic biopsy. After biopsy, FA values were compared to assess the cellularity and vascularity of tumor tissue. Although mean FA values trended to differ among histological types, all mean tumor FA values were lower than those of normal brain regions. Positive correlation was observed between FA values and both cellularity (r = 0.65, p < 0.05) and vascularity (r = 0.45, p < 0.05). We had hypothesized that the FA value of an astrocytic tumor would be determined by a balance between factors increasing the directionality of water diffusion, such as high cellularity and/or vascularity, and factors decreasing the directionality of water diffusion, such as fiber destruction. However, our results suggest that the FA values of glioblastoma, anaplastic astrocytoma, diffuse astrocytoma and pilocytic astrocytoma are largely affected by cellularity and/or vascularity, whereas that of gliomatosis cerebri are largely affected by the preservation of nerve fibers. Measurement of FA value using DTI will allow prediction of histological characteristics such as cellularity, vascularity and/or fiber structure in astrocytic tumors.     

Is whole-brain irradiation necessary for primary central nervous system lymphoma? Patterns of recurrence after partial-brain irradiation

BACKGROUND: Neurotoxicity after whole-brain irradiation remains a major problem in the treatment of primary central nervous system lymphoma (PCNSL). To clarify whether whole-brain radiation is necessary for PCNSL, the authors retrospectively analyzed the outcome of patients treated with partial-brain irradiation. METHODS: A nationwide survey was performed regarding the treatment of PCNSL. Among 62 institutions surveyed, 7 were identified in which whole-brain irradiation was not necessarily employed. Questionnaires were sent to these institutions and 43 patients who had been treated using partial-brain fields since 1985 were collected. Thirty-two patients had solitary lesions and 11 had multiple lesions. Patterns of recurrence could be identified in 38 patients. RESULTS: The cumulative in-field and out-field recurrence rates at 5 years were 57% and 49%, respectively. Of 14 out-field recurrences, 2 occurred at the safety margin of the previous radiation field. The out-field recurrence rate was 45% in patients with a single lesion and 67% in those with multiple tumors (P = 0.79). The out-field recurrence rate was 22% for patients treated with safety margins of > or = 4 cm and 83% for those treated with safety margins of < 4 cm (P = 0.0079). The median survival time and the 5-year survival rate were 28.5 months and 20%, respectively, in the former group of patients and 15 months and 11%, respectively, in the latter group (P = 0.057). CONCLUSIONS: Focal radiotherapy with safety margins of < 4 cm appears to be associated with a very high rate of out-field recurrence, but the use of a radiation field with generous safety margins (> or = 4 cm) appears to be worth further investigation.     

Increased expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 is correlated with poor prognostic variables in patients with thymic epithelial tumors

BACKGROUND: A distinction between noninvasive, invasive, and metastatic thymoma on the basis of the cytologic features is difficult. The current study investigated whether the expression of MMP and TIMP was correlated with tumor invasiveness and prognosis in patients with thymoma. METHODS: Tumor tissue samples were obtained from 42 patients with thymic epithelial tumors between 1974 and 2001 at Tokushima University Hospital. Three-micrometer-thick, formalin-fixed, paraffin-embedded tissue sections were immunostained using specific antibodies against MMP-2, MMP-9, TIMP-1, and TIMP-2. RESULTS: MMP-2 expression was detected in 30 tumors (71%), and TIMP-2 expression was detected in 31 tumors (74%). MMP-9 expression was detected in 22 of 36 tumors (61%), and TIMP-1 expression was detected in only 7 tumors (19%). MMP-2 and TIMP-2 expression levels were very low (10% and 0%, respectively) in noninvasive tumors but were very high (91% and 97%, respectively) in invasive tumors. In thymic epithelial tumors, the more progressive the clinical stage of tumor, the higher the strongly positive rate of MMP-2 and TIMP-2 expression. There was no correlation between positivity for MMP-9 and stage. Twenty-five percent of Type AB thymomas and 50% of Type B1 thymomas expressed MMP-2 and TIMP-2. Most of Type A, Type B2, Type B3, and Type C thymomas expressed MMP-2 and TIMP-2. There were significant differences in disease-free survival at 5 years between patients without and with MMP-2 expression (91% vs. 55%, respectively) and patients without and with TIMP-2 expression (100% vs. 53%, respectively). CONCLUSIONS: MMP-2 and TIMP-2 are key enzymes for invasiveness of thymic epithelial tumors. The expression of these proteins can predict a poor outcome in patients with thymoma.     

