Soluble Vegfr3 gene therapy suppresses multi-organ metastasis in a mouse mammary cancer model.

Accumulating evidence on the association of VEGF-C with lymphangiogenesis and lymph node metastasis implicates lymphatic vessels as a potential target in anti-cancer therapy. To evaluate whether blocking VEGF-C and VEGFR-3 signaling can inhibit multi-organ metastases, a mouse metastatic mammary cancer model was subjected to gene therapy using a soluble VEGFR-3 expression vector (psVEGFR-3). We showed that psVEGFR-3 significantly diminished cell growth in vitro with or without added VEGF-C, and significantly reduced primary tumor growth and tumor metastases to wide-spectrum organs in vivo. Although apoptotic cell death and angiogenesis levels did not differ between the control and psVEGFR-3 groups, cell proliferation and lymphangiogenesis in the mammary tumors were significantly decreased in the psVEGFR-3 group. Furthermore, lymphatic vessel invasion was significantly inhibited in this group. Real-time RT-PCR analysis revealed significantly high expression of the Vegfr3 gene due to gene therapy, and the transcriptional levels of Pcna and Lyve1 tended to decrease in the psVEGFR-3 group. Immunofluorescence staining indicated that phospho-tyrosine expression was considerably lower in tumor cells of psVEGFR-3-treated mammary carcinomas than those of control tumors. Double immunofluorescence staining indicated that phospho-tyrosine+ /LYVE-1+ (a lymphatic vessel marker) tended to decrease in psVEGFR-3-treated mammary carcinomas compared with control mice, indicating a decline in the activity of the VEGF-C/VEGFR-3 axis. These findings showed that a blockade of VEGF-C/VEGFR-3 signaling caused by sVEGFR-3 sequestered VEGF-C and prevented the side-effects of anti-angiogenesis and suppressed overall metastases, suggesting their high clinical significance.

Urethane-induced Mammary Carcinogenesis Susceptibility in Transgenic Mice Expressing a Dominant-negative TGF-ß Type II Receptor.

BACKGROUND/AIM: Transforming growth factor-ß (TGF-ß) plays dual suppressive and oncogenic roles in mammary carcinogenesis. MATERIALS AND METHODS: To analyze whether TGF-ß exerts suppressive or oncogenic actions on mammary carcinogenesis, transgenic mice overexpressing a dominant-negative mutant type II TGF-ß receptor (TßRII-DNR) driven by the mouse mammary tumor virus (MMTV) promoter were treated with a low dose of urethane, a carcinogen present in fermented food products and alcoholic beverages. RESULTS: Lobular proliferative lesions, showing high ß-casein expression, developed in the mammary glands of TßRII-DNR+/+ mice aged >61 weeks. Compared with wild-type mice, TßRII-DNR+/+ mice administered with urethane showed significant increases in dysplastic hyperplasias and adenocarcinomas of the mammary glands. CONCLUSION: The functional decline of TGF-ß signaling in mammary glands led to a high susceptibility to urethane-induced mammary carcinogenesis. TGF-ß signaling may act as a tumor suppressor during mammary tumor development.

Crude α-Mangostin Suppresses the Development of Atherosclerotic Lesions in Apoe-Deficient Mice by a Possible M2 Macrophage-Mediated Mechanism.

Lifestyle choices play a significant role in the etiology of atherosclerosis. Male Apoe-/- mice that develop spontaneous atherosclerotic lesions were fed 0%, 0.3%, and 0.4% mangosteen extracts, composed largely of α-mangostin (MG), for 17 weeks. Body weight gains were significantly decreased in both MG-treated groups compared to the control, but the general condition remained good throughout the study. The levels of total cholesterol (decreased very-low-density lipoprotein in lipoprotein profile) and triglycerides decreased significantly in the MG-treated mice in conjunction with decreased hepatic HMG-CoA synthase and Fatty acid transporter. Additionally, increased serum lipoprotein lipase activity and histopathology further showed a significant reduction in atherosclerotic lesions at both levels of MG exposure. Real-time PCR analysis for macrophage indicators showed a significant elevation in the levels of Cd163, an M2 macrophage marker, in the lesions of mice receiving 0.4% MG. However, the levels of Nos2, associated with M1 macrophages, showed no change. In addition, quantitative immunohistochemical analysis of macrophage subtypes showed a tendency for increased M2 populations (CD68⁺/CD163⁺) in the lesions of mice given 0.4% MG. In further analysis of the cytokine-polarizing macrophage subtypes, the levels of Interleukin13 (Il13), associated with M2 polarization, were significantly elevated in lesions exposed to 0.4% MG. Thus, MG could suppress the development of atherosclerosis in Apoe-/- mice, possibly through an M2 macrophage-mediated mechanism.

