Edmund Chinchar

EGCG, a major green tea catechin suppresses breast tumor angiogenesis and growth via inhibiting the activation of HIF-1α and NFκB, and VEGF expression

The role of EGCG, a major green tea catechin in breast cancer therapy is poorly understood. The present study tests the hypothesis that EGCG can inhibit the activation of HIF-1α and NFκB, and VEGF expression, thereby suppressing tumor angiogenesis and breast cancer progression. Sixteen eight-wk-old female mice (C57BL/6 J) were inoculated with 10^6 E0771 (mouse breast cancer) cells in the left fourth mammary gland fat pad. Eight mice received EGCG at 50–100 mg/kg/d in drinking water for 4 weeks. 8 control mice received drinking water only. Tumor size was monitored using dial calipers. At the end of the experiment, blood samples, tumors, heart and limb muscles were collected for measuring VEGF expression using ELISA and capillary density (CD) using CD31 immunohistochemistry. EGCG treatment significantly reduced tumor weight over the control (0.37 ± 0.15 vs. 1.16 ± 0.30 g; P < 0.01), tumor CD (109 ± 20 vs. 156 ± 12 capillary #/mm^2; P < 0.01), tumor VEGF expression (45.72 ± 1.4 vs. 59.03 ± 3.8 pg/mg; P < 0.01), respectively. But, it has no effects on the body weight, heart weight, angiogenesis and VEGF expression in the heart and skeletal muscle of mice. EGCG at 50 μg/ml significantly inhibited the activation of HIF-1α and NFκB as well as VEGF expression in cultured E0771 cells, compared to the control, respectively. These findings support the hypothesis that EGCG, a major green tea catechin, directly targets both tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of breast cancer, which is mediated by the inhibition of HIF-1α and NFκB activation as well as VEGF expression.

Increased plasma levels of soluble vascular endothelial growth factor receptor 1 (sFlt-1) in women by moderate exercise and increased plasma levels of vascular endothelial growth factor in overweight/obese women.

The incidence of breast cancer is increasing worldwide, and this seems to be related to an increase in lifestyle risk factors, including physical inactivity and overweight/obesity. We have reported previously that exercise induced a circulating angiostatic phenotype characterized by increased soluble fms-like tyrosine kinase-1 (sFlt-1) and endostatin and decreased unbound vascular endothelial growth factor (VEGF) in men. However, there are no data on women. The present study determines the following: (a) whether moderate exercise increased sFlt-1 and endostatin and decreased unbound VEGF in the circulation of adult female volunteers and (b) whether overweight/obese women have a higher plasma level of unbound VEGF than lean women. A total of 72 African American and White adult women volunteers ranging in age from 18 to 44 years were enrolled in the exercise study. All the participants walked on a treadmill for 30 min at a moderate intensity (55–59% heart rate reserve), and oxygen consumption (VO2) was quantified utilizing a metabolic cart. We obtained blood samples before and immediately after exercise from 63 participants. ELISA assays showed that the plasma levels of sFlt-1 were 67.8±3.7 pg/ml immediately after exercise (30 min), significantly higher than the basal levels, 54.5±3.3 pg/ml, before exercise (P<0.01; n=63). There was no significant difference in the % increase in the sFlt-1 levels after exercise between African American and White (P=0.533) women or between lean and overweight/obese women (P=0.892). There was no significant difference in the plasma levels of unbound VEGF (35.28±5.47 vs. 35.23±4.96 pg/ml; P=0.99) or endostatin (111.12±5.48 vs. 115.45±7.15 ng/ml; P=0.63) before and after exercise. The basal plasma levels of unbound VEGF in overweight/obese women were 52.26±9.6 pg/ml, significantly higher than the basal levels of unbound VEGF in lean women, 27.34±4.99 pg/ml (P<0.05). The results support our hypothesis that exercise-induced plasma levels of sFlt-1 could be an important clinical biomarker to explore the mechanisms of exercise training in reducing the progression of breast cancer and that VEGF is an important biomarker in obesity and obesity-related cancer progression.

EGCG Suppresses Melanoma Tumor Angiogenesis and Growth without Affecting Angiogenesis and VEGF Expression in the Heart and Skeletal Muscles in Mice

Melanoma is a highly malignant cancer with a potent capacity to metastasize distantly and has a higher mortality. There is no effective therapy for high risk melanoma patients to prevent relapse or distant metastasis. Therefore effective chemoprevention strategies are needed. The present study mainly evaluates the effects of EGCG on melanoma angiogenesis, growth, and capillary density (CD) in the heart and skeletal muscles of mice. 5 x 10^5 B16F10 cells were inoculated into the right proximal dorsal of the back in the eight week old male mice (n=12). Then, 6 mice received EGCG at 50-100 mg/kg/d in drinking water for 4 weeks and 6 control mice received drinking water only. Tumor size was monitored using dial calipers. At the end of the experiment, blood samples, tumors, hearts, and limb muscles were collected and measured for VEGF expression using ELISA and capillary density (CD) using CD31 immunohistochemistry. Compared to the control, EGCG treatment significantly reduced tumor weight (2.9±0.5 vs. 5.9±1.1 g; P<0.01; n=6), melanoma CD (117±9 vs. 167±23; P<0.01), and melanoma VEGF expression (32±1.5 vs. 42±2 pg/mg; P < 0.01), respectively. Also EGCG had no effects on body weight, heart weight, angiogenesis or VEGF expression in the heart and skeletal muscle of mice. EGCG (20-50 � g/ml) significantly inhibited the proliferation, migration, VEGF expression, and the activation of HIF-1� and NFB in cultured B16F10 cells, respectively. These findings support the hypothesis that EGCG, a major green tea polyphenol, directly targets tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of melanoma, and that the down- regulation of VEGF expression by EGCG is associated with the inhibition of HIF-1� and NFB activation. EGCG has great potential as a chemopreventive agent because it has no effect on angiogenesis in normal tissue and has low toxicity.