Antitumor effects of a soluble insulin-like growth factor I receptor in human ovarian cancer cells: advantage of recombinant protein administration in vivo

Antitumor effects of a soluble form dominant negative of the type I insulin-like growth factor receptor (IGF-IR) designated as 486/STOP were evaluated in CaOV-3 human ovarian cancer cells by establishing stable transformants overexpressing 486/STOP and by administration of 486/STOP recombinant protein. Expression of 486/STOP was detected from total cell lysates, as well as conditioned media collected from stable transformants. In stable transformants, growth in monolayer was slightly retarded, and anchorage-independent growth in vitro and tumorigenicity in vivo were markedly inhibited. Addition of conditioned media from 486/STOP cells inhibited anchorage-independent growth of parental cells. Although tumorigenicity of parental cells in vivo was abrogated when they were cocultured in monolayer with 486/STOP cells over 48 h before injection to nude mice, coinjection of parental cells and 486/STOP cells without preculture was not successful. In contrast, administration of 486/STOP partially purified recombinant protein inhibited tumorigenicity of parental cells in vivo. Because 486/STOP cells result in massive apoptosis in vivo within 48 h, usage of a recombinant protein has a great advantage to use its unique bystander effect in vivo for clinical application.     

Imatinib inhibits various types of activating mutant kit found in gastrointestinal stromal tumors

Mutations of proto-oncogene c-KIT in gastrointestinal stromal tumors (GISTs) are considered to cause a constitutive activation of KIT responsible for their oncogenesis. Imatinib has therapeutic potential for GISTs because of its inhibitory effect on KIT kinase activity. To investigate the effect of Imatinib on various c-KIT mutations found in GISTs, we examined kinase activity of KIT, cell proliferation and tumorigenicity of transfectants with various c-KIT mutations. Murine lymphoid Ba/F3 cells transfected with one of the three types of mutants (KIT(del559-560), KIT(642Glu), and KIT(820Tyr)) or wild-type KIT were used for the experiments. Phosphorylation of KIT, mitogen-activated protein (MAP) and Akt was studied by immunoblotting with or without immunoprecipitation. In vitro studies on cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylcetrazolium bromide colorimetric assay and in vivo tumorigenicity assay using nude mice were also carried out. Imatinib could inhibit the KIT, MAP and Akt phosphorylation of all the transfectants but had a weaker effect on KIT(820Tyr). Imatinib potently inhibited the proliferation of cells transfected with KIT(820Tyr) at the concentration of 10 microM whereas it inhibited the other 3 types at 1 microM. Moreover, Imatinib could inhibit the tumor formation in nude mice transplanted with transfectants. In various types of activating mutant KIT, Imatinib could inhibit the constitutive activation of KIT signal transduction and cell proliferation both in vitro and in vivo although the effect of Imatinib on KIT(820Tyr) was weaker than that on KIT(del559-560) or KIT(642Glu).     

Immuno-gene therapy with adenoviruses expressing fms-like tyrosine kinase 3 ligand and CD40 ligand for mouse hepatoma cells in vivo

Introduction of genes encoding immuno-stimulatory cytokines into cancer cells is known to enhance antitumor immunity. CD40 ligand (CD40L, CD154) and fms-like tyrosine kinase 3 ligand (Flt3L) are recently identified cytokines, which have been demonstrated to stimulate antitumor immunity in several cancer models. However little is known about antitumor activity of Ftl3L and CD40L against hepatocellular carcinoma (HCC). In the present study, we constructed replication-defective adenoviruses expressing Flt3L and CD40L and examined their therapeutic efficacy on mouse HCC, MH134 cells. Subcutaneous injection of MH134 cells genetically engineered to express Flt3L and/or CD40L developed tumors in all the syngeneic immunocompetent mice, but tumor growth was significantly delayed as compared to control mice. Partial inhibition of this antitumor effect in athymic nude mice suggests that both innate and adaptive immunity appear to play a role. It was shown by immunodepletion of NK cells with anti-asialo-GM1 antibody that the effector cells involved in innate immunity are NK cells. In a therapeutic setting, however, injection of adenovirus expressing Flt3L or CD40L into pre-established MH134 tumors exhibited no efficacy. These data demonstrate that Flt3L and CD40L induce significant, but only weak, antitumor immunity against MH134 cells presumably through both innate and adaptive immunity. Our results suggest that immuno-gene therapy with Flt3L and CD40L may need adjuvant modalities to achieve strong immune response.     

