Mouse macrophage metalloelastase generates angiostatin from plasminogen and suppresses tumor angiogenesis in murine colon cancer

Previous studies showed that the mouse macrophage metalloelastase (MME) generates the angiogenesis inhibitor angiostatin from plasminogen in vitro. This study aimed to determine whether tumor cells engineered with MME could generate angiostatin and suppress tumor angiogenesis in vivo. Murine CT-26 colon cancer cells stably transfected with MME were inoculated subcutaneously. A radioisotope tracer, immunoblotting, immunofluorescence and immunohistochemistry were used to explore the pathway of the angiostatin generation. The results showed that tumors derived from MME-transfected cells demonstrated a less microvessel density compared with control tumors derived from vector-transfected and non-transfected cells (P<0.001). The expression of vascular endothelial growth factor (VEGF) was significantly lower in the MME-transfected group compared with that of the controls. The growth of MME-transfected tumors was significantly retarded compared with the control tumors (P<0.001). Western blot analysis, using a specific anti-mouse plasminogen (1-4 Kringle) antibody, demonstrated two strong immunoreactive bands (38- and 35-kDa) in MME-transfected tumors. gamma-ray counting data demonstrated that plasminogen cleavage occurred mostly in tumors formed by cells forced to express. We concluded that MME was demonstrated to be an efficient angiostatin-producing MMP and its presence was negatively correlated with the growth of colon cancer in tumor-bearing mice. These findings provide direct evidence that MME generates angiostatin in tumor-bearing mice and the therapeutic application of MME against tumors.     

Transfection of mouse macrophage metalloelastase gene into murine CT-26 colon cancer cells suppresses orthotopic tumor growth, angiogenesis and vascular endothelial growth factor expression

A cDNA fragment coding for domains I and II of mouse macrophage metalloelastase (MME) was transfected into murine CT-26 colon cancer cells that are MME deficient. An orthotopic implantation model was established by using MME-transfected cells. In MME-transfected primary tumors, it demonstrated that tumor growth and microvessel formation were significantly inhibited compared with the controls. The expression of vascular endothelial growth factor (VEGF) mRNA and protein was significantly lower in MME-transfected group compared with those in the controls. Our data show that both MME and VEGF gene expression is highly associated with the vascularity of tumors, which may depend on a balance between MME and VEGF expression.     

Mouse macrophage metalloelastase gene delivery by HVJ-cationic liposomes in experimental antiangiogenic gene therapy for murine CT-26 colon cancer

We previously demonstrated that gene replacement of mouse macrophage metalloelastase (MME) into murine melanoma cells that grow rapidly and are MME deficient suppresses the primary tumor growth in vivo by halting angiogenesis. The aim of the present study was to evaluate the effectiveness of gene therapy against cancer using a cDNA-encoding MME gene. In a subcutaneous tumor model of CT-26 mouse colon cancer cells that are MME deficient, syngeneic mice repetitively treated with direct injections into the tumors of MME- hemagglutinating virus of Japan (HVJ), a type of HVJ-cationic liposome encapsulating a plasmid expressing MME, developed smaller tumors (210 +/- 47.2 mm(3) versus 925 +/- 156 mm(3) mean +/- SEM; p = 0.0004) with fewer microvessels (10.25 +/- 1.03 vs. 17.25 +/- 2.14; p = 0.03) than control mice. TUNEL staining revealed a significant increase of apoptotic cells in the MME-HVJ liposomes-treated tumors compared with control tumors. MME was effectively expressed in the s.c. tumors treated with MME-HVJ liposomes, inducing angiostatin generation in those tumors, as demonstrated by Western blot analysis. In conclusion, our study demonstrated that repeated in vivo transduction of the MME gene directly into the tumors using HVJ-cationic liposomes suppressed the tumor growth by an antiangiogenic mechanism, providing, then, a feasible strategy for gene therapy of cancer.     

