Hyerim Song

Inhibition of tumor progression by oral piceatannol in mouse 4T1 mammary cancer is associated with decreased angiogenesis and macrophage infiltration.

Piceatannol, a polyphenol which exhibits anticancer activities, is found in grapes, red wine and berries. It has been shown to inhibit several transcription factor pathways. The present study was conducted to determine whether oral administration of piceatannol inhibits mammary tumor progression. 4T1 mammary carcinoma cells were injected into the mammary fat pad of syngeneic female BALB/c mice. Starting 1 day later, piceatannol (10- or 20-mg/kg body weight/day) was administered by oral gavage for 30 days. Piceatannol treatment reduced tumor growth. In tumor tissues, piceatannol treatment reduced the expression of transcription factors P-NFκB p65, P-STAT3 and HIF-1α and multiple proteins involved in regulation of cell cycle progression (Ki67, cyclin D1, cyclin A, CDK2, CDK4), angiogenesis (VEGF-A, VEGFR-2, VE-cadherin, CD31) and lymphangiogenesis (VEGF-C, LYVE-1), as well as macrophage infiltration. Piceatannol significantly increased apoptotic cells and expression of both Bax and cleaved caspase-3 but reduced Bcl-2 expression in tumor tissues. In addition, piceatannol reduced the number and volume of pulmonary tumor nodules and expression of MMP-9 in both lung and tumor. It also reduced tissue levels of cytokines/chemokines, including M-CSF and MCP-1. In vitro results revealed that piceatannol inhibited migration of 4T1 cells and monocytes, as well as secretion of MCP-1 and M-CSF by 4T1 cells. 4T1 cell-conditioned medium stimulated monocyte migration, which was suppressed by a CCR2 antibody. These results indicate that alteration in tumor microenvironment (macrophages, transcription factors, etc.) is an important mechanism by which piceatannol inhibits tumor proliferation, angiogenesis and lymphangiogenesis, leading to suppression of mammary tumor growth and metastasis.

Carnosic Acid Inhibits the Epithelial-Mesenchymal Transition in B16F10 Melanoma Cells: A Possible Mechanism for the Inhibition of Cell Migration

Carnosic acid is a natural benzenediol abietane diterpene found in rosemary and exhibits anti-inflammatory, antioxidant, and anti-carcinogenic activities. In this study, we evaluated the effects of carnosic acid on the metastatic characteristics of B16F10 melanoma cells. When B16F10 cells were cultured in an in vitro Transwell system, carnosic acid inhibited cell migration in a dose-dependent manner. Carnosic acid suppressed the adhesion of B16F10 cells, as well as the secretion of matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1, urokinase plasminogen activator (uPA), and vascular cell adhesion molecule (VCAM)-1. Interestingly, secretion of TIMP-2 increased significantly in B16F10 cells treated with 10 μmol/L carnosic acid. Additionally, carnosic acid suppressed the mesenchymal markers snail, slug, vimentin, and N-cadherin and induced epithelial marker E-cadherin. Furthermore, carnosic acid suppressed phosphorylation of Src, FAK, and AKT. These results indicate that inhibition of the epithelial-mesenchymal transition may be important for the carnosic acid-induced inhibition of B16F10 cell migration.

Plasma marker proteins associated with the progression of lung cancer in obese mice fed a high‐fat diet

Recent studies have indicated that obesity increases the risk of developing several types of cancers including lung cancer, which is the leading cause of cancer death worldwide. In the present study, we attempted to discover marker proteins associated with lung cancer progression mediated by treatment of a high‐fat diet (HFD) using 2DE combined with MALDI‐TOF‐MS. Image analysis and further statistical analysis allowed for the detection and identification of 14 proteins, which consequently were classified into two groups based on their regulation patterns in response to diet and tumor. Interestingly, the protein abundances of ten proteins exhibited a synergistic effect when treated with HFD in tumor‐bearing mice (Group I). Proteins that had a higher abundance in the plasma of tumor‐bearing mice included FGB, Tf, Hpx, Cp, and Hp and the proteins that had a lower abundance included A1AT precursor, PON1, TTRt, and α2‐M. These proteins can be used as molecular markers that contribute simultaneously to both obesity and cancer. Four other proteins showed an increase (complement C3 and FGA) or decrease (Apo H and AT III precursor) in the only tumor‐bearing mice independently of diet (Group II). The marker proteins identified here may lead to the development of new therapeutics for obesity‐causative treatment of lung cancer.

