The antitumor effect of postoperative treatment with genistein alone or combined with cyclophosphamide in mice bearing transplantable tumors.
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Genistein has been shown to be an inhibitor of tumor growth as well in vitro as in vivo. In addition to its antitumor effect, genistein reveals the antimetastatic and antiangiogenic properties. In this paper we described the results of our studies on the antimetastatic activity of genistein alone or combined with cyclophosphamide (CY) in mice which before this treatment were exposed to surgical excision of the primary tumor. Three transplantable subcutaneously growing mouse tumors were applied: Lewis lung cancer (LL2), B16F-10 melanoma and 16/C mammary cancer. The antitumor and antimetastatic effect was evaluated by the estimation of a number of lung colonies and a number of primary tumor recurrence as compared to the control mice exposed to the s.c. tumor extirpation only. Twenty days after the surgery, an average of 52 lung tumor colonies per mouse were detected in control mice bearing LL2 cancer. The treatment with genistein resulted in the reduction of the lung colonies to 24 per mouse. The treatment with CY reduced the number of lung colonies to 12 (p < 0.05) and combined treatment with both agents to 4 (p < 0.05). The percentage of primary tumor recurrence was 25, 86, 67 and 80% in the control, genistein treated, CY treated, and genistein + CY treated mice, respectively. Twenty days after the surgery, no lung metastases in the control mice bearing B16F-10 melanoma were observed. The percentage of primary tumor recurrence in the control, genistein treated, CY treated and genistein + CY treated mice was: 86, 29, 57 and 67% respectively. Two different protocols of the treatment with genistein were applied in 16/C mammary cancer model. In the first one genistein was injected before and in the other after surgical excision of tumor. The histological examination revealed the presence of lung metastases in all, untreated and treated, according to both protocols groups of mice. The percentage of primary tumor recurrence in the control mice, genistein treated according to the protocol I, and II was: 100, 40, and 40%, respectively. (+info)
Tumor progression despite efficient tumor antigen cross-presentation and effective "arming" of tumor antigen-specific CTL.
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To determine whether APC function or "arming" of CTL for lytic function are the points at which Ags from a nonimmunogenic tumor fail to induce an effective immune response, we established a murine tumor model that expressed intracellular OVA and selected a clone (cOVA-9) that remained susceptible to lysis by specific CD8(+) T cells throughout tumor growth. Viable cOVA-9 tumor cells grew in normal mice at a rate similar to the parental tumor, and vaccination with irradiated cOVA-9 cells did not induce protection against itself or the parental line, confirming its nonimmunogenic status. In vivo evaluation during tumor growth demonstrated persisting tumor Ag cross-presentation accompanied by the generation of potent, specific CTL which were detectable when tumors were barely palpable. Despite the presence of highly active CTL in the tumor-draining lymph nodes, there was no apparent lysis of tumor-associated APC. These data show that tumor-draining APC are not dysfunctional with regard to two crucial processes, in vivo tumor Ag cross-presentation and specific CTL arming, and that failure to prevent tumor growth is not in the induction phase, but in the effector phase and occurs within the tumor itself before the tumor matrix is established. (+info)
Anti-metastatic gene therapy utilizing subcutaneous inoculation of EC-SOD gene transduced autologous fibroblast suppressed lung metastasis of Meth-A cells and 3LL cells in mice.
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We have previously reported that superoxide stimulates the motility of tumor cells and the administration of Cu-Zn superoxide dismutase (SOD) significantly suppresses metastasis. However, ideally, anti-metastatic therapy should be long-lasting, systemically effective and have low toxicity. The half-life of Cu-Zn SOD in plasma is so short that it cannot provide long-lasting effects. Therefore, in this study we have developed a gene therapy in a mouse model utilizing extracellular SOD (EC-SOD), which is the most prevalent SOD isoenzyme in extracellular fluids. We retrovirally transfected fibroblasts (syngeneic) with the EC-SOD gene and established EC-SOD-secreting fibroblasts. Inoculation of EC-SOD-secreting fibroblasts suppressed both artificial and spontaneous metastatic lung nodules in mouse metastasis models. These data indicate the feasibility of anti-metastatic gene therapy utilizing the EC-SOD gene. (+info)
Nonviral in vivo gene delivery into tumors using a novel low volume jet-injection technology.