Inhibition of tumor growth through suppression of angiogenesis by brain-specific angiogenesis inhibitor 1 gene transfer in murine renal cell carcinoma

This study was designed to elucidate the therapeutic effect of transfering the brain-specific angiogenesis inhibitor 1 (BAI1) gene to a mouse renal cell carcinoma cell line (Renca). Female BALB/c mice were inoculated subcutaneously with wild-type Renca (Renca/Wild) cells or Renca cells transfected with the BAI-1 (Renca/BAI-1) or LacZ (Renca/LacZ) gene. Tumor growth was observed every other day from 3 to 35 days after implantation. Moreover, the intratumoral injection of the adenovirus vector containing the gene encoding BAI1 was conducted at two-day intervals from 11 to 31 days after implantation of the Renca/Wild or Renca/BAI1 tumor. Tumor blood flow was measured by colorimetric angiogenesis assay (CAA). The concentration of the vascular endothelial growth factor (VEGF) in the cell culture supernatants was determined by enzyme-linked immunoassay. The size of the Renca/BAI1 tumor was significantly (p<0.01) suppressed compared to the Renca/Wild and Renca/LacZ tumors 21 days after tumor implantation. The injection of the BAI1 viral vector at 2-day intervals significantly inhibited the growth of both the Renca/Wild and Renca/BAI1 tumors. The blood volume measured by CAA and microvessel density was significantly lower in the Renca/BAI1 than in the Renca/Wild and Renca/LacZ tumors (p<0.01 and p<0.05, respectively). A significant (p<0.01) reduction in VEGF concentration in the supernatant was demonstrated in the Renca/BAI1 compared with the Renca/Wild and Renca/LacZ cell cultures. These observations suggest that the transfer of the BAI1 gene to Renca can suppress the tumor growth via the inhibition of angiogenesis. The down-regulation of VEGF production in tumor cells contributes to this anti-tumor effect.     

LCRG1 suppresses tumor growth in vivo by liposome-mediated gene transfer

Objective: To investigate whether LCRG1 (Laryngeal Carcinoma Related Gene 1) has tumor suppressor function. Methods: The recombinant plasmid pcDNA3.1(+)/LCRG1 was successfully constructed. The biological effects of LCRG1 on Hep-2 cell line were studied by cell transfection, cell growth observation colony formation analysis and tumorigenicity experiments. Results: The LCRG1 gene potently inhibited tumorgenesis in vitro and in vivo, as showed by dramatic growth arrest observed in cell growth analysis and suppression of anchorage-independent growth and tumorigenicity in nude mice. Conclusion: Our results suggested that LCRG1 may be a candidate of tumor suppressor gene. Foundation item: This work was supported by the National Natural Science Foundation of China(No. 39900052) and a grant from the Ministry of Health of PR China (No. 98-1-118). Biography: Li You-jun(1972–), doctor of medicine, assistant professor, Xiangya Medical School, Central South University, majors in pathology and pathophysiology.     

Suppression of tumor angiogenesis and growth by gene transfer of a soluble form of vascular endothelial growth factor receptor into a remote organ

Antiangiogenic therapy shows promise as a strategy for cancer treatment. We constructed an adenovirus (AdVEGF-ExR) expressing the entire extracellular domain of the human vascular endothelial growth factor (VEGF) receptor (flt-1) fused to the Fc portion of human IgG. The soluble receptor secreted from AdVEGF-ExR-infected cells bound to VEGF and inhibited VEGF-induced DNA synthesis in endothelial cells. When human lung cancer cell line H157, which produces not only VEGF but also fibroblast growth factor 2 and interleukin 8 at substantial levels, was infected with AdVEGF-ExR, cell growth in vitro was not affected. However, when H157 cells infected with AdVEGF-ExR were injected s.c. into nude mice, tumor formation stopped on the 10th day after reaching a certain size (about 100 mm3), and tumor size declined gradually thereafter. When AdVEGF-ExR was injected into skeletal muscle and uninfected H157 cells were injected s.c., the soluble receptor was detectable in the circulating blood for 3 weeks, tumor growth ceased after 10 days, and tumor size declined thereafter. Histological examination revealed that intratumor angiogenesis was markedly suppressed, and apoptosis was enhanced. Using the same experimental protocol, a significant suppression of tumor growth was also seen in four of five other lung cancer cell lines, some of which secreted VEGF at nominal levels, at least under normoxic conditions in vitro. Our results demonstrate that adenovirus-mediated expression of a soluble VEGF receptor in a remote organ could inhibit tumor angiogenesis and enhance apoptosis and thereby suppress tumor growth in vivo. Adenovirus-mediated overexpression of a soluble VEGF receptor in a remote organ may have the potential to be a feasible and effective strategy for cancer treatment.     