A High-Fat Diet Containing Lard Accelerates Prostate Cancer Progression and Reduces Survival Rate in Mice: Possible Contribution of Adipose Tissue-Derived Cytokines

To examine the effects of high-fat diet (HFD) containing lard on prostate cancer development and progression and its underlying mechanisms, transgenic adenocarcinoma mouse prostate (TRAMP) and TRAMP-C2 allograft models, as well as in vitro culture models, were employed. In TRAMP mice, HFD feeding increased the incidence of poorly differentiated carcinoma and decreased that of prostatic intraepithelial neoplasia in the dorsolateral lobes of the prostate, which was accompanied by increased expression of proteins associated with proliferation and angiogenesis. HFD feeding also led to increased metastasis and decreased survival rate in TRAMP mice. In the allograft model, HFD increased solid tumor growth, the expression of proteins related to proliferation/angiogenesis, the number of lipid vacuoles in tumor tissues, and levels of several cytokines in serum and adipose tissue. In vitro results revealed that adipose tissue-conditioned media from HFD-fed mice stimulated the proliferation and migration of prostate cancer cells and angiogenesis compared to those from control-diet-fed mice. These results indicate that the increase of adipose tissue-derived soluble factors by HFD feeding plays a role in the growth and metastasis of prostate cancer via endocrine and paracrine mechanisms. These results provide evidence that a HFD containing lard increases prostate cancer development and progression, thereby reducing the survival rate.

Benzyl Isothiocyanate Inhibits Prostate Cancer Development in the Transgenic Adenocarcinoma Mouse Prostate (TRAMP) Model, Which Is Associated with the Induction of Cell Cycle G1 Arrest

Benzyl isothiocyanate (BITC) is a hydrolysis product of glucotropaeolin, a compound found in cruciferous vegetables, and has been shown to have anti-tumor properties. In the present study, we investigated whether BITC inhibits the development of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) mice. Five-week old, male TRAMP mice and their nontransgenic littermates were gavage-fed with 0, 5, or 10 mg/kg of BITC every day for 19 weeks. The weight of the genitourinary tract increased markedly in TRAMP mice and this increase was suppressed significantly by BITC feeding. H and E staining of the dorsolateral lobes of the prostate demonstrated that well-differentiated carcinoma (WDC) was a predominant feature in the TRAMP mice. The number of lobes with WDC was reduced by BITC feeding while that of lobes with prostatic intraepithelial neoplasia was increased. BITC feeding reduced the number of cells expressing Ki67 (a proliferation marker), cyclin A, cyclin D1, and cyclin-dependent kinase (CDK)2 in the prostatic tissue. In vitro cell culture results revealed that BITC decreased DNA synthesis, as well as CDK2 and CDK4 activity in TRAMP-C2 mouse prostate cancer cells. These results indicate that inhibition of cell cycle progression contributes to the inhibition of prostate cancer development in TRAMP mice treated with BITC.

Dietary oleuropein inhibits tumor angiogenesis and lymphangiogenesis in the B16F10 melanoma allograft model: a mechanism for the suppression of high-fat diet-induced solid tumor growth and lymph node metastasis

