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.     

Comprehensive evaluation of canine renal papillary necrosis induced by nefiracetam,a neurotransmission enhancer

The effects of nefiracetam, a neurotransmission enhancer, on renal biochemistry and morphology with toxicokinetic disposition were investigated in both in vivo and in vitro systems. In the in vivo studies with rats, dogs, and monkeys, only the dog exhibited renal papillary necrosis. Namely, when beagle dogs were orally administered with 300 mg/kg/day of nefiracetam over 11 weeks, decreased urinary osmotic pressure was noted from week 5, followed by increases in urine volume and urinary lactate dehydrogenase from week 8. The first morphological change was necrosis of ductal epithelia in the papilla in week 8. In toxicokinetics after 3 weeks of repeated oral administration to dogs, nefiracetam showed somewhat high concentrations in serum and the renal papilla as compared with rats and monkeys. As for metabolites, although metabolite-18 (M-18) concentration in the renal papilla of dogs was between that in rats and monkeys, the concentration ratios of M-18 in the papilla to cortex and papilla to medulla were remarkably high. In the in vitro studies, while nefiracetam itself showed no effects on the synthesis of prostaglandin E2 and 6-keto-prostaglandin F1alpha, a stable metabolite of prostaglandin I2, in canine renal papillary slices, only M-18 among the metabolites clearly decreased both prostaglandin syntheses. The basal prostaglandin synthesis in canine renal papillary slices was extremely low relative to those in rats and monkeys. Taken together, certain factors such as basal prostaglandin synthesis, M-18 penetration into the renal papilla leading to an intrarenal gradient, and inhibitory potential of M-18 on prostaglandin synthesis were considered to be crucial for the occurrence of renal papillary necrosis in dogs.     

Novel anti-inflammatory actions of nobiletin,a citrus polymethoxy flavonoid,on human synovial fibroblasts and mouse macrophages

We previously reported that nobiletin (5,6,7,8,3',4'-hexamethoxy flavone), a citrus polymethoxy flavonoid, effectively interferes with the production of promatrix metalloproteinase (proMMP)-9/progelatinase B in rabbit synovial fibroblasts [J. Rheumatol. 27 (2000) 20]. In this paper, we further examine the effects of nobiletin on the production of cyclooxygenases (COXs), prostaglandin (PG) E(2), and proinflammatory cytokines in human synovial fibroblasts and the mouse macrophage J774A.1 cell line. Nobiletin suppressed the interleukin (IL)-1-induced production of PGE(2) in human synovial cells in a dose-dependent manner (<64 microM). Additionally, it selectively downregulated COX-2, but not COX-1 mRNA expression. Nobiletin also interfered with the lipopolysaccharide-induced production of PGE(2) and the gene expression of proinflammatory cytokines including IL-1alpha, IL-1beta, TNF-alpha and IL-6 in mouse J774A.1 macrophages. In addition, nobiletin downregulated the IL-1-induced gene expression and production of proMMP-1/procollagenase-1 and proMMP-3/prostromelysin-1 in human synovial fibroblasts. In contrast, production of the endogenous MMP inhibitor, TIMP-1, was augmented by nobiletin. These anti-inflammatory actions of nobiletin are very similar to those of anti-inflammatory steroids such as dexamethasone, and the upregulation of TIMP-1 production is a unique action of nobiletin. Therefore, these results further support the notion that nobiletin is likely to be a candidate for characterization as a novel immunomodulatory and anti-inflammatory drug.     

Evaluation of nonthreshold leukemogenic response to methyl nitrosourea in p53-deficient C3H/He mice

The classic controversy of whether genotoxic chemicals induce cancers with or without a certain low-dose limit, i.e., the threshold, is revisited because of a number of current publications available addressing the plausibility of "practical" thresholds even for genotoxic carcinogens, the mechanism of which may be hypothesized to be due, in part, to a repair system composed of ordinarily available various defense mechanisms under the steady-state DNA damage. The question of whether an absolute nonthreshold or a relative nonthreshold, i.e., a "practical" threshold specifically in the low-dose level, is present may not be answered even with the use of a prohibitively large number of wild-type mice. Could the excessive incidence of tumorigenesis in p53-deficient mice contribute to our understanding of the threshold vs nonthreshold issue in genotoxic carcinogenesis? This is considered because an exaggeration of tumorigenesis in p53-deficient mice is hypothesized to reduce or eliminate the range of threshold due to the p53-deficiency-mediated reduction of DNA repair and apoptosis. The present study of chemical leukemogenesis in p53-deficient mice by transplantation assay was designed to answer this question. Briefly, 218 C3H/He mice were lethally irradiated and repopulated with bone marrow cells from wild-type, heterozygous p53-deficient, and homozygous p53-deficient C3H/He mice. This was followed by treatment with a single and graded dose of methyl nitrosourea at 6.6, 14.8, 33.3, 50.0, and 75.0 mg/kg body wt, with the vehicle-treated control groups treated with zero dose for each genotype. Whereas mice repopulated with p53-deficient bone marrow cells showed a marked reduction of the threshold for leukemogenicity, mice repopulated with wild-type bone marrow cells did not exhibit leukemia at a dose of 33.3 mg/kg body wt and showed a curve with a high probability for the linear regression model with a positive dose intercept, predicting a threshold by the likelihood ratio test. Thus, the failure of wild-type mice to show an increase in incidence of leukemogenesis at low doses of genotoxic carcinogens may be due not to a statistical rarity, but to various p53-related pharmacophysiological functions, possibly including DNA repair and apoptosis that may account for a threshold.