Lentiviral vector neutral endopeptidase gene transfer suppresses prostate cancer tumor growth

Neprilysin (neutral endopeptidase, NEP) is a cell surface peptidase whose expression is lost in approximately 50% of prostate cancers (PC). NEP normally functions to inactivate peptides such as bombesin and endothelin-1, and potentiates the effects of the PTEN tumor suppressor via a direct protein-protein interaction. NEP loss contributes to PC progression. We investigated the therapeutic efficacy of using a lentiviral vector system to restore NEP expression in PC cells. Third-generation lentiviral vectors encoding wild-type NEP (L-NEP) or green fluorescent protein (L-GFP) were introduced into NEP-deficient 22RV1 PC cells. Cells infected with L-NEP or L-GFP at a multiplicity of infection of 10 demonstrated NEP enzyme activity of 1171.2+/-4.9 and 17.2+/-5.3 pmol/microg/min (P<0.0001), respectively. Cell viability, proliferation and invasion were each significantly inhibited in 22RV1 cells expressing NEP compared with control cells infected with L-GFP (P<0.01). Analysis of known downstream effects of NEP showed NEP-expressing cells exhibiting decreased Akt and focal adhesion kinase phosphorylation and increased PTEN protein expression. Finally, injection of L-NEP into established 22RV1 xenograft tumors significantly inhibited tumor growth (P<0.01). These experiments demonstrate that lentiviral NEP gene transfer is a novel targeted strategy for the treatment of NEP-deficient PC.     

Myriocin, a serine palmitoyltransferase inhibitor, suppresses tumor growth in a murine melanoma model by inhibiting de novo sphingolipid synthesis

Advanced melanoma is the most virulent form of cancer and has a poor prognosis. In a previous study, myriocin, an inhibitor of serine palmitoyltransferase, was found to suppress melanoma cell proliferation by cell cycle arrest at the G 2/M phase through decreased sphingolipid levels and increased p53 and p21 (waf1/cip1) expression. ( 1) In the present study, myriocin (1 mg/kg, every other day for 3 weeks) was administered intradermally or intraperitoneally to melanoma mice. Tumor formation was significantly inhibited by intradermal and intraperitoneal administration of myriocin. The expression of Cdc25C, Cdc2 and cyclin B1 was decreased in tumor tissues from myriocin-treated mice, while the expression of p53 and p21 (waf1/cip1) was increased compared with that of the controls. The levels of sphingolipids in serum, liver and tumor tissue from myriocin-treated mice were decreased compared with those of controls. The decreased levels of sphingolipids in serum and liver of melanoma mice treated with myriocin suggests that myriocin may be accessible to tumor tissues of advanced melanoma. Taken together, the suppression of sphingolipid synthesis by myriocin inhibits the expression of Cdc25C or activates the expression of p53 and p21 (waf1/cip1) . This is followed by Cdc2 and cyclin B1 inhibition which results in the suppression of tumor growth.     