Previously, we reported that high-fat-diet (HFD)-induced obesity stimulates melanoma progression in the B16F10 allograft model. In this study, we examined whether oleuropein (OL), the most abundant phenolic compound in olives, inhibits HFD-induced melanoma progression. Four-week-old male C57BL/6N mice were fed a HFD-diet with or without OL. After 16 weeks of feeding, B16F10-luc cells were subcutaneously injected and the primary tumor was resected 3 weeks later. OL suppressed HFD-induced solid tumor growth. In the tumor tissues, OL reduced HFD-induced expression of angiogenesis (CD31, VE-cadherin, VEGF-A, and VEGFR2), lymphangiogenesis (LYVE-1, VEGF-C, VEGF-D, and VEGFR3), and hypoxia (HIF-1α and GLUT-1) markers as well as HFD-induced increases in lipid vacuoles and M2 macrophages (MΦs). All animals were euthanized 2.5 weeks after tumor resection. OL suppressed HFD-induced increases in lymph node (LN) metastasis; expression of VEGF-A, VEGF-C, and VEGF-D in the LN; and M2-MΦs and the size of adipocytes in adipose tissues surrounding LNs. Co-culture results revealed that the crosstalk between B16F10s, M2-MΦs, and differentiated 3T3-L1 cells under hypoxic conditions increased the secretion of VEGF-A and -D, which stimulated tube formation and migration of endothelial cells (HUVECs) and lymphatic endothelial cells (LEC), respectively. Additionally, OL directly inhibited the differentiation of 3T3-L1 preadipocytes and tube formation by HUVECs and LECs. The overall results indicated that dietary OL inhibits lipid and M2-MΦ accumulation in HFD-fed mice, which contributes to decreases in VEGF secretion, thereby leading to inhibition of angiogenesis and lymphangiogenesis.

Abstract 911: Dietary oleuropein (OL) improves high-fat diet-induced metabolic stress and suppresses solid tumor growth and lymph node (LN) metastasis in the B16F10 melanoma allograft model

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Epidemiological studies indicate that obesity-associated metabolic stress increases the risk of developing several cancers. We previously reported that a high-fat diet (HFD)-induced obesity stimulates tumor growth and metastasis of B16F10 melanoma cancer cells in C57BL/6 mice. OL, the most abundant phenolic compound in olives, was reported to exert several physiological activities, including antioxidative, anticancer, and antimicrobial activities. In the present study, we examined whether OL inhibits HFD-induced melanoma progression in the B16F10 allograft model. Four-week old, male C57BL/6N mice were fed a diet [control diet (10 kcal% fat), HFD (60 kcal% fat), HFD + 0.02% OL, or HFD + 0.04% OL]. Twelve weeks after the initiation of feeding, fasting blood glucose, plasma insulin, and HOMA-IR were increased in HFD-fed mice, which was suppressed by dietary OL. Dual-energy X-ray absorptiometry results revealed that the proportion of lean body mass was decreased, whereas that of fat mass was increased in HFD-fed mice, and these changes were suppressed by dietary OL. After 16 weeks of feeding, B16F10-luc cells were subcutaneously injected into the animals’ left flank; bioluminescence imaging was conducted; and the primary tumor was resected 3 weeks later. OL suppressed HFD-induced solid tumor growth; HFD-induced increases in the expression of Ki67, cyclin D1, VEGF-A, VEGF-C, VEGF-D, CD31 (platelet endothelial cell adhesion molecule-1) and LYVE (lymphatic vessel endothelial receptor)-1; and HFD-induced decreases in TUNEL-positive apoptotic cells and cleaved PARP levels in the tumor tissues. OL also suppressed HFD-induced changes in the levels of many cytokines/chemokines in tumor tissues. All animals were killed 3 weeks after the tumor resection. OL suppressed HFD-induced increases in the size of LN; LN metastasis; and expression of VEGF-A and VEGF-C in the LN. OL suppresses HFD-induced increases in the size of adipocytes and accumulation of F4/80+ macrophages in adipose tissues surrounding the LN. In vitro culture results revealed that OL inhibited lipid accumulation in 3T3-L1 preadipocytes and the migration and viability of B16F10 cells. The present results demonstrated that dietary OL suppresses tumor growth and LN metastasis in HFD-fed mice. These effects are probably mediated, at least in part, via the inhibition of lipid accumulation, thereby reducing obesity-associated metabolic stress. Additionally, in vitro results indicate that OL may directly inhibit melanoma cell growth and migration, which may be one of the mechanisms by which dietary OL suppress melanoma progression in these mice. Citation Format: Hyerim Song, Jae In Jung, Gyoo Taik Kwon, So Young Park, Han Jin Cho, Jung Han Yoon Park. Dietary oleuropein (OL) improves high-fat diet-induced metabolic stress and suppresses solid tumor growth and lymph node (LN) metastasis in the B16F10 melanoma allograft model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 911. doi:10.1158/1538-7445.AM2015-911