Pathological and molecular analyses of atherosclerotic lesions in ApoE-knockout mice.

The establishment of consistent and reliable methods for the analysis of atherosclerosis molecular pathways and for testing the efficiency of new therapeutics is of utmost importance. Here, we fed ApoE-knockout (KO) mice with high-fat diet to for 16 weeks to induce atherosclerosis. Atherosclerotic lesions in mice were methodically investigated using pathologic analyses and molecular biology tools. These lesions were histopathologically classified into three categories: early, progressive, and combined lesions. Immunohistochemical analyses showed that both F4/80 (macrophage marker) and tenascin-C are expressed in these lesions. Real-time PCR analysis conducted using formalin-fixed paraffin-embedded tissues with atherosclerotic lesions demonstrated an increase in the levels of many inflammatory chemokines, including Cxcl16, while antibody arrays performed using frozen atherosclerotic tissue samples showed elevated TIMP-1 expression. Subsequent immunohistochemical analyses showed that the expression of CXCL16, TIMP-1, MMP-9, MMP-8, and LOX-1 is localized in the atherosclerotic lesions. We confirmed that the expression of these proteins is localized to atherosclerotic lesion, which suggests their roles in the development of the lesions in ApoE-KO mice. Therefore, this mouse model represents an appropriate tool for elucidating molecular mechanisms underlying the development of atherosclerosis, and a model for the evaluation of therapeutic efficiency of novel drugs.

Locked nucleic acid antisense inhibitor targeting apolipoprotein C-III efficiently and preferentially removes triglyceride from large very low-density lipoprotein particles in murine plasma.

A 20-mer phosphorothioate antisense oligodeoxyribonucleotide having locked nucleic acids (LNA-AON) was used to reduce elevated serum triglyceride levels in mice. We repeatedly administered LNA-AON, which targets murine apolipoprotein C-III mRNA, to high-fat-fed C57Bl/6J male mice for 2 weeks. The LNA-AON showed efficient dose-dependent reductions in hepatic apolipoprotein C-III mRNA and decreased serum apolipoprotein C-III protein concentrations, along with efficient dose-dependent reductions in serum triglyceride concentrations and attenuation of fat accumulation in the liver. Through precise lipoprotein profiling analysis of sera, we found that serum reductions in triglyceride and cholesterol levels were largely a result of decreased serum very low-density lipoprotein (VLDL)-triglycerides and -cholesterol. It is noteworthy that larger VLDL particles were more susceptible to removal from blood than smaller particles, resulting in a shift in particle size distribution to smaller diameters. Histopathologically, fatty changes were markedly reduced in antisense-treated mice, while moderate granular degeneration was frequently seen the highest dose of LNA-AON. The observed granular degeneration of hepatocytes may be associated with moderate elevation in the levels of serum transaminases. In conclusion, we developed an LNA-based selective inhibitor of apolipoprotein C-III. Although it remains necessary to eliminate its potential hepatotoxicity, the present LNA-AON will be helpful for further elucidating the molecular biology of apolipoprotein C-III.

Therapy with siRNA for Vegf-c but not for Vegf-d suppresses wide-spectrum organ metastasis in an immunocompetent xenograft model of metastatic mammary cancer.