Lysis and growth stimulation of a murine melanoma determined by density of macrophage populations

Activated macrophages were found to induce opposite effects on the growth of the F10 variant of B16 melanoma in both in vivo and in vitro systems. Low macrophage numbers had lytic effects and high numbers stimulated the growth of the melanoma. In fact, the number of macrophages per area (namely, their density) was found to determine whether lysis or growth stimulation would occur. Low density macrophages were found to be structurally and functionally different from high density macrophages. Macrophages in sparse cultures were round, separate, and lysed tumor cells, while crowded macrophages appeared spread, with pseudopodia, and caused enhancement of tumor growth.     

Adeno-associated virus-mediated gene transfer of endostatin inhibits angiogenesis and tumor growth in vivo

A variety of approaches has demonstrated that interfering with tumor-induced angiogenesis may be an effective strategy in cancer therapy. However, it is likely that to be most effective such strategies will require extended suppression of the angiogenic process. Gene therapy offers a possible approach to achieve sustained release of a therapeutically potent transferred gene product. In the present study the angiogenesis inhibitor endostatin was expressed through a recombinant adeno-associated viral (rAAV) vector and shown to be biologically active in vitro and in vivo. Intramuscular injection of rAAV-HuEndo (1 x 10(9) i.u.) led to a sustained serum endostatin level of approximately 35-40 ng/mL. This endostatin level was sufficient to inhibit tumor cell-induced angiogenesis and to suppress both the initiation and subsequent growth of a human colorectal cancer model.     

Adenoviral gene transfer of stromal cell-derived factor-1 to murine tumors induces the accumulation of dendritic cells and suppresses tumor growth

The human CXC chemokine, stromal cell-derived factor 1 (SDF-1alpha), is known to function in vitro as a chemotactic factor for lymphocytes, monocytes, and dendritic cells. In the context that dendritic cells are powerful antigen-presenting cells, we hypothesized that adenoviral gene transfer of SDF-1alpha to tumors might inhibit growth of preexisting tumors through attracting dendritic cells to the tumor. AdSDF-1alpha mediated the expression of SDF-1alpha mRNA and protein in A549 cells in vitro, and the supernatant of the AdSDF-1alpha-infected A549 cells showed chemotactic activity for dendritic cells. When syngeneic murine CT26 colon carcinoma tumors (BALB/c) and B16 melanoma and Lewis lung cell carcinoma (C57Bl/6) were injected with AdSDF-1alpha (5 x 10(8) plaque-forming units), there was an accumulation of dendritic cells and CD8(+) cells within the tumor and significant inhibition of tumor growth compared with tumors injected with PBS or AdNull (control vector). The injection of AdSDF-1alpha into tumors induced the inflammatory enlargement and the accumulation of dendritic cells in the draining lymph node. Intratumoral AdSDF-1alpha administration elicited tumor-specific CTLs and adoptive transfer of splenocytes from AdSDF-1alpha-treated mice resulted in the elongation of survival after tumor challenge. Interestingly, in wild-type and CD4(-/-) mice but not in CD8(-/-) mice, AdSDF-1alpha inhibited the growth of the tumor. These observations suggest that adenoviral gene transfer of SDF-1alpha may be a useful strategy to accumulate dendritic cells in tumors and evoke antitumor immune responses to inhibit tumor growth.     

Glipizide,an antidiabetic drug,suppresses tumor growth and metastasis by inhibiting angiogenesis

Angiogenesis is involved in the development, progression and metastasis of various human cancers. Herein, we report the discovery of glipizide, a widely used drug for type 2 diabetes mellitus, as a promising anticancer agent through the inhibition of tumor angiogenesis. By high-throughput screening (HTS) of an FDA approved drug library utilizing our in vivo chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) models, glipizide has been identified to significantly inhibit blood vessel formation and development. Moreover, glipizide was found to suppress tumor angiogenesis, tumor growth and metastasis using xenograft tumor and MMTV-PyMT transgenic mouse models. We further revealed that the anticancer capability of glipizide is not attributed to its antiproliferative effects, which are not significant against various human cancer cell lines. To investigate whether its anticancer efficacy is associated with the glucose level alteration induced by glipizide application, glimepiride, another medium to long-acting sulfonylurea antidiabetic drug in the same class, was employed for the comparison studies in the same fashion. Interestingly, glimepiride has demonstrated no significant impact on the tumor growth and metastasis, indicating that the anticancer effects of glipizide is not ascribed to its antidiabetic properties. Furthermore, glipizide suppresses endothelial cell migration and the formation of tubular structures, thereby inhibiting angiogenesis by up-regulating the expression of natriuretic peptide receptor A. These findings uncover a novel mechanism of glipizide as a potential cancer therapy, and also for the first time, provide direct evidence to support that treatment with glipizide may reduce the cancer risk for diabetic patients.     

