Gyoo Taik Kwon

Bone marrow-derived, alternatively activated macrophages enhance solid tumor growth and lung metastasis of mammary carcinoma cells in a Balb/C mouse orthotopic model

IntroductionTumor-associated macrophages, which are derived from the infiltration of circulating bone marrow-derived monocytes, consist primarily of a polarized M2 macrophage (M2-Mϕ) population and are associated with poor prognosis in various cancers. In the present study, we attempted to assess whether M2-Mϕs derived from bone marrow stimulate the promotion and progression of mammary tumors.Methods4T1 murine mammary carcinoma cells were injected either alone or coupled with M2-Mϕs into the mammary fat pads of syngeneic female Balb/C mice. M2-Mϕs were prepared by treating monocytes isolated from female Balb/C mouse bone marrow with IL-4. Tumor cell growth was determined using an in vivo imaging system and the expression of cell proliferation-related, angiogenesis-related, and lymphangiogenesis-related proteins in tumor tissues was immunohistochemically analyzed. To evaluate the effects of the crosstalk between 4T1 cells and M2-Mϕs on the secretion and mRNA expression of cytokines and the migration of monocytes, 4T1 cells and M2-Mϕs were co-cultured and cytokine antibody array, real-time RT-PCR, and trans-well migration assays were conducted.ResultsThe co-injection of M2-Mϕs into the mammary fat pads of mice increased solid tumor growth and lung metastasis of 4T1 cells as well as the infiltration of CD45+ leukocytes into tumor tissues. The proportions of Ki-67+ proliferating cells and the expression of hypoxia inducible factor-1α, vascular endothelial cell growth factor A, CD31, vascular endothelial cell growth factor C, and lymphatic vessel endothelial receptor-1 were increased significantly in the tumor tissues of mice co-injected with 4T1 cells and M2-Mϕs. The in vitro results revealed that the proliferation of 4T1 cells, the migration of monocytes, and the secretion of granulocyte colony-stimulating factor, IFNγ, IL-1α, IL-2, IL-16, IFNγ-induced protein-10, keratinocyte-derived chemokine, macrophage colony-stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1α, and RANTES were increased when 4T1 cells were co-cultured with M2-Mϕs, as compared with when the 4T1 cells were cultured alone.ConclusionThe crosstalk between 4T1 cells and M2-Mϕs increased the production of cytokines, which may have induced immune cell infiltration into tumor tissues, tumor cell proliferation, angiogenesis, and lymph angiogenesis, thereby increasing solid tumor growth and lung metastasis.

A high-fat diet increases angiogenesis, solid tumor growth, and lung metastasis of CT26 colon cancer cells in obesity-resistant BALB/c mice.

We evaluated whether high‐fat diet (HFD), in the absence of increased calorie intake, increases colon cancer growth and metastasis. Four‐week‐old male BALB/c mice were fed on an HFD (60 kcal% fat) or control diet (10 kcal% fat) for 16 wk, after which CT26 colon cancer cells were subcutaneously injected into the right flank. Solid tumor growth and the number and volume of tumor nodules in the lung were increased markedly in the HFD group with only a slight increase in body weight (5.9%). HFD feeding increased tumor tissue levels of Ki67, cyclin A, cyclin D1, CDK2, Bcl‐xL, and Bcl‐2; reduced p53 levels and TUNEL‐positive apoptotic cells; increased the levels of CD45, CD68, CD31, VEGF, P‐VEGF receptor‐2, iNOS, and COX‐2 as well as hemoglobin content; and increased the levels of HIF‐1α, P‐STAT3‐Y705, P‐STAT3‐S727, P‐IκB‐α, P‐p65, p65, P‐c‐Jun, P‐Akt, P‐ERK1/2, P‐p38, and P‐SAPK/JNK. HFD feeding increased the serum levels of EGF, insulin, IGF‐I, IFN‐γ, leptin, RANTES, MCP‐1, IL‐1ra, and SDF‐1α and media conditioned by epididymal fat tissue explants from HFD‐fed mice caused an increase in microvessel outgrowth from the mouse aorta and tube formation of human umbilical vein endothelial cells. These results indicate that the chronic consumption of an HFD increases colon cancer cell proliferation, tumor angiogenesis, and lung metastasis in mice in the absence of discernible weight gain. HFD feeding increases the levels of growth factors which activate transcription factors, thereby inducing the expression of many genes involved in the stimulation of inflammation, angiogenesis, and cellular proliferation.