Abstract 2078: High-fat diet feeding increases the expression of chemokines in the target organs and their corresponding receptors in tumor tissues of mice injected with highly metastatic prostate cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Prostate cancer is the third most common cancer in men worldwide. Recently, approximately 1 billion people in the world are overweight and obese, and epidemiological evidence indicates that overweight and obesity increase risks of developing several types of cancers. We previously noted that high-fat diet (HFD) feeding increased prostate cancer development and lung metastasis in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. When TRAMP-C2 prostate cancer cells which were derived from a TRAMP mouse were subcutaneously injected into C57BL/6 mice, they grew in a solid tumor. However, distant metastasis was not detected. When these poorly metastatic TRAMP-C2 cells were injected into the tail vein, lung metastasis was detected at 15 weeks after the injection. In order to obtain highly metastatic clones, we isolated TRAM-C2 cells metastasized to the lung, cultured, injected again into the tail vein, and the process was repeated one more time. When the resulting highly metastatic cells (MTC2) were injected into the tail vein, metastasis was detected in the lung at 4.5 weeks. Compared to poorly metastatic TRAMP-C2, transcripts of the chemokine receptors CCR2 and CXCR2 were tremendously upregulated in highly metastatic MTC2. The ligands of these receptors, MCP-1 and CXCL1/CXCL2, stimulated the invasion of MTC2, and neutralizing antibodies against CCR2 and CXCR2 almost completely blocked the ligand-induced invasion indicating that these receptors are functional in MTC2. When MTC2 were injected into the tail vein, HFD-feeding stimulated lung metastasis. The transcripts of CXCL1 and CXCL2 were significantly increased in the lungs of HFD-fed mice. When MTC2 were subcutaneously injected into HFD-fed C57BL/6 mice, solid tumor growth and lymph node metastasis as well as tumor angiogenesis and lymphangiogenesis were significantly increased in HFD-fed mice. HFD feeding increased the transcripts of MCP-1, CXCL1, and CXCL2 in the lymph node as well as those of CXCR2 and CCR2 in tumor tissues. Additionally, the transcripts of CCR2, CCR7 and CXCR2 were increased markedly in MTC2 cells when co-cultured with mature adipocytes. In conclusion, we demonstrate that HFD-feeding accelerates the solid tumor growth, metastasis and lymphangiogenesis in C57BL/6 mice injected with prostate cancer cells. These results indicate that MCP-1, CXCL1, and CXCL2 in the metastatic target organs draw prostate tumor cells expressing their corresponding receptors. HFD feeding stimulates metastasis by not only increasing the levels of these ligands in the target organs but also increasing the levels of the receptors on tumor cells. Citation Format: Gyoo Taik Kwon, Hyerim Song,, Jung Han Yoon Park. High-fat diet feeding increases the expression of chemokines in the target organs and their corresponding receptors in tumor tissues of mice injected with highly metastatic prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2078. doi:10.1158/1538-7445.AM2014-2078

Abstract 606: Inhibitory effects of piceatannol on tumor angiogenesis and lung metastasis in the 4T1 mammary cancer model