内皮抑素基因治疗对小鼠肿瘤组织内新生血管形成抑制作用的实验研究

目的:研究腺病毒介导的内皮抑素基因治疗对小鼠肺肿瘤组织内部血管的抑制作用。方法:皮下注射2×106LLC细胞至C57BL/6小鼠背部建立小鼠肺癌种植瘤模型,2周后瘤内注射2×109pfu内皮抑素腺病毒载体治疗。免疫组化检测内皮抑素在肿瘤组织的原位表达,观察治疗对肿瘤生长的影响。免疫组化标记计数肿瘤内部血管密度,观察治疗对肿瘤血管的影响。结果:Admendostatin治疗从第2周起就可明显减缓肿瘤生长速度,P<0.05。免疫组化显示治疗组肿瘤组织内皮抑素蛋白强阳性表达,对照组阴性或痕量表达。Admendostatin治疗组、空载体对照组和阴性对照组肿瘤血管密度用CD31和CD105抗原标记分别为37.5±4.6、65.2±5.8、68.5±4.5和10.5±3.2、39.7±5.6、42.4±4.8。治疗组与两对照组相比差异有统计学意义,P<0.05;空载体对照与阴性对照相比差异无统计学意义,P>0.05。结论:腺病毒介导的内皮抑素基因治疗可抑制小鼠皮下种植瘤的生长,其作用机制是抑制肿瘤内部新生血管的生成,应用CD105抗原标记可准确反映新生血管的变化。     

Polyethylenimine-mediated in vivo gene transfer of a transmembrane superantigen fusion construct inhibits B16 murine melanoma growth

Immunotherapy has been proposed as a therapeutic strategy in advanced-stage melanomas in which other therapeutic options have little effect. The Staphylococcus enterotoxin A (SEA) has been used to stimulate an antitumoral immune response but its use is hampered by severe systemic side effects. Here, we show that SEA can be targeted to melanoma cells to limit these side effects. More specifically, we used a nonviral vector, the cationic polymer, polyethylenimine (PEI), to express a transmembrane SEA fusion construct (pSEA-TM) in B16F10-induced subcutaneous melanoma in mice. The efficacy of this in vivo transfection was enhanced by concomitant infusion of epinephrine to induce local vasoconstriction. In these conditions, repeated injections of pSEA-TM/PEI complexes elicited a significant response, as evidenced by tumor growth inhibition, without systemic adverse effects. T cell infiltration of the tumors, together with positive lymphocyte proliferation tests, suggested local and systemic immune responses. Altogether, PEI-mediated targeting of SEA to melanoma tumor cells in vivo efficiently stimulates the antitumor immune response without inducing the side effects observed with systemic administration of SEA.     

Cystemustine induces redifferentiation of primary tumors and confers protection against secondary tumor growth in a melanoma murine model