Cancer metastasis contributes significantly to cancer mortality and is facilitated by lymphangiogenesis and angiogenesis. Vascular endothelial growth factors-C and D (VEGF-C and VEGF-D) are heavily involved in lymphangiogenesis. Using small interfering RNA (siRNA) against mouse Vegf-c, and Vegf-d, we sought to inhibit metastasis in a model of metastatic murine mammary cancer. BJMC3879Luc2 cell-induced mammary carcinomas received direct intratumoral injections in vivo of either plasmid VEGF-C/D siRNA (psiVEGF-C, psiVEGF-D) or a vector control followed by in vivo gene electrotransfer weekly for seven weeks. Treatment with psiVEGF-C and with psiVEGF-D resulted in lower tumor volumes as compared to the controls. Treatment with psiVEGF-C suppressed wide-spectrum organ metastasis involving lung and lymph nodes. Treatment with psiVEGF-C further reduced the number of dilated lymphatic vessels with invading cancer cells and inhibited tumor blood microvessel density. In contrast, although treatment with psiVEGF-D was not effective and gave equivocal results, it did induce some insignificant reduction in tumor volume increment, average numbers of lymph node metastases and average number of intratumoral dilated lymphatic vessels. In conclusion, specific silencing of the Vegf-c gene suppresses wide-spectrum organ metastasis, including the lung and lymph nodes. However, therapy with siRNA for Vegf-d was not adequately effective in this murine system.

Mammary cancer gene therapy targeting lymphangiogenesis: VEGF-C siRNA and soluble VEGF receptor-2, a splicing variant.

Metastasis contributes significantly to cancer mortality, and the most common pathway of initial dissemination is via the afferent ducts of the lymphatics. Overexpression of vascular endothelial growth factor (VEGF)-C has been associated with lymphangiogenesis and lymph node metastasis in a multitude of human neoplasms, including breast cancers. We recently reported that both VEGF-C siRNA and endogenous soluble vascular endothelial growth factor receptor-2 (esVEGFR-2, a new splicing variant) inhibit VEGF-C function and metastasis in a mouse model of metastatic mammary cancer. Here we briefly review our previous experimental work, specifically targeting tumor lymphangiogenesis, in which metastatic mouse mammary cancers received direct intratumoral injections of either expression vectors VEGF-C siRNA or esVEGFR-2, or the empty plasmid vector, once a week for 6 or 8 weeks, followed by in vivo gene electrotransfer of the injected tumors. Throughout our study, both tumor lymphangiogenesis and the multiplicity of lymph node metastasis were significantly inhibited, with an overall reduction in tumor growth, by both VEGF-C siRNA and esVEGFR-2; further, a significant reduction in the number of dilated lymphatic vessels containing intraluminal cancer cells was observed with both treatments. Thus, therapeutic strategies targeting lymphangiogenesis may have great clinical significance for the treatment of metastatic human breast cancer.

The endogenous soluble VEGF receptor-2 isoform suppresses lymph node metastasis in a mouse immunocompetent mammary cancer model.

BACKGROUND: Cancer metastasis contributes significantly to cancer mortality and is facilitated by lymphangiogenesis and angiogenesis. A new splicing variant, endogenous soluble vascular endothelial growth factor receptor-2 (esVEGFR-2) that we recently identified is an endogenous selective inhibitor of lymphangiogenesis. To evaluate the antimetastatic potential of esVEGFR-2, gene therapy with vector expressing esVEGFR-2 (pesVEGFR-2) or endostatin (pEndo) as a positive control was conducted on murine metastatic mammary cancer. METHODS: Syngeneic inoculated metastatic mammary cancers received direct intratumoral injection of pesVEGFR-2, pEndo or pVec as control, once a week for six weeks. In vivo gene electrotransfer was performed on the tumors after each injection. RESULTS: Deaths from metastasis were much lower in the pesVEGFR-2 and pEndo groups than in those of the pVec. Tumor volume was significantly lower in the pesVEGFR-2 and the pEndo groups throughout the study. Multiplicity of lymph node and lung metastatic nodules was significantly suppressed in the pesVEGFR-2 and pEndo groups. Moreover, the total number of overall metastasis including the other organs was also decreased in these groups. However, pesVEGFR-2 was not able to decrease the number of lungs, ovaries, kidneys and adrenals with metastasis as counted by unilateral or bilateral metastasis. The number of CD34+/Lyve-1⁻ blood microvessels was significantly decreased in the pEndo group, while the number of CD34⁻/Lyve-1+ lymphatic vessels was significantly decreased in the pesVEGFR-2 and pEndo groups. In addition, a significant reduction in the number of dilated lymphatic vessels containing intraluminal cancer cells was observed in the pesVEGFR-2 and pEndo groups. Levels of apoptosis were significantly increased in the pEndo group, whereas the rates of cell proliferation were significantly decreased in the pesVEGFR-2 and pEndo groups. CONCLUSIONS: Our data demonstrate that esVEGFR-2 can inhibit mainly lymph node metastasis. The antimetastatic activity of esVEGFR-2 may be of high clinical significance in the treatment of metastatic breast cancer because lymph node involvement is a most important prognostic factor in cancer patients.

