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.

Synthetic α-mangostin dilaurate strongly suppresses wide-spectrum organ metastasis in a mouse model of mammary cancer.

We previously reported that, in a mouse model of mammary cancer, α-mangostin alone exhibits anti-metastatic properties. To enhance this anti-metastatic effect, we examined the efficacy of synthetic α-mangostin dilaurate (MGD), prepared by adding lauric acid to α-mangostin, in the same experimental system wherein mice bearing mammary tumors are exposed to dietary MGD at 0, 2000 and 4000 ppm. Lauric acid has a high propensity for lymphatic absorption, which is the most common pathway of initial dissemination of many solid malignancies. Both mammary tumor volumes and wide-spectrum organ metastasis were markedly reduced at 2000 and 4000 ppm: furthermore, survival in the 4000-ppm group was significantly greater than in control mice. Apoptosis in mammary carcinomas was also significantly increased in the 4000-ppm group, whereas blood microvessel density and lymphatic vessel invasion were markedly reduced. In real-time PCR analyses of tumor samples, increased p21 and decreased Pcna expression were observed with 4000 ppm but values were not statistically significant when compared to expression in control tumors. However, exposure to 4000 ppm significantly decreased expression of phospho-Akt (Ser473/Thr308) as compared to the control, indicating a role in the anti-tumorigenic effects of MGD. These findings suggest that MGD may be useful for adjuvant therapy and chemoprevention and that conjugated medium-chain fatty acids may enhance the efficacy of certain chemotherapeutic agents.

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.

α-Mangostin extracted from the pericarp of the mangosteen (Garcinia mangostana Linn) reduces tumor growth and lymph node metastasis in an immunocompetent xenograft model of metastatic mammary cancer carrying a p53 mutation.

BACKGROUND: The mangosteen fruit has a long history of medicinal use in Chinese and Ayurvedic medicine. Recently, the compound α-mangostin, which is isolated from the pericarp of the fruit, was shown to induce cell death in various types of cancer cells in in vitro studies. This led us to investigate the antitumor growth and antimetastatic activities of α-mangostin in an immunocompetent xenograft model of mouse metastatic mammary cancer having a p53 mutation that induces a metastatic spectrum similar to that seen in human breast cancers. METHODS: Mammary tumors, induced by inoculation of BALB/c mice syngeneic with metastatic BJMC3879luc2 cells, were subsequently treated with α-mangostin at 0, 10 and 20 mg/kg/day using mini-osmotic pumps and histopathologically examined. To investigate the mechanisms of antitumor ability by α-mangostin, in vitro studies were also conducted. RESULTS: Not only were in vivo survival rates significantly higher in the 20 mg/kg/day α-mangostin group versus controls, but both tumor volume and the multiplicity of lymph node metastases were significantly suppressed. Apoptotic levels were significantly increased in the mammary tumors of mice receiving 20 mg/kg/day and were associated with increased expression of active caspase-3 and -9. Other significant effects noted at this dose level were decreased microvessel density and lower numbers of dilated lymphatic vessels containing intraluminal tumor cells in mammary carcinoma tissues. In vitro, α-mangostin induced mitochondria-mediated apoptosis and G1-phase arrest and S-phase suppression in the cell cycle. Since activation by Akt phosphorylation plays a central role in a variety of oncogenic processes, including cell proliferation, anti-apoptotic cell death, angiogenesis and metastasis, we also investigated alterations in Akt phosphorylation induced by α-mangostin treatment both in vitro and in vivo. Quantitative analysis and immunohistochemistry showed that α-mangostin significantly decreased the levels of phospho-Akt-threonine 308 (Thr308), but not serine 473 (Ser473), in both mammary carcinoma cell cultures and mammary carcinoma tissues in vivo. CONCLUSIONS: Since lymph node involvement is the most important prognostic factor in breast cancer patients, the antimetastatic activity of α-mangostin as detected in mammary cancers carrying a p53 mutation in the present study may have specific clinical applications. In addition, α-mangostin may have chemopreventive benefits and/or prove useful as an adjuvant therapy, or as a complementary alternative medicine in the treatment of breast cancer.

Sunitinib suppresses tumor growth and metastases in a highly metastatic mouse mammary cancer model.