Licochalcone E Present in Licorice Suppresses Lung Metastasis in the 4T1 Mammary Orthotopic Cancer Model

We investigated whether licochalcone E (LicE), a phenolic constituent of licorice, inhibits mammary tumor growth and metastasis using animal and cell culture models. 4T1 mammary carcinoma cells were injected into the mammary fat pads of syngeneic BALB/c mice. Starting 7 days after the injection, the mice received LicE (7 or 14 mg/kg body weight/day) via oral gavage for 25 days. LicE suppressed solid tumor growth and lung metastasis, but did not exhibit kidney or liver toxicity. In tumor tissues, LicE treatment induced a reduction in the expression of Ki67, cyclins, and cyclin-dependent kinases and stimulated apoptosis with increased expression of Bax and cleaved caspase-3 but decreased expression of Bcl-2. In addition, LicE decreased expression of CD31, vascular endothelial growth factor (VEGF)-A and C, VEGF-receptor 2, lymphatic vessel endothelial receptor-1, CD45, cyclooxygenase-2, inducible nitric oxide synthase, and hypoxia inducible factor-1α in tumor tissues. In lung tissues, LicE reduced the levels of proinflammatory cytokines and angiogenesis/metastasis-related proteins. In mammary cancer cell cultures, LicE (5–20 μmol/L) dose dependently inhibited cell migration and invasion. LicE inhibited secretion of matrix metalloproteinase-9, urokinase-type plasminogen activator and VEGF-A, and stimulated secretion of tissue inhibitor of metalloproteinase-2 in MDA-MB-231 cells. In addition, LicE inhibited tube formation of vascular endothelial cells. We show that LicE administration suppressed tumor growth and lung metastasis in the mouse model in conjunction with LicE inhibition of cell migration, invasion, and tube formation in vitro. Reduced tumor growth and metastasis in LicE-treated mice may be, at least in part, attributed to reduced inflammation and tumor angiogenesis. Cancer Prev Res; 6(6); 603–13. ©2013 AACR.

The Hexane Extract of Saussurea lappa and Its Active Principle, Dehydrocostus Lactone, Inhibit Prostate Cancer Cell Migration

Saussurea lappa has been used in Chinese traditional medicine for the treatment of abdominal pain, tenesmus, nausea, and cancer; previous studies have shown that S. lappa also induces G(2) growth arrest and apoptosis in gastric cancer cells. In this study, we investigated the effects of hexane extracts of S. lappa (HESLs) on the migration of DU145 and TRAMP-C2 prostate cancer cells. DU145 and TRAMP-C2 cells were cultured in the presence of 0-4 μg/mL HESL with or without 10 ng/mL epidermal growth factor (EGF). HESL inhibited the basal and EGF-induced migration of prostate cancer cells in a dose-dependent manner, whereas HESL did not influence the viability of these cancer cells under the conditions used in this study. Active fractions of HESL were separated via column chromatography, and the structure of the active principle was determined using (1)H and (13)C nuclear magnetic resonance spectroscopy. The active compound, dehydrocostus lactone (DHCL), in fraction 7 dose-dependently inhibited the basal and EGF-induced migration of prostate cancer cells. HESL and DHCL reduced matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 secretion but increased TIMP-2 levels in both the absence and presence of EGF. Our results demonstrate that the inhibition of MMP-9 secretion and the stimulation of TIMP-2 secretion contribute to reduced migration of DU145 cells treated with HESL and DHCL. These results indicate that HESL containing its active principle, DHCL, has potential as an antimetastatic agent for the treatment of prostate 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 Suppresses High-Fat Diet- Stimulated Mammary Tumor Progression via the Alteration of Tumor Microenvironments in Obesity-Resistant BALB/c Mice