Abstract 2093: Sunitinib suppresses the proliferation, migration, apoptosis resistance, tumor angiogenesis and growth in MDA-MB-468 cell cultures and xenografts .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC We previously reported the anti-tumor efficacy of sunitinib (VEGF receptor tyrosine kinase inhibitor) in murine ER-positive breast cancer xenograft model, in which sunitinib suppressed both autocrine and paracrine effects in breast cancer progression. The investigations of sunitinib on triple-negative breast cancer are very limited. The clinical utility of sunitinib is less understood in the setting of breast cancer. The present study determines: 1) whether VEGF is highly expressed in MDA-MB-468 cells, compared to MCF-7 and MDA-MB-231 cells; 2) whether sunitinib inhibits the proliferation, migration, apoptosis resistance of cultured MDA-MB-468 cells; 3) whether oral sunitinib treatment suppresses tumor angiogenesis and growth in MDA-MB-468 xenografts; and 4) whether sunitinib changes % of breast cancer stem cells in the xenografts. MDA-MB-231, MDA-MB-468, MCF-7 cells were cultured using RPMI 1640 media with 10% FBS. VEGF protein levels were detected using ELISA (R&D Systams). MDA-MB-468 cells were exposed to sunitinib for 18 hours for measuring proliferation (3H-thymidine incorporation), migration (BD BioCoat Matrigel Invasion Chamber), and apoptosis (Millipore ApopTag). 10ˆ6 MDA-MB-468 cells inoculated into the left fourth mammary gland fat pad in athymic nude-foxn1 mice. When the tumor volume reached 100 mmˆ3, sunitinib was given by gavage at 80 mg/kg/2 days for 4 weeks. Tumor angiogenesis was determined by CD31 immunohistochemistry. Breast cancer stem cells were determined by flow cytometry analysis using CD44+/CD24- or low. VEGF protein levels in MDA-MB-468 cells were 10257±292 pg/ml that are 3-folds higher than those (3428±74 pg/ml) in MDA-MB-231 cells and 31-folds higher than those (335±4 pg/ml) in MCF-7 cells (P<0.01, n=6). 1, 5 and 10 μmol/L of sunitinib caused 24, 41 and 59% reduction in the proliferation of MDA-MB-468 cells, respectively, compared to the control (P<0.01, n=6). Sunitinib at 1 μmol/L significantly inhibited the migration of MDA-MB-468 cells by 45% compared to the control (P<0.01, n=6). Sunitinib (5 μmol/L) increased 31% apoptosis of MDA-MB-468 cells (P<0.01). Oral sunitinib treatment significantly inhibited tumor angiogenesis and tumor growth, and slightly increased % of breast cancer stem cells in the xenografts. These findings suggest that VEGF is highly expressed in triple negative breast cancer (TNBC) cells especially in MDA-MB-468 cells. VEGF receptor tyrosine kinase inhibitor (sunitinib) can inhibit autocrine effects (proliferation, migration, and apoptosis resistance) and paracrine effects (tumor angiogenesis) in triple negative breast cancer progression. However, the possibility should be considered of sunitinib increasing breast cancer stem cells. Sunitinib or other anti-VEGF agents in TNBC therapy may have to be combined with cancer stem cell-targeting drugs. Citation Format: Edmund Chinchar, Kristina L. Makey, John Gibson, Lucio Miele, Jian-Wei Gu. Sunitinib suppresses the proliferation, migration, apoptosis resistance, tumor angiogenesis and growth in MDA-MB-468 cell cultures and xenografts . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2093. doi:10.1158/1538-7445.AM2013-2093

Abstract P4-06-04: Gamma-secretase inhibitors suppress the activation of NFκB and the expression of TNFα, IL-6 and IL-8 in triple negative breast cancer cells

