Toxicodendron vernicifluum Stokes extract inhibits solid tumor growth and lung metastasis of 4T1 murine mammary carcinoma cells in BALB/c mice

Abstract

Toxicodendron vernicifluum Stokes has long been used as a food supplement and traditional herbal medicine in East Asia. We applied a new extraction method to produce Toxicodendron vernicifluum Stokes extract (TVSE), that doesn't contain urushiol (an allergenic toxin) but dose have higher levels of some flavonoids such as fustin and fisetin. This study was conducted to investigate the anticancer effects of TVSE in an in vivo system. Fifty BALB/c mice were acclimated for one week and then injected with 4T1 murine mammary carcinoma cells in mammary fat pads. After 7 days, the mice were randomly divided into 5 groups, and orally administered with 0, 50, 100, 200 or 400 mg of TVSE/kg body weight (BW)/day for 20 days. TVSE reduced tumor volume and weight dose-dependently. The expression of Ki67 was significantly reduced and the number of TUNEL-positive apoptotic cells was significantly increased in the TVSE-treated group over 100 mg/kg BW/day. While tumor nodules were not found in the liver, but only in lungs, the number of tumor nodules was reduced in a dose-dependent manner in the TVSE treated groups compared to the control group. In breast tumors, expression of platelet endothelial cell adhesion molecule (PECAM-1) and vascular endothelial growth factor (VEGF) was reduced by TVSE treatment. TVSE treatment significantly suppressed mRNA expression in tumors of matrix metalloproteinase (MMP)-2, tissue inhibitor of metalloproteinase (TIMP)-1, urokinase-type plasminogen activator (uPA), intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 while increasing plasminogen activator inhibitor (PAI)-1. These results suggest that TVSE is potentially beneficial for the suppression of breast cancer growth and its-associated lung metastasis.

Fig 1. Oral administration of TVSE inhibits tumor growth in BALB/c mice injected with 4T1 mammary cancer cells.

4T1 cells (1 × 10⁵ cells/ mice) were injected into the mammary fat pads of six-week-old female BALB/c mice. After the tumor size reached 50 mm³ (7 days after 4T1 cell injection), TVSE was administered by oral gavage for 20 days. (A) The tumor volume was measured using calipers and calculated using the formula (0.52 × long diameter × short diameter²), n = 10. (B) At the end of the administration period, all mice were sacrificed. The tumors were excised and photographed. (C) Tumors weights. Each bar represents the mean ± SEM (n = 10). Means without a common letter differ, P < 0.05. CON, 0 mg of TVSE/kg body weight (BW)/day; T50, 50 mg of TVSE/ kg BW/day; T100, 100 mg of TVSE/ kg BW/day; T200, 200 mg of TVSE/ kg BW/day; T400, 400 mg of TVSE/ kg BW/day.

Fig 2. Oral administration of TVSE inhibits the lung metastasis of 4T1 cells in BALB/c mice.

Mice were injected with 4T1 cells and administered with TVSE as described. After 20 days administration, the lungs were excised and fixed in Bouin’s solution. (A) Lung photographs. (B) Metastatic tumor nodules in the lungs were counted. Each bar represents the mean ± SEM (n = 10). Means without a common letter differ, P < 0.05. CON, 0 mg of TVSE/kg body weight (BW)/day; T50, 50 mg of TVSE/ kg BW/day; T100, 100 mg of TVSE/ kg BW/day; T200, 200 mg of TVSE/ kg BW/day; T400, 400 mg of TVSE/ kg BW/day.

Fig 3. Oral administration of TVSE suppresses cell proliferation and induces apoptosis in 4T1 tumors in BALB/c mice.

Mice were injected with 4T1 cells and administered with TVSE as described. (A) Tumor sections were stained with Ki67 antibody. Representative images of IF staining are shown. Scale bar, 100 μm (upper panel). The Ki67-positive cells were counted (lower panel). (B) Tumor sections were stained with TUNEL. Images of TUNEL staining are shown. Scale bar, 100 μm (upper panel). TUNEL-positive apoptotic cells were counted (lower panel). Each bar represents the mean ± SEM (n = 4). Means without a common letter differ significantly, P < 0.05. CON, 0 mg of TVSE/kg body weight (BW)/day; T50, 50 mg of TVSE/ kg BW/day; T100, 100 mg of TVSE/ kg BW/day; T200, 200 mg of TVSE/ kg BW/day; T400, 400 mg of TVSE/ kg BW/day.

Fig 4. Oral administration of TVSE decreases angiogenesis in 4T1 tumors in BALB/c mice.

Mice were injected with 4T1 cells and administered with TVSE as described. (A) Tumor sections were stained with PECAM-1 and VEGF antibodies. Representative images of IF staining are shown. Scale bar, 50 μm. (B) The staining intensity of the indicated protein was quantified. Each bar represents the mean ± SEM (n = 4). (C) Blood samples were collected from mice at sacrifice, and sera were prepared. Serum levels of VEGF were measured with a VEGF ELISA kit. Each bar represents the mean ± SEM (n = 10). Means without a common letter differ significantly, P < 0.05. CON, 0 mg of TVSE/kg body weight (BW)/day; T50, 50 mg of TVSE/ kg BW/day; T100, 100 mg of TVSE/ kg BW/day; T200, 200 mg of TVSE/ kg BW/day; T400, 400 mg of TVSE/ kg BW/day.

Fig 5. Oral administration of TVSE alters the mRNA expression of metastasis-related genes in 4T1 tumors in BALB/c mice.

Mice were injected with 4T1 cells and administered with TVSE as described. The total RNA in tumors was extracted, reverse transcribed, and real-time PCR was conducted. The amount of each mRNA was normalized to the amount of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA and the control levels were set at 100. Each bar represents the mean ± SEM (n = 10). Means without a common letter differ significantly, P < 0.05. CON, 0 mg of TVSE/kg body weight (BW)/day; T50, 50 mg of TVSE/ kg BW/day; T100, 100 mg of TVSE/ kg BW/day; T200, 200 mg of TVSE/ kg BW/day; T400, 400 mg of TVSE/ kg BW/day.