BACKGROUND: Sunitinib is an inhibitor that blocks tyrosine phosphorylation (p-Tyr) of receptors including vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor. Sunitinib suppresses angiogenesis and cell proliferation and is an effective treatment for renal cell carcinoma and gastrointestinal stromal tumors. In the present study, we examined the antitumor and antimetastatic activities of sunitinib in mouse metastatic mammary cancer. MATERIALS AND METHODS: Mammary tumors induced by inoculation of BJMC3879 cells into mice were treated with sunitinib using mini-osmotic pumps. At 1 week and 7 weeks after initiation of drug administration, cancer tissue was removed and carried out histopathological and immunohistochemical examination. RESULTS: Tumor growth, as well as metastasis to the lungs and other organs, was significantly inhibited in sunitinib-treated mice. Cell death areas in mammary carcinomas were much larger in the sunitinib-treated groups than in the control group. In addition, sunitinib induced necrotic cell death rather than apoptosis. Although microvessel density was significantly lower in the sunitinib-treated mammary tumors, numbers of metastases to lymph nodes and the number of lymphatic vessels in the mammary tumors were not significantly different among groups. Cell proliferation, as assessed by BrdU-labeling indices, was significantly lower in mammary carcinomas of sunitinib-treated mice. The amounts of p-VEGFR-2 and p-Tyr, as determined by immunohistochemistry, were greatly reduced in sunitinib-treated mice. CONCLUSION: In a mouse model of mammary cancer, sunitinib inhibited tumor growth and metastasis (with the exception of lymph node metastasis), angiogenesis, and cell proliferation possibly due to reduced levels of p-VEGFR-2 and p-Tyr.

Lymphangiogenesis and Axillary Lymph Node Metastases Correlated with VEGF-C Expression in Two Immunocompetent Mouse Mammary Carcinoma Models.

Lymphangiogenesis and the expression of vascular endothelial cell growth factor C (VEGF-C) in tumors have been considered to be causally promoting lymphatic metastasis. There are only a few studies on lymphatic metastasis in immunocompetent allograft mouse models. To study the relationship between VEGF-C-mediated lymphangiogenesis and axillary lymph node metastasis, we used two mouse mammary carcinoma cell lines; the BJMC338 has a low metastatic propensity, whereas the BJMC3879 has a high metastatic propensity although it originated from the former cell line. Each cell line was injected separately into two groups of female BALB/c mice creating in vivo mammary cancer models. The expression level of VEGF-C in BJMC3879 was higher than BJMC338. As the parent cell line, BJMC3879-derived tumors showed higher expression of VEGF-C compared to BJMC338-derived tumors. This higher expression of VEGF-C in BJMC3879-derived tumors was associated with marked increase in infiltrating macrophages and enhanced expression of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) reflecting increased tumoral lymphatic density and subsequent induction of axillary lymph node metastasis. Our mouse mammary carcinoma models are allotransplanted tumors showing the same axillary lymph node metastatic spectrum as human breast cancers. Therefore, our mouse models are ideal for exploring the various molecular mechanisms of cancer metastasis.

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.

Antimetastatic effect of suicide gene therapy for mouse mammary cancers requires T-cell-mediated immune responses.

These experiments were conducted to investigate whether the antimetastatic effects of HSVtk/GCV therapy involve T-cell-mediated immune responses. In the first experiment, immunocompetent syngeneic mice were inoculated with metastatic mammary cancers, then given a direct intratumoral injection of a plasmid vector containing a suicide gene (pHSVtk) or control vector once a week for 8 weeks. Gene electrotransfer treatment was applied to the tumors, and mice were administered ganciclovir (GCV) using a mini-osmotic pump. At the end of the experiment, tumor volume was significantly lower in the pHSVtk/GCV group. Macrophage accumulations were frequently observed in the peripheries of the necrotic regions in pHSVtk-transfected mice. Levels of CD4 and CD8 proteins in tumors were higher in the pHSVtk/GCV group than in the control group. Interleukin (IL)-12 mRNA levels tended to be higher in tumors in the pHSVtk/GCV group, but there were large variations. Tumor microvessel density was significantly lower in the pHSVtk/GCV group. The numbers of dilated lymphatic vessels containing intraluminal tumor cells tended to be higher in the pHSVtk/GCV group. However, vascular endothelial growth factor (VEGF)-A and VEGF-C mRNA levels in tumors were similar in the control and pHSVtk/GCV groups. In the second experiment, tumor volume and metastatic parameters were compared for immunocompetent syngeneic mice and immunodeficient athymic mice (without an intact T-cell system) given pHSVtk/GCV therapy. Although tumor volumes were significantly smaller in both syngeneic and athymic mice given pHSVtk/GCV therapy, the inhibition ratios (relative to control mice) were much greater in syngeneic mice than in athymic mice. No suppression of metastasis to the lymph nodes and lungs was observed for athymic mice given pHSVtk/GCV therapy. Our data suggest that HSVtk/GCV suicide gene therapy exerts an antimetastatic effect via a T-cell-mediated immune response.