N'-(2-Chloroethyl)-N-(2-(methylsulfonyl)-ethyl)-N'-nitrosourea (cystemustine) is a chloroethylnitrosourea that has been used in the treatment of human melanoma. Its main antitumor effect is DNA damage to malignant melanocytes. Although unreported at present, other effects may also account for its cytotoxicity, some of them could be more or less delayed with respect to its administration. In this report, we have developed a model of secondary tumor with B16 melanoma in syngeneic C57B16 recipients to investigate the impact of cystemustine treatment of primary B16 melanoma tumors on the fate of secondary implanted untreated tumors. The data presented in this report indicate that cystemustine-treated cells or the administration of cystemustine provoke an important growth delay of primary melanoma tumors, together with an increase in cell pigmentation and cell morphology changes. Data also show that prime treatment induces a dramatic decrease in tumor weight of secondary untreated tumors accompanied by an increase in melanin content and an alteration of cell morphology. Finally, 1H-NMR spectroscopy was performed on treated B16 cells, showing an alteration in the phospholipid derivatives of melanocytes, suggesting subsequent modifications of membrane phospholipid composition. In conclusion, the data highlight two important findings: (a) cystemustine produces modifications other than DNA damage, i.e., cell morphology changes, pigmentation, and phospholipid metabolism alterations, indicating an interference with cell cycle, cell redifferentiation, and proliferation programs; and (b) cystemustine-treated tumors appear to confer a protective effect against the development of secondary untreated tumors that may be mediated by cytokines or an immune response.     

Suppression of tumor growth by galectin-7 gene transfer

Galectin-7 is a beta-galactoside-binding animal lectin specifically expressed in stratified epithelia. Its expression is inducible by p53 and is down-regulated in squamous cell carcinomas. Other investigators previously showed that galectin-7 is a proapoptotic protein, and we showed that ectopic expression of galectin-7 in HeLa cells renders the cells more sensitive to a variety of apoptotic stimuli. In the present study, we showed that ectopic expression of galectin-7 in the human colon carcinoma cell line DLD-1 also made the cells more sensitive to apoptosis under various conditions. We also found that galectin-7-transfected DLD-1 (DLD-1-Gal7) cells grew significantly more slowly than control transfectants (DLD-1-V) under normal culture conditions in the absence of apoptosis. Moreover, a significantly lower number of colonies were formed from DLD-1-Gal7 cells than from DLD-1-V cells under anchorage-independent cell growth conditions. Most importantly, tumor formation from DLD-1-Gal7 cells was dramatically reduced compared with DLD-1-V cells when these cells were inoculated s.c. into severe combined immunodeficient mice. DLD-1-Gal7 tumors showed a significantly lower proliferation rate than DLD-1-V tumors as determined by in vivo 5-bromo-2'-deoxyuridine incorporation. DLD-1-Gal7 tumors also contained a lower density of blood vessels than DLD-1-V tumors, suggesting that ectopic expression of galectin-7 suppresses angiogenesis. This may partially account for the greater suppressive effect of galectin-7 on tumor growth in vivo than in vitro. Our results show that galectin-7 has a suppressive effect on tumor growth, suggesting that galectin-7 gene transfer or other means of specifically inducing galectin-7 expression may be a new approach for management of cancers.     

Human growth hormone gene transfer into tumor cells may improve cancer chemotherapy

Chemotherapy remains the main tool for the treatment of cancers, but is often hampered by tumor cell resistance. In this context, the transfer of genes able to accentuate the effect of anticancer drugs may constitute a useful approach, as exemplified by inactivation of nuclear factor (NF)-kappa B via direct transfer of a gene encoding a negative dominant of its natural inhibitor I kappa B, leading to improved response to cancer chemotherapy. Following our previous report that transfection of human growth hormone (hGH) gene into human monocytic cell lines may also inactivate NF-kappa B in another situation, we decided to test the consequences of hGH gene transfer on cancer treatments. We demonstrated that hGH-transfected human myeloid leukemia U937 cells were sensitized to an apoptotic signal mediated by the anticancer drugs. In parallel, we found that, by inhibiting degradation of I kappa B, hGH gene transfer diminished NF-kappa B entry into the nuclei of U937 cells exposed to daunorubicin. Finally, we report that hGH-transfected tumor cells engrafted in nude mice responded in vivo to chemotherapy with nontoxic doses of daunorubicin whereas, under the same conditions, control tumor cells remained insensitive. Overall, this study therefore suggests that hGH gene transfer may offer new therapeutic prospects in cancer therapy.