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The jet-injection technology has developed as an applicable alternative to viral or liposomal gene delivery systems. In this study a novel, low-volume, 'high-speed jet injector' hand-held system was used for the direct gene transfer of naked DNA into tumors. Lewis-lung carcinoma bearing mice were jet-injected with the beta-galactosidase (LacZ), the green fluorescence (GFP) or the human tumor necrosis factor alpha (TNF-alpha) gene carrying vector plasmids. The animals received five jet injections into the tumor at a pressure of 3.0 bar, delivering 3--5 microl plasmid DNA (1 microg DNA/microl in water) per single jet injection. The jet injection of DNA leads to a widespread expression pattern within tumor tissues with penetration depths of 5--10 mm. Analysis of tumor cryosections revealed moderate LacZ or GFP expression at 48 h and strong reporter gene expression 72 h and 96 h after jet injection. The simultaneous jet injection of the TNF-alpha and LacZ carrying vectors demonstrated efficient expression and secretion of both the cytokine, as well as LacZ expression within the tumor 24 h, 48 h, 72 h, 96 h and 120 h after jet injection. These studies demonstrate the applicability of jet injection for the efficient in vivo gene transfer into tumors for nonviral gene therapy of cancer using minimal amounts of naked DNA. (+info)
Isolation of an antitumor compound from Agaricus blazei Murill and its mechanism of action.
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The Basidiomycete fungus Agaricus blazei Murill has traditionally been used as a health food for the prevention of cancer, diabetes, hyperlipidemia, arteriosclerosis and chronic hepatitis. In the present study, we examined the antitumor activities of various substances isolated from the lipid fraction of A. blazei. Tumor growth was retarded by the oral administration of the lipid fraction extracted from A. blazei with a chloroform/methanol mixture in sarcoma 180-bearing mice. The substance with the antitumor activity in the lipid fraction was isolated via silica gel column chromatography, eluted with an acetonitrile/methanol (3:2) mixture and identified as ergosterol by direct comparison of the (1)H NMR and mass spectrometry spectral data of an authentic sample. The oral administration of ergosterol to sarcoma 180-bearing mice significantly reduced tumor growth at doses of 400 and 800 mg/kg administered for 20 d without side effects, such as the decreases in body, epididymal adipose tissue, thymus, and spleen weights and leukocyte numbers induced by cancer chemotherapy drugs. Ergosterol had no cytotoxicity against tumor cells. To clarify the antitumor activity of ergosterol, we examined the effects of ergosterol on tumor-induced angiogenesis using two in vivo models. Intraperitoneal administration of ergosterol at doses of 5, 10 and 20 mg/kg for 5 consecutive d inhibited the neovascularization induced by Lewis lung carcinoma cell-packed chambers, suggesting that either ergosterol or its metabolites may be involved in the inhibition of tumor-induced neovascularization. Therefore, we further examined the inhibitory effects of ergosterol on Matrigel-induced neovascularization. Female C57BL/6 mice were subcutaneously inoculated with Matrigel containing acidic fibroblast growth factor and heparin with or without ergosterol. Ergosterol inhibited the Matrigel-induced neovascularization, suggesting that ergosterol directly inhibits Matrigel-induced neovascularization. From these results, it seems likely that the antitumor activity of ergosterol might be due to direct inhibition of angiogenesis induced by solid tumors. This is the first report of ergosterol as an antiangiogenic substance. (+info)
Suppression of integrin expression and tumorigenicity by sulfation of lactosylceramide in 3LL Lewis lung carcinoma cells.
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To investigate the cellular functions of sulfated glycosphingolipids, we introduced the cerebroside sulfotransferase (CST) gene into J5 cells, a subclone of 3LL Lewis lung carcinoma cells. The J5 cells lack acidic glycosphingolipids but accumulate their common biosynthetic precursor, lactosylceramide. We established the stable CST transfectants, J5/CST-1 and J5/CST-2 clones, highly expressing sulfated lactosylceramide (SM3). Both clones exhibited more spherical morphology in comparison to mock transfectant, and their adhesiveness to fibronectin and laminin was significantly lower. The loss of cell-substratum interactions in these SM3-expressing cells could be attributed to decreased expression of integrins (alpha(5), alpha(6), and beta(1)) on the cell surface and their whole cellular levels. However, the levels of H-2K(b) and H-2D(b) antigens remained unchanged. Reverse transcriptase-polymerase chain reaction and Northern blot analyses for these integrins exhibited significant decrease of beta(1) gene expression in J5/CST-1 and 2, but there was no change in the levels of alpha(5) and alpha(6) transcripts. Deglycosylation by endoglycosidase H treatment clearly demonstrated that the precursor form of beta(1) integrin, possessing high mannose oligosaccharide chains, was preferentially decreased in the CST transfectants. These results demonstrate that endogenous SM3 negatively regulates beta(1) integrin expression at the transcriptional level, and the decrease of alpha integrin proteins in the CST transfectants was due to the post-transcriptional modification. We suggest the putative importance of the intracellular pre-beta(1) integrin pool for normal integrin maturation and subsequent function. Although the rates of cell proliferation in vitro for mock and CST transfectants were similar, tumorigenicity of J5/CST-1 and -2 cells inoculated into syngeneic C57/BL6 mice was greatly decreased or even absent. This was probably due to global loss of the efficient cell-matrix interactions, which are essential for the development of malignant tumors in vivo. Thus, we showed the evidence that cellular SM3 negatively regulates the cell-substratum interaction, resulting in the loss of tumorigenicity. (+info)
Resveratrol isolated from Polygonum cuspidatum root prevents tumor growth and metastasis to lung and tumor-induced neovascularization in Lewis lung carcinoma-bearing mice.