Targeted gene delivery to human osteosarcoma cells with magnetic cationic liposomes under a magnetic field

Gene delivery using cationic liposomes results in relatively low transfection, especially under in vivo conditions. This system, however, can overcome some of the problems associated with viral delivery systems. The present study was carried out in order to improve the transfection efficiency of cationic liposomes by preparing magnetic cationic liposomes (MCL). Small MCL approximately 40 nm in diameter and incorporating one or two magnetite particles were prepared with phosphatidylethanolamine and 3beta-[N-(N', N'-dimethylaminoethane)-carbamoyl] cholesterol. The efficiency of MCL in gene delivery was evaluated by using plasmid DNA containing a luciferase reporter gene and human osteosarcoma Saos-2 cells. Without a magnetic field, maximum luciferase activity was observed when DNA was mixed with MCL at a 1:5 ratio and incubated with cells for 6 h. Under a magnetic field, maximum luciferase activity was achieved by 30-min magnetic induction. This improvement in transfection efficiency by magnetic induction was approximately 3.5-fold. The feasibility of this active transgenic system was further shown by measuring apoptosis rates after transfection of the p53 gene to Saos-2 cells. Apoptosis rates increased to 18.9% from 2.4% by magnetic induction. In conclusion, a gene delivery system including MCL and magnetic induction was found to achieve rapid and enhanced gene delivery in vitro. Such a gene delivery system may be applicable under in vivo conditions, and is expected to offer numerous clinical advantages.     

Suppression of cell migration in ovarian cancer cells mediated by PTEN overexpression

Peritoneal dissemination is the major progression pathway of ovarian cancer, and its control is important for improvement of the prognosis. PTEN is a tumor suppressor gene, and is known to inhibit cancer cell growth and migration. To investigate the possibility of gene therapy using PTEN for ovarian cancer, we introduced PTEN cDNA into an ovarian cancer cell line HRA carrying wild-type PTEN, and examined the effects in vitro and in vivo. Using PTEN cDNA cloned from a human liver cDNA library, a PTEN expression vector was constructed. This vector was introduced into HRA cells by the standard calcium phosphate precipitation method, and an HRA cell line overexpressing PTEN (HRA/PTEN) was established. On the cell migration test by in vitro scratch wound healing assay, the number of migrating cells was 6.3+/-0.9 cells/mm(2) in HRA/PTEN, which was significantly smaller than that in the control (39.7+/-3.2 cells/mm(2)) (p<0.01). No significant differences were observed in the in vitro cell growth or in vivo tumor growth between HRA/PTEN and the control. The findings described above, show that enhanced expression of PTEN inhibits ovarian cancer cell migration, suggesting that gene therapy approaches using PTEN for control of peritoneal dissemination of ovarian cancer are possible.     

Uptake of cadmium in meals from the digestive tract of young non-smoking Japanese female volunteers

OBJECTIVES: To estimate rates of cadmium (Cd) uptake from the digestive tract and changes in Cd in biological specimens after intake of Cd mainly in rice. METHODS: Twenty-five young non-smoking Japanese female volunteers (20-23 in age) were recruited and a 20-d experimental study was conducted. With polished rice containing 0.004 ppm and 0.340 ppm of Cd, Meal L and Meal H were prepared. Approximately 12% of total Cd in Meal L and 92% of total Cd in Meal H originated in rice. The volunteers ate Meal L for 11 d to achieve a stable intake-output balance of Cd. Fifteen of the 25 volunteers ate Meal H on the 12(th) day (Group D1), and the remaining 10 ate Meal H on the 12(th), 13(th) and 14(th) day (Group D3). All 25 subjects then resumed the consumption of Meal L to the end of the study (20(th) day). All meals, feces and urine were collected during the study, and Cd intake from the daily meals (Cd-I), Cd in feces (Cd-F) and Cd in urine (Cd-U) were determined. For measurement of Cd in blood (Cd-B), venous blood was collected from all volunteers on the day before the study and again on the 12(th) and 20(th) day; venous blood was also collected from 4-8 volunteers at additional time points. RESULTS: Mean Cd-I was 4.51 microg/d (range: 1.85-6.93) or 48.48 microg/d (range: 27.98-56.27) when they ate Meal L or Meal H. Cd-F and Cd-B exhibited faster responses to the change in Cd-I than did Cd-U. The Cd(uptake) rate, defined as (1-Cd-F(excess) /Cd-I(excess)) (Fig. 1), was 47.2% (range: -9.4-83.3%) in Group D1 and 36.6% (range: -9.2-73.5%) in Group D3, and the Cd(balance) rate, defined as (1-Cd-F(output) /Cd-I(intake)), was 23.9% (range: -4.0-37.7%) in Group D1 and 23.7% (range: -8.2-56.9%) in Group D3. CONCLUSIONS: Cd-F and Cd-B are better biological monitoring parameters for assessing change in Cd-I than Cd-U. The Cd(uptake) and Cd(balance) rates appeared to be higher than those in previous papers when ingested Cd mainly originated in rice.