Emily Young

Chronic alcohol consumption stimulates VEGF expression, tumor angiogenesis and progression of melanoma in mice.

The mechanisms of alcohol-induced cancer in humans are unclear. We used the immunocompetent mice implanted with B16F10 cells to evaluate the effects of physiologically relevant EtOH intake on tumor growth and angiogenesis of melanoma. 6-wk-old male mice (C57BL/6J) were given 1% EtOH in drinking water for 12-hrs during the night which was then replaced with regular water during the remaining 12-hrs each day for 4 wks (n=10). The control mice received regular drinking water only. In the 2nd wk, all mice were inoculated subcutaneously on the right proximal dorsal with ~5×105 B16F10 cells. In the end, the tumors were isolated for measuring tumor size, average microvascular density (AMVD) using CD31 immunohistochemistry, and the expression of VEGF and its receptor (Flt-1) using Northern blot, ELISA, and immunohistochemistry. EtOH intake caused a 2.16-fold increase in tumor weight over the control (4.81±0.39 vs. 2.23±0.48 g; n=10; P=0.003), a 2.02-fold increase in AMVD (60.63±5.56 vs. 30.01±7.41/mm2; P=0.0014), and a significant increase in VEGF mRNA and protein expression plus Flt-1 protein levels in melanoma compared to the control group (P

Postmenopausal obesity promotes tumor angiogenesis and breast cancer progression in mice

Obese postmenopausal women have a 50% higher risk of breast cancer than non-obese women. There is not an animal model that mimics postmenopausal obesity related to breast cancer progression. 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, then followed by a low-fat (5%, LF, n=6) or a high-fat (60%, HF, n=6) diet for 12 weeks. In the eighth week of the dietary program, 10^6 E0771 (mouse breast cancer) cells were injected in the left fourth mammary gland. Tumor size was monitored for 4 weeks. 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. 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 < 0.01). There was a two-fold increase in the ratio of visceral fat/BW in OVX/HF mice, compared to those in OVX/LF group (0.062±0.005 vs. 0.032±0.003; p < 0.01). Postmenopausal obesity significantly increased breast tumor weight over the control (4.62±0.63 vs. 1.98±0.27 g; p < 0.01) and IMD (173±3.7 vs. 139±4.3 IM#/mm^2; p < 0.01). Tumor VEGF levels were higher in OVX/HF mice, compared to OVX/LF group (73.3±3.8 vs. 49.5±4.3 pg/mg protein; p < 0.01). Plasma VEGF levels (69±7.1 vs. 48±3.5 pg/ml) and visceral fat VEGF levels (424.4±39.5 vs. 208.5±22.4 pg/mg protein) were significantly increased in OVX/HF mice, compared to OVX/LF group, respectively (n=6; p < 0.01). Interestingly, adipose tissue primary culture showed that subcutaneous fat released more VEGF, compared to visceral fat (6.77±1.14 vs. 0.94±0.16 pg/mg tissue; n=6; p < 0.01). 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.

Vascular endothelial growth factor receptor inhibitor enhances dietary salt-induced hypertension in Sprague-Dawley rats

Clinical evidence links the inhibition of VEGF to hypertension. However, the mechanisms by which VEGF affects the pathogenesis of hypertension remain in question. We determined 1) whether administration of VEGF receptor inhibitor SU5416 enhances dietary salt-induced hypertension in Sprague-Dawley (SD) rats, and 2) whether VEGF or SU5416 directly affects proliferation of cultured human renal proximal tubular epithelial cells (HRPTEC) and endothelial nitric oxide synthase (eNOS) expression in cultured human glomerular microvessel endothelial cells (HGMEC). Ten 10-wk-old male SD rats received a high sodium diet (HS; 8%) and the other 10 SD rats received a normal sodium diet (NS; 0.5%) for 4 wks. After 2 wks of the dietary program, five rats were administered with SU5416 at 10 mg x kg(-1) x day(-1) ip or DMSO (vehicle) for 14 days in HS and NS groups. Mean arterial pressure was significantly higher in rats treated with SU5416, as opposed to those treated with DMSO and fed with HS for 4 wk (157.6 +/- 3.9 vs. 125.9 +/- 4.3 mmHg, P < 0.01). Increased proteinuria and albuminuria were associated with marked renal histological abnormalities in HS group with SU5416 administration, compared with those in the vehicle HS group. 3H-thymidine incorporation assay showed that SU5416 blocked the actions of both exogenous and endogenous VEGF on the proliferation of HRPTEC. VEGF (10 ng/ml) significantly increased eNOS protein levels by 29% in cultured HGMEC, but its action was completely abolished by SU5416. These results suggest that VEGF receptor inhibition enhances dietary salt-induced hypertension and kidney injury, possibly by direct damage on renal cells and decreasing NO production by eNOS.