Piceatannol (trans-3,4,3′,5′-tetrahydroxystilbene) is a polyphenol found in Rheum undulatum, grapes, red wine, and berries and has been shown to exert anticarcinogenic effects. In the present study, we determined whether oral administration of piceatannol inhibits solid tumor growth, angiogenesis, and lung metastasis of mammary cancer cells. 4T1 cells (5 x 10 4 cells) were injected into the mammary fat pad of syngeneic, 6-week old, female BALB/c mice. One day after, the mice were subjected to gavage for 30 days with piceatannol (0, 10, or 20 mg/kg body weight/day). Oral piceatannol treatment induced a significant reduction in the growth of solid tumors. Immunohistochemical staining of tumor tissues revealed that the expression of Ki67 (prototypic cell cycle related nuclear antigen), cyclin D1, cyclin A, cyclin-dependent kinase (CDK)4, CDK2, hypoxia-inducible factor 1, vascular endothelial growth factor, CD31 (platelet endothelial cell adhesion molecule-1) and LYVE-1 (lymphatic vessel endothelial receptor 1) was significantly decreased in the mice received 20 mg/kg piceatannol as compared to mice received vehicle. Piceatannol significantly increased the numbers of TUNEL-positive, apoptotic cells in tumor tissues, which was accompanied by increased Bax and cleaved caspase-3 expression and reduced Bcl-2 expression. Additionally, piceatannol feeding reduced the number and volume of pulmonary tumor nodules, which was accompanied by reduced expression of matrix metalloproteinase-9 in lung tissues. Piceatannol induced a significant reduction in the blood levels of chemokine ligand (CXCL) 16, CXCL13 (BCA-1), interleukin-16, macrophage colony-stimulating factor, monocyte chemoattractant protein-1, triggering receptor expressed on myeloid cells-1, stromal cell-derived factor-1, and plasminogen activator inhibitor-1 compared to the vehicle-fed mice. The present results indicate that piceatannol has an ability to inhibit breast cancer development and metastasis and this effect is attributable to its ability to inhibit tumor angiogenesis, lymphangiogenesis and reduce the blood levels of a broad variety of cytokines and chemokines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 606. doi:1538-7445.AM2012-606

Abstract 1824: Effects of high-fat diet feeding on solid tumor growth and lung metastasis in the Lewis lung cancer allograft model

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Epidemiological studies indicate that overweight and obesity are associated with increased risk of developing several cancers. In the present study, we determined whether a chronic consumption of a high-fat diet increases solid tumor growth and metastasis in the C57BL/6N mouse lung cancer allograft model. Four-week old, male C57BL/6N mice were fed a purified diet containing 34.9% (w/w) fat (high fat diet) or 4.3 % fat (control diet) for 16 weeks, and then Lewis lung carcinoma cells were subcutaneously injected at 5 × 104 cells per mouse into the animals’ left flank. The primary tumor was resected 3 weeks later, and all animals were killed 21 days after the surgery. The high-fat diet feeding significantly increased the body weights and energy intakes as compared with those in the control diet group. The weights of the liver, spleen, epididymal fat pad, and mesenteric fat pad were increased in the high-fat diet group compared to the control group. The high fat diet increased solid tumor growth as well as the number and the size of colonies in the lung. Immunohistochemical staining of tumor tissues revealed that the expression of Ki67 (prototypic cell cycle related nuclear antigen), CD45 (phospho-tyrosine phosphatase), CD31 (platelet endothelial cell adhesion molecule-1), vascular endothelial growth factor (VEGF), cyclin-dependent kinase 4, cyclin A, and cyclin D1 was significantly increased in the high-fat diet group compared to the control group. The high-fat diet feeding significantly increased the blood levels of leptin, monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), granulocyte-colony stimulating factor (G-CSF), triggering receptor expressed on myeloid cells-1 (TREM-1), angiopoietin-1 (Ang-1), chemokine ligand 16 (CXCL16), plasminogen activator inhibitor-1 (PAI-1), and stromal cell-derived factor-1 (SDF-1) compared to the control group. Results of the present study indicate that the prolonged consumption of a high-fat diet enhances the secretion of cytokines, chemokines, matrix metalloproteinases, and angiogenesis-related proteins and increases the infiltration of macrophages into tumor tissues which, in turn, stimulate the proliferation and metastasis of lung cancer cells. The increased angiogenesis probably contributes to increased tumor growth and metastasis in mice fed on a high-fat diet. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1824. doi:10.1158/1538-7445.AM2011-1824