Background: Inflammation mediators, such as NF-kappa B (NFκB) and tumor necrosis factor alpha (TNFα), play major role in breast cancer pathogenesis, progression, and relapse. Triple negative breast cancer (TNBC) is a heterogeneous group of aggressive breast cancers and often has a poor outcome, in which Notch pathways are highly activated. However, role of crosstalk between Notch and inflammation mediators in TNBC progression and recurrence is poorly understood. The present study determines: 1) whether NFκB is highly activated in TNBC cells such as MDA-MB-231 (claudin-low-like) and MDA-MB-468 (basal-like), compared to ER-positive cells (MCF-7); 2) whether TNFα, IL-6 and IL-8 are highly expressed in TNBC cells, compared to MCF-7 cells; 3) whether Notch inhibition by gamma-secretase inhibitors (GSIs) significantly decrease NFκB activation and the expression of TNFα, IL-6 and IL-8 in TNBC cells; 4) whether GSI inhibits TNBC cell migration. Material and Methods: MDA-MB-231, MDA-MB-468, MCF-7 cells were cultured using RPMI 1640 media with 10% FBS. The cells were exposed to GSIs such as DAPT and RO4929097 for 18 hours. Nuclear NFκB activation was determined by using the TransAM NFκB p65 kit (Active Motif). The expressions of TNFα, IL-6, IL-8, and IFNγ were determined by the ELISA kits (RD and 2) Notch inhibition by GSIs significantly decrease NFκB activation and the expression of TNFα, and IL-8 in TNBC cells. These results support the hypothesis that the crosstalk between Notch and NFκB lead to activation of inflammation mediators such as TNFα and IL 8, which contribute to TNBC progression and recurrence. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-04.

P4-03-02: High Fat Diet-Induced Postmenopausal Obesity Promotes Tumor Angiogenesis and Breast Cancer Progression in Age-Relevant Ovariectomized Mice.

Background: Obese postmenopausal women have 50% higher risk of breast cancer than non-obese women. The mechanisms of postmenopausal obesity-induced breast cancer are poorly understood due to lack of the established animal model that mimics postmenopausal obesity related to breast cancer progression. Material and Methods: Using age-relevant C57BL/6 mice, this study determined whether postmenopausal obesity increases VEGF expression, tumor angiogenesis, and breast tumor growth. Ovariectomy (OVX) was performed in 12 sixty week-old female mice (life span is about 140 weeks), then followed by a low-fat (5%, LF, n=6) or a high-fat (60%, HF, n=6) diet for 12 weeks. In the 8th wk of the dietary program, 10^6 E0771 (mouse breast cancer) cells were injected in the left fourth mammary gland. Tumor size was monitored using dial calipers for 4 wk. Body weights were monitored weekly. At the end of the experiment, blood samples, visceral fat, and tumors were collected for measuring VEGF expression using ELISA and intratumoral microvessel density (IMD) using CD31 immunochemistry. Results: Body weight was significantly increased in OVX/HF mice, compared to OVX/LF group (55.3±1.7 vs. 41.5±1.5 g; P Discussion: Our observations indicate that ovariectomy plus a high fat diet with the inoculation of E0771 (mouse ER+ breast cancer) cells in female wild type >60 week old mice can mimic human obesity-induced postmenopausal breast cancer. The increased tumor angiogenesis in postmenopausal obese mice was correlated with increased breast tumor growth, adipose tissue mass, and adipokines such as VEGF. These findings support the hypothesis that postmenopausal obesity promotes tumor angiogenesis and breast cancer progression, possibly through increased adipose tissue mass and adipokines such as VEGF that could systemically and locally affect breast cancer progression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-03-02.

P3-17-07: EGCG, a Green Tea Antioxidant Suppresses Breast Tumor Angiogenesis and Growth Via Inhibiting the Activation of HIF-1a and NFκB, and VEGF Expression.

Background Cancer cells are under greater oxidative stress than normal cells. Limited studies have showed that epigallocatechin-3-gallate (EGCG), a green tea antioxidant can afford protection against a variety of cancer types. The role of EGCG in breast cancer therapy is poorly understood. The present study tests the hypothesis that EGCG as an antioxidant can inhibit the activation of HIF-1α and NFκB, and VEGF expression, thereby suppressing tumor angiogenesis and breast cancer progression. Material and Methods: 16 eight-wk-old female mice (C57BL/6J) were inoculated with 10^6 E0771 (mouse breast cancer) cells in the left fourth mammary gland fat pad. 8 mice received EGCG at 50–100 mg/kg/d in drinking water for 4 weeks. 8 control mice received drinking water only. Tumor size was monitored using dial calipers. At the end of the experiment, blood samples, tumors, heart and limb muscles were collected for measuring VEGF expression using ELISA and capillary density (CD) using CD31 immunochemistry. Cultured E0771 cells were used for determining the direct effects of EGCG on proliferation (3H-thymidine incorporation), migration (Matrigel assay), VEGF expression (ELISA), the activation of HIF-1α and NFκB (motif binding assays, Active Motif). MCF-7 and MDA-MB-231 cells were also used for 3H-thymidine incorporation. Results: Oral EGCG treatment significantly reduced tumor weight over the control (0.37±0.15 vs. 1.16±0.30 g; P Discussion: These findings support the hypothesis that EGCG, a green tea antioxidant, directly targets both tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of breast cancer, which is mediated by the inhibition of HIF-1α and NFκB activation as well as VEGF expression. Interestingly, oral EGCG treatment has no effects on the body weight, heart weight, angiogenesis and VEGF expression in the heart and skeletal muscle of mice. This work will have important implications for translating EGCG therapy to human breast cancer treatment and prevention. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-07.