Raloxifene inhibits tumor growth and lymph node metastasis in a xenograft model of metastatic mammary cancer.

BACKGROUND: The effects of raloxifene, a novel selective estrogen receptor modulator, were studied in a mouse metastatic mammary cancer model expressing cytoplasmic ERα. METHODS: Mammary tumors, induced by inoculation of syngeneic BALB/c mice with BJMC3879luc2 cells, were subsequently treated with raloxifene at 0, 18 and 27 mg/kg/day using mini-osmotic pumps. RESULTS: In vitro study demonstrated that the ERα in BJMC3879luc2 cells was smaller (between 50 and 64 kDa) than the normal-sized ERα (66 kDa) and showed cytoplasmic localization. A statistically significant but weak estradiol response was observed in this cell line. When BJMC3879luc2 tumors were implanted into mice, the ERα mRNA levels were significantly higher in females than in males. In vitro studies showed that raloxifene induced mitochondria-mediated apoptosis and cell-cycle arrest in the G1-phase and a decrease in the cell population in the S-phase. In animal experiments, tumor volumes were significantly suppressed in the raloxifene-treated groups. The multiplicity of lymph node metastasis was significantly decreased in the 27 mg/kg group. Levels of apoptosis were significantly increased in the raloxifene-treated groups, whereas the levels of DNA synthesis were significantly decreased in these groups. No differences in microvessel density in tumors were observed between the control and raloxifene-treated groups. The numbers of dilated lymphatic vessels containing intraluminal tumor cells were significantly reduced in mammary tumors in the raloxifene-treated groups. The levels of ERα mRNA in mammary tumors tended to be decreased in the raloxifene-treated groups. CONCLUSION: These results suggest that the antimetastatic activity of raloxifene in mammary cancer expressing cytoplasmic ERα may be a crucial finding with clinical applications and that raloxifene may be useful as an adjuvant therapy and for the chemoprevention of breast cancer development.

An immunocompetent murine model of metastatic mammary cancer accessible to bioluminescence imaging.

BACKGROUND: Many areas of research, including gene and pharmacological therapeutics, would benefit from longitudinal in vivo monitoring methodologies. To investigate the feasibility of one such methodology, we developed a murine mammary cancer model amenable to sequential bioluminescent imaging of tumor growth and metastasis in living animals. MATERIALS AND METHODS: Metastatic mouse mammary carcinoma BJMC3879 cells were transfected to stably express firefly luciferase and inoculated into immunocompetent female BALB/c mice. RESULTS: Sequential analysis using bioluminescent imaging showed increasing photon counts correlated to expanding mammary tumor volumes; in addition, strong signals from axillary, mandibular, femoral, thoracic and abdominal regions in mice were histopathologically determined to be due to metastases, the majority of which occurred in lymph nodes and lungs. CONCLUSION: The bioluminescent mouse mammary cancer model we established provides a method for quantifiable longitudinal in vivo imaging that can be used in gene and pharmacological therapy applications.

Panaxanthone isolated from pericarp of Garcinia mangostana L. suppresses tumor growth and metastasis of a mouse model of mammary cancer.

BACKGROUND: The antitumor growth and antimetastatic activity of panaxanthone (approximately 80% alpha-mangostin and 20% gamma-mangostin) were studied in a mouse metastatic mammary cancer model that produces a metastatic spectrum similar to that seen in human breast cancer. MATERIALS AND METHODS: Mammary tumors, induced by inoculation of syngeneic BALB/c mice with BJMC3879 cells, were subsequently treated with panaxanthone at 0, 2,500, or 5,000 ppm in their diet. In vitro studies were also conducted to evaluate the effects of alpha-mangostin, the main component of panaxanthone, on BJMC3879 cells. RESULTS: In the in vivo study, tumor volumes were significantly suppressed in mice treated with 2,500 and 5,000 ppm panaxanthone in their diet. The multiplicity of lung metastasis was significantly lower in the 5,000 ppm group. Lymph node metastasis also tended to decrease in the 5,000 ppm group but not significantly. The antitumor effects of panaxanthone were associated with elevation of apoptotic cell death, antiproliferation (inhibition of PCNA) and antiangiogenesis (inhibition of microvessel density). The in vitro study demonstrated that alpha-mangostin induced apoptosis, as evidenced by increased numbers of TUNEL-positive cells, elevated activities of caspases and a decrease in mitochondrial membrane potential, cell cycle arrest in the G(1)-phase and decreases in the cell population in the S- and G(2)/M-phases. CONCLUSION: These results suggest that the observed antimetastatic activity of panaxanthone may be of clinical significance as adjuvant therapy in metastatic human breast cancer, and may also be useful as a chemopreventative of breast cancer development.