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Resveratrol is a naturally occurring phytoalexine found in medicinal plants. We found that resveratrol, at doses of 2.5 and 10 mg/kg, significantly reduced the tumor volume (42%), tumor weight (44%) and metastasis to the lung (56%) in mice bearing highly metastatic Lewis lung carcinoma (LLC) tumors, but not at a dose of 0.6 mg/kg. Resveratrol did not affect the number of CD4(+), CD8(+) and natural killer (NK)1.1.(+) T cells in the spleen. Therefore, the inhibitory effects of resveratrol on tumor growth and lung metastasis could not be explained by natural killer or cytotoxic T-lymphocyte activation. In addition, resveratrol inhibited DNA synthesis most strongly in LLC cells; its 50% inhibitory concentration (IC(50)) was 6.8 micromol/L. Resveratrol at 100 micromol/L increased apoptosis to 20.6 +/- 1.35% from 12.1 +/- 0.36% (P < 0.05) in LLC cells, and decreased the S phase population to 22.1 +/- 1.03% and 29.2 +/- 0.27% from 35.2 +/- 1.72% (P < 0.05) at concentrations of 50 and 100 micromol/L, respectively. Resveratrol inhibited tumor-induced neovascularization at doses of 2.5 and 10 mg/kg in an in vivo model. Moreover, resveratrol significantly inhibited the formation of capillary-like tube formation from human umbilical vein endothelial cells (HUVEC) at concentrations of 10-100 micromol/L; the degree of the inhibition of capillary-like tube formation by resveratrol was 45.5% at 10 micromol/L, 50.2% at 50 micromol/L and 52.6% at 100 micromol/L. Resveratrol inhibited the binding of vascular endothelial growth factor (VEGF) to HUVEC at concentrations of 10-100 micromol/L, but not at concentrations of 1 and 5 micromol/L. The degree of inhibition of VEGF binding to HUVEC by resveratrol was 16.9% at 10 micromol/L, 53.2% at 50 micromol/L and 47.8% at 100 micromol/L. We suggest that the antitumor and antimetastatic activities of resveratrol might be due to the inhibition of DNA synthesis in LLC cells and the inhibition of LLC-induced neovascularization and tube formation (angiogensis) of HUVEC by resveratrol (+info)
A precise and efficient stereological method for determining murine lung metastasis volumes.
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We have developed a computer-assisted stereological method based on unbiased principles for estimating metastasis volumes in mouse lungs. We evaluated this method using the transplantable Lewis lung carcinoma. Twenty-one days after subcutaneous inoculation of 10(6) Lewis lung cells into C57BL/6J mice, the mice had primary tumors with an average volume of 2300 mm(3). After perfusion fixation, the lungs were removed, embedded in OCT compound, snap-frozen, and processed for stereology. The metastasis volumes were estimated by application of the Cavalieri principle after evaluation of single sections from several evenly distributed tissue levels. The metastasis volume in a group of nine mice varied between 0.01 and 14.4 mm(3), with an average of 6.1 mm(3). The coefficient of variation was 0.9. The coefficient of error of the volume estimation was determined in five cases and varied from 0.08 to 0.23. Thus, the variation on the metastasis volumes that is achieved by this method contributes very little, 2.5%, to the total variance within the group of mice. In conclusion, we have developed an efficient and unbiased method to determine the metastasis burden in mouse lungs. (+info)