Long-term high-salt diet causes hypertension and decreases renal expression of vascular endothelial growth factor in Sprague-Dawley rats

We seek to determine: 1) whether a long-term high salt diet induces hypertension and renal injury in Sprague-Dawley (SD) rats and 2) whether the high salt diet-induced hypertension and renal injury are associated with decreased renal VEGF expression. Twelve 10-wk-old male SD rats received a high salt diet (HS, 8%) and twelve SD rats received a normal salt diet (NS, 0.5 %) for 8 weeks. Using a tail cuff, weekly monitoring showed that blood pressure increased significantly after 6, 7, & 8 wks in HS group, compared to NS group (P<0.01). At 4 wks and 8 wks of diet, mean arterial pressure (MAP) was determined in conscious rats by continuous monitoring through a catheter placed in the carotid artery. MAP was not significantly different between HS and NS group in 4 wks, but was significantly higher in HS than NS group (140+/-5.3 vs.112+/-2.2 mmHg; P<0.01) in 8 wks. Increased proteinuria and albuminuria were associated with marked renal histological abnormalities in HS group, compared to those in NS group. Northern blot and ELISA demonstrated that 8 wks of HS diet significantly decreased renal expression of VEGF mRNA and protein, compared to NS group (P<0.01). In 8 wks, total urinary excretion of sFlt-1 was significantly higher in HS than NS group (9.28+/-1.05 vs. 2.05+/-0.55 ng/day; P<0.01) whereas the plasma levels of sFlt-1 remained stable. These results suggest that a long-term HS diet induces renal injury and hypertension, which are associated with decreased renal VEGF expression in normotensive rodent animals.

SU11248, A selective tyrosine kinases inhibitor suppresses breast tumor angiogenesis and growth via targeting both tumor vasculature and breast cancer cells

SU11248 is a selective inhibitor of certain protein kinases including VEGFR types 1-3 that are expressed in human breast cancer. The present study determines whether the anti-tumor activity of SU11248 results from the inhibition of angiogenesis, as well as direct anti-proliferation and anti-migration effects on breast tumors. Eight-wk old female mice (C57BL/6) were given SU11248 at 20-40 mg/kg/d in drinking (distilled) water for 4 wks. Control mice received drinking water only. In the 2nd wk, 10^6 E0771 (mouse breast cancer) cells were injected in the left fourth mammary gland. Tumor size was monitored using dial calipers. At the end, tumors were isolated for measuring tumor size and intratumoral microvessel density (IMD) using CD31 immunohistochemistry. SU11248 significantly reduced tumor weight over the control (1.220.28 vs. 3.280.31 g; n=8; P<0.01) and IMD (11110 vs. 1556 IM#/mm^2; P<0.01). RT-PCR indicated that VEGFR1 and R2 were expressed in cultured E0771 cells. VEGF (10 ng/ml) caused a 42% increase in proliferation of E0771 cells, compared to the control (P<0.01; n = 8), and there was a significant decrease in proliferation of E0771 cells treated with VEGF plus SU11248 (10 μmol/L) vs. the control (65%, P<0.01). VEGF caused a 2-fold increase in the proliferation of HUVEC vs. the control (P<0.01; n = 8), but its action was completely eradicated by SU11248. Neither VEGF nor SU11248 had any effect on the proliferation of cultured HASMC. Migration assay showed that SU11248 (10 μmol/L) significantly inhibited the migration of cultured E0771 cells. SU11248 significantly inhibited the proliferation of MCF-7 and MDA-MB-231 cells in a dose-related manner. These findings support the hypothesis that the anti-tumor activity of SU11248 on breast cancer is possibly mediated by targeting the paracrine and autocrine effects of VEGF on breast cancer to suppress tumor angiogenesis, proliferation, and migration.

Oral administration of Pyrrolidine Dithiocarbamate (PDTC) inhibits VEGF expression, tumor angiogenesis, and growth of breast cancer in female mice

The progression of breast cancer is associated with oxidative stress. However, the effects of pyrrolidine dithiocarbamate (PDTC), a known antioxidant, on the development of breast cancer are poorly understood. The present study evaluates the effects of PDTC on tumor growth, the expression of vascular endothelial growth factor (VEGF), and angiogenesis of breast cancer in female mice. Eight week old female mice (C57BL/6J) were given PDTC at 100 to 200 mg/kg/day for 3 weeks (n=10). The control mice received regular drinking water only. In the 2nd wk, 5x10^5 E0771 (mouse breast cancer) cells were injected in the pad of the fourth mammary gland of the mice. Tumor size was monitored using dial calipers. At the end of the experiment, the tumors were isolated and measured for tumor size, intratumoral microvessel (IM) density using CD31 immunohistochemistry staining, NFκB activation using EMSA, and VEGF protein levels using ELISA. PDTC treatment caused a significant decrease in tumor weight compared to the control (0.64±0.22 vs. 1.43±0.31 g; n=8; P