Vaticanol C, a novel resveratrol tetramer, reduces lymph node and lung metastases of mouse mammary carcinoma carrying p53 mutation.

PURPOSE: The effects of vaticanol C (Vat-C), a novel resveratrol tetramer, were studied in a mouse metastatic mammary cancer model carrying mutations in p53 that produce a metastatic spectrum similar to that seen in human breast cancers. METHODS: Mammary tumors, induced by inoculation of syngeneic BALB/c mice with BJMC3879 cells, were subsequently treated with Vat-C at 0, 100 and 200 ppm in their diet. RESULTS: The in vitro study demonstrated that Vat-C induced apoptosis, as inferred by morphological changes, nucleosomal DNA fragmentation and elevated activities of caspases. Although tumor volumes were not apparently suppressed in mice treated with Vat-C, the multiplicity of lymph node metastasis was significantly decreased in the 200-ppm group. Furthermore, the multiplicity of lung metastasis was also significantly lower in the 200-ppm group. In any category of organ metastasis, the number of organs with metastasis tended to be lower in the 200-ppm group, but these findings were not statistically significant. The levels of apoptosis were significantly higher in the 200-ppm group, but DNA synthesis only a tended to be lower in this group. Microvessel density in tumors also tended to be lower in the Vat-C-treated groups. Moreover, the numbers of lymphatic vessels having intraluminal tumor cells was significantly lower in mammary tumors of mice given 100 and 200-ppm Vat-C, indicating a reduction in migrating tumor cells into the lymphatic vessels of tumor tissue. CONCLUSIONS: These results suggest that the observed antimetastatic activity of Vat-C may be of clinical significance as an adjuvant therapy in metastatic human breast cancer having p53 mutations, and may also be useful as a chemopreventative of breast cancer development.

Comparative effects of lovastatin on mammary and prostate oncogenesis in transgenic mouse models.

The effects of lovastatin, a potent inhibitor of HMG CoA reductase, on experimental mammary and prostate oncogenesis, were studied in vitro and in vivo. Lovastatin inhibited cell growth in vitro in a dose-dependent manner for both mammary and prostate cancer cell lines, which was associated with p53-independent apoptosis. Flow cytometric analyses of lovastatin-treated mammary and prostate cancer cells demonstrated cell-cycle G(1) arrest, as well as decreases in S and G(2)/M fractions. p21(Waf1) and p27(Kip1) were induced by lovastatin in both types of cancer cells. Gene expression profiling of cells treated with lovastatin, however, was remarkable for a paucity of transcriptional changes induced by lovastatin. Treatment with lovastatin for 4 weeks did inhibit the formation of pre-neoplastic mammary intraepithelial neoplasias (MIN) in vivo, but not invasive carcinomas in the C3(1)/SV40 TAg transgenic model of mammary cancer. The decreased multiplicity of MIN lesions was associated with increased levels of apoptosis in these lesions. However, cell proliferation in the mammary lesions was not significantly different between lovastatin-treated and control mice 1 day after lovastatin treatment. In female mice treated with lovastatin for 12 weeks, there was a tendency for reduced tumor volume, which did not reach statistical significance. However, lovastatin did not suppress any lesion formation in the prostate of C3(1)/SV40 TAg transgenic male mice. Our results suggest that as lovastatin exerts an inhibitory effect on the development of early mammary lesions of mammary carcinogenesis, this compound may be useful for the chemoprevention of mammary cancer and might have utility as an adjuvant in breast cancer therapy. The chemopreventive effects of lovastatin in vivo, however, may be tissue-specific.