Easy stable transfection of a human cancer cell line by electrogene transfer with an Epstein-Barr virus-based plasmid vector.

We report an easy and stable transfection technique using electrogene transfer with a nonviral Epstein-Barr (EB) virus-based vector. To achieve stable transfection of human breast cancer cells, we conducted electrogene transfer of an EB virus-based plasmid vector (reduced size of oriP) containing the enhanced green fluorescence protein (eGFP) gene. Because the EB virus-based vector exhibits high transfer efficiency and strong persistent transgene expression as a result of autonomous replication in human cells, and as Nucleofector electrogene transfer can achieve highly efficient gene transfection, this method is particularly suitable for generation of stably transfected cell lines.

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.

Expression of Vicia villosa agglutinin (VVA)-binding glycoprotein in primary breast cancer cells in relation to lymphatic metastasis: is atypical MUC1 bearing Tn antigen a receptor of VVA?

Aberrant carbohydrate expression frequently occurs in breast cancer and may endow cells with metastatic potential. Here we first studied the relationship between expression of Vicia villosa agglutinin (lectin) (VVA)-binding carbohydrates and aggressive breast cancer. We then investigated the molecular characteristics of these glycoproteins and compared them with those of glycoproteins recognized by the mouse anti-Tn monoclonal antibody (MAb) HB-Tn1. Histochemical studies of samples from 322 cases of invasive ductal carcinoma demonstrated that VVA-binding carbohydrate expression correlated with tumor stage, lymphatic invasion, and lymph node metastasis (p=0.0385, p=0.0019, and p=0.0430. respectively). Western blotting analysis of frozen materials from 39 cases, under denaturing and reducing conditions, revealed that the major cancer cell-specific VVA-binding proteins were molecules of about 30, 33, and >200 kDa. Cases expressing the approximately 33 kDa molecule had significant lymphatic invasion more frequently than did cases not expressing this molecule (p=0.0076). Binding of VVA to the approximately 30 and approximately 33 kDa molecules was completely lost by preincubation of VVA with 1 mM Tn antigen (N-acetylgalactosamine alpha1-O-serine). The VVA-binding molecules appeared to react with VU-3C6 anti-MUC1 MAb. Expression of HB-Tn1 in breast cancer cells showed significant correlation with expression of VVA-binding carbohydrate(s) (p<0.0001) but HB-Tn1 reactivity was not clearly related to breast cancer aggressiveness. Because anti-Tn MAbs bound to Tn antigen clusters, we concluded that atypical MUC1 bearing the noncluster form of Tn antigen is implicated in aggressive growth of primary breast cancer cells, particularly in lymphatic metastasis.

COX-2 inhibitor celecoxib suppresses tumor growth and lung metastasis of a murine mammary cancer.

BACKGROUND: The antitumor growth and antimetastatic actions of celecoxib [a selective cyclooxygenase-2 (COX-2) inhibitor] were investigated in a metastatic murine mammary cancer model. MATERIALS AND METHODS: Mice bearing mammary tumors, developed after inoculation of syngeneic BALBIc mice with a mammary carcinoma cell line carrying a p53 mutation, were treated with celecoxib at 0, 7.5 and 15 mg/kg five times a week for seven weeks. RESULTS: Tumor volumes were significantly reduced in association with an increase in apoptosis and a decrease in DNA synthesis in tumor tissues. In vitro studies demonstrated a significant increase in the number of cells undergoing apoptosis, with significantly elevated activities of caspase-3 and caspase-9, but not caspase-8, and a dose-dependent decrease in mitochondrial membrane potential, indicating the mitochondrial pathway of apoptosis. In addition, treatment with celecoxib showed cell cycle arrest in the G -phase and decreased cell population in the S- and G2/M-phases. Furthermore, tumor microvessel formation and mRNA levels for VEGF-A and COX-2 were markedly decreased. CONCLUSION: Celecoxib may be useful as an adjuvant therapy for breast cancer containing p53 mutations due to its ability to both induce p53-independent mitochondria-mediated apoptosis and exert anti-angiogenic potential.

In vivo electrogene transfer of interleukin-12 inhibits tumor growth and lymph node and lung metastases in mouse mammary carcinomas.

BACKGROUND: Human breast cancer metastasizes mainly to lymph nodes, lungs, liver, and bone; in the majority of cases, it is the development of metastases which leads to death. In order to suppress mammary cancer metastasis, we applied in vivo electrogene transfer (non-viral method) as a means of interleukin-12 (IL-12) gene therapy on highly metastatic murine mammary cancer model. METHODS: Metastatic mammary tumors induced by inoculation in BALB/c female mice were treated by intratumoral injections of either a plasmid vector containing IL-12 or empty vector and then subjected to in vivo electrogene transfer once a week for 8 weeks. RESULTS: Treatment with IL-12 resulted in elevation of both IL-12 and IFNgamma levels in mammary tumors and in serum and intratumoral levels of CD4 and CD8 proteins were also increased. Tumor volumes and lymphatic and pulmonary metastases were significantly reduced. The histopathological changes induced by IL-12 characteristically included marked inflammation, increased apoptosis, decreased DNA synthesis, peripheral influx of significantly greater numbers of active macrophages, and reduced blood microvessel density, and apoptotic vascular endothelial cells were frequently seen. Western blotting showed decreases in VEGFR-3 of tumors exposed to IL-12 gene therapy. In adjuvant immunofluorescence studies, the CD31-positive endothelial cells of microvessels showed decreased VEGFR-3 expression in IL-12-treated tumors. However, apparent alterations in VEGFR-3 expression of podoplanin-positive lymphatic endothelial cells were not observed in IL-12-treated tumors. Although recombinant IL-12 did not inhibit tubular formation of human umbilical vein endothelial cells in a Matrigel assay, recombinant IFNgamma did completely suppress the tubular formation. CONCLUSIONS: In vivo electrogene transfer of IL-12 exerts strong anti-tumorigenic and anti-metastatic effects likely due to T-cell-mediated immune responses as well as anti-angiogenic action.

Lack of polymorphism in MUC1 tandem repeats in cancer cells is related to breast cancer progression in Japanese women.

MUC1 is a transmembrane molecule characterized by a repeated sequence of 20 amino acid (TAP PAHGVTSAPDTRPAPGS). Abnormal overexpression of MUC1 in cancer cells is thought to contribute to their aggressive growth, but molecular mechanisms associated with this effect are still unclear. Our current study aimed to clarify whether MUC1 expression as recognized by VU-3C6 anti-MUC1 mouse IgG monoclonal antibody (MAb) with a dominant epitope of 12 amino acids: GVTSAPDTRPAP, correlated with aggressive properties of human breast cancer. Immunohistochemical studies of 309 samples of formalin-fixed and paraffin-embedded materials showed no statistical correlation between MUC1 expression and clinicopathological parameters, as well as several breast cancer aggressiveness-related markers. Expression or nonexpression of MUC1 in 50 frozen samples, as determined by Western blotting, demonstrated no correlation with aggressive properties of breast cancer. However, samples with one MUC1-positive band more often had lymphatic vessel invasion and lymph node metastasis than those with more than two or three MUC1-positive bands (p<0.014 and p<0.043, respectively). Because VU-3C6 MAb recognizes MUC1 with short branches of O-glycosylated core carbohydrates, we used immunohistological methods to examine Tn antigen (precursor antigen: GalNAcalpha-O-Ser/Thr), Thomsen-Friedenreich (T) antigen, and sialyl-Tn antigen (STn) antigen. We found a strong correlation between expression of MUC1 and Tn antigen (p<0.0006), and samples with Tn antigen expression had more lymphatic metastasis than those with no such expression (p<0.08). We concluded that the lack of polymorphic MUC1 expression with Tn antigen is one characteristic related to aggressive breast cancer.

Electrogene transfer of an Epstein-Barr virus-based plasmid replicon vector containing the diphtheria toxin A gene suppresses mammary carcinoma growth in SCID mice.

Experimental mammary cancer therapy in mice was conducted using electrogene transfer of a non-viral EBV-based plasmid vector (reduced size of the oriP gene), containing the DT-A gene. Because the EBV-based plasmid vector exhibits high transfer efficiency and strong persistent transgene expression due to autonomous replication in human cells, it is particularly suitable as a tool for cancer gene therapy. In vitro, 79% of MDA-MB231 human mammary carcinoma cells died as a result of the EBV-based vector containing DT-A (pEB-DTA) by 48 h after transfection. DNA synthesis was also significantly decreased as compared to levels with a control vector. In vivo, mammary tumors induced by inoculation of SCID mice with MDA-MB231 cells were subsequently treated by direct injection of pEB-DTA vector or pEB-GFP vector as a control once a week for 5 weeks. After each injection, the tumors were subjected to in vivo electrogene transfer. Significantly reduced tumor volumes were observed for the pEB-DTA group in experimental week 1 and thereafter throughout the study. At necropsy, strong and extent expression of GFP was still observed in tumors receiving pEB-GFP 6 days after the last electrogene transfer. The ratio of histological necrotic area to viable area was significantly increased in the pEB-DTA-treated tumors, where levels of apoptosis were significantly higher than those observed in the pEB-GFP group. DNA synthesis showed a tendency to decrease in the pEB-DTA group but this was not significant. The incidence and multiplicity of lung metastasis were similar between the groups. There was also no difference in the density of microvessels between groups. We therefore conclude that the EBV-based plasmid vector system combined with in vivo electrogene transfer can result in efficient gene transfection and that the non-viral replicon vector containing DT-A suppresses tumor growth due to apoptotic cell death in this model.

Roles of CXC chemokines and macrophages in the recruitment of inflammatory cells and tumor rejection induced by Fas/Apo-1 (CD95) ligand-expressing tumor.

The role of CD95 ligand (FasL/Apo-1L)-expressing tumors in immunosuppression or immunopotentiation is controversial. CD95L-transfected tumors induce immunopotentiation after vigorous neutrophil infiltration. Thus, the induction of neutrophil infiltration by CD95L seems to play an important role in tumor rejection. The mechanism by which CD95L-expressing tumors cause neutrophil infiltration and antitumor immunity has not been well understood. CXC chemokine receptor 2 (CXCR2) knockout (KO) mice are a powerful tool for studying CXC chemokine-mediated neutrophil infiltration. We investigated the roles of CD95L and chemokines in CD95L-induced antitumor activity by using CXCR2 KO mice and CD95LcDNA-transfected MethA (MethA + CD95L) fibrosarcoma. MethA + CD95L cells were completely rejected in wild-type (WT) and even in KO mice. MethA + CD95L cells injected intraperitoneally (i.p.) induced the recruitment of both neutrophils and macrophages in WT but only macrophages in KO mice, although CXC and CC chemokines were released in both mice. Macrophages incubated with MethA + CD95L cells released CXC and CC chemokines. Macrophages derived from WT and KO but not neutrophils from WT mice induced the recruitment of neutrophils when adoptively i.p. transferred with MethA + CD95L cells into CD95L/CD95-deficient mice. The different recruitment of inflammatory cells between WT and KO mice was attributed to bone marrow (BM) cells by BM transfer experiment. Our results demonstrated that CXC chemokines are essential for neutrophil recruitment and that macrophages but not neutrophils play a critical role in the CD95L-induced infiltration of inflammatory cells and the eradication of CD95L-expressing tumor cells.

Experimental gene therapy in mammary and urinary bladder cancer using electrogene transfer.

We investigated the effectiveness of in vivo electrogene transfer as a means of therapy in rat urinary bladder carcinoma and in mammary carcinoma models in both athymic and syngeneic mice using the herpes simplex virus 1 thymidine kinase (HSVtk) or IL-12 genes in combination with ganciclovir (GCV). A significant increase in the levels of tissue apoptosis and necrosis was induced with a single injection of HSVtk vector directly into bladder and mammary tumors followed by in vivo transfection and a regimen of intraperitoneal GCV injection. This procedure induced significant selective tumor cell death, characterized by marked inflammation and peripheral macrophage influx. Active caspase-3 was also strongly expressed in areas of cell death, indicating the initiation of apoptosis. This result was confirmed in corollary in vitro studies on a mouse bladder carcinoma cell line in which elevated caspase-3, -8, and -9 activities and decreased mitochondrial membrane potential were observed as a result of transfection with HSVtk and addition of GCV to the medium. In the syngeneic mouse mammary cancer model, we additionally found both tumor volume and metastasis to lymph nodes and lungs to be significantly reduced throughout the 2-month experiment. However, in contrast to their syngeneic counterparts, HSVtk/GCV therapy did not effectively inhibit mammary tumor growth/metastasis in an athymic mouse model, leading us to believe that T-cell-mediated immune responses may participate via the bystander effect in HSVtk/GCV experimental therapy. We subsequently evaluated the antitumor activity of IL-12, which can activate T-cell-mediated immune responses involving macrophages, in the syngeneic mammary tumors and found that IL-12 also significantly suppressed mammary tumor growth and metastasis. We thus suggest that in vivo electrogene transfer is a useful transfection tool in cancer gene therapy and, in addition, we show that T-cell-mediated immune responses may be a critical factor in cancer gene therapy using HSVtk/GCV and IL-12.