We previously reported that a high‐fat diet (HFD) and M2‐macrophages induce changes in tumor microenvironments and stimulate tumor growth and metastasis of 4T1 mammary cancer cells in BALB/c mice. In this study, we attempted to determine whether benzyl isothiocyanate (BITC) inhibits HFD‐induced changes in tumor progression and in tumor microenvironments. Four groups of female BALB/c mice (4‐week‐old) were fed on a control diet (CD, 10 kcal% fat) and HFD (60 kcal% fat) containing BITC (0, 25, or 100 mg/kg diet) for 20 weeks. Following 16 weeks of feeding, 4T1 cells (5 × 104 cells) were injected into the mammary fat pads, and animals were killed 30 d after the injection. HFD feeding increased solid tumor growth and the number of tumor nodules in the lung and liver, as compared to the CD group, and these increases were inhibited by BITC supplementation. The number of lipid vacuoles, CD45+ leukocytes and CD206+ M2‐macrophages, expression of Ki67, levels of cytokines/chemokines, including macrophage‐colony stimulating factor (M‐CSF) and monocyte chemoattractant protein‐1, and mRNA levels of F4/80, CD86, Ym1, CD163, CCR2, and M‐CSF receptor were increased in the tumor tissues of HFD‐fed mice, and these increases were inhibited by BITC supplementation. In vitro culture results demonstrated that BITC inhibited macrophage migration as well as lipid droplet accumulation in 3T3‐L1 cells. These results suggest that suppression of lipid accumulation and macrophage infiltration in tumor tissues may be one of the mechanisms by which BITC suppresses tumor progression in HFD‐fed mice.

β-Caryophyllene potently inhibits solid tumor growth and lymph node metastasis of B16F10 melanoma cells in high-fat diet-induced obese C57BL/6N mice

We reported previously that high-fat diet (HFD) feeding stimulated solid tumor growth and lymph node (LN) metastasis in C57BL/6N mice injected with B16F10 melanoma cells. β-caryophyllene (BCP) is a natural bicyclic sesquiterpene found in many essential oils and has been shown to exert anti-inflammatory activities. To examine whether BCP inhibits HFD-induced melanoma progression, 4-weeks old, male C57BL/6N mice were fed a control diet (CD, 10 kcal% fat) or HFD (60 kcal% fat + 0, 0.15 or 0.3% BCP) for the entire experimental period. After 16 weeks of feeding, B16F10s were subcutaneously injected into mice. Three weeks later, tumors were resected, and mice were killed 2 weeks post-resection. Although HFD feeding increased body weight gain, fasting blood glucose levels, solid tumor growth, LN metastasis, tumor cell proliferation, angiogenesis and lymphangiogenesis, it decreased apoptotic cells, all of which were suppressed by dietary BCP. HFD feeding increased the number of lipid vacuoles and F4/80+ macrophage (MΦ) and macrophage mannose receptor (MMR)+ M2-MΦs in tumor tissues and adipose tissues surrounding the LN, which was suppressed by BCP. HFD feeding increased the levels of CCL19 and CCL21 in the LN and the expression of CCR7 in the tumor; these changes were blocked by dietary BCP. In vitro culture results revealed that BCP inhibited lipid accumulation in 3T3-L1 preadipocytes; monocyte migration and monocyte chemoattractant protein-1 secretion by B16F10s, adipocytes and M2-MΦs; angiogenesis and lymphangiogenesis. The suppression of adipocyte and M2-cell accumulation and the inhibition of CCL19/21-CCR7 axis may be a part of mechanisms for the BCP suppression of HFD-stimulated melanoma progression.

Kaempferol Downregulates Insulin-like Growth Factor-I Receptor and ErbB3 Signaling in HT-29 Human Colon Cancer Cells

Background: Novel dietary agents for colon cancer prevention and therapy are desired. Kaempferol, a flavonol, has been reported to possess anticancer activity. However, little is known about the molecular mechanisms of the anticancer effects of kaempferol. The aim of this study was to determine the inhibitory effect of kaempferol on growth factor-induced proliferation and to elucidate its underlying mechanisms in the HT-29 human colon cancer cell line. Methods: To assess the effects of kaempferol and/or growth factors [insulin-like growth factor (IGF)-I and heregulin (HRG)-β], cells were cultured with or without 60 μmol/L kaempferol and/or 10 nmol/L IGF-I or 20 μg/L HRG-β. Cell proliferation, DNA synthesis, and apoptosis were determined by a cell viability assay, a [3H]thymidine incorporation assay, and Annexin-V staining, respectively. Western blotting, immunoprecipitation, and an in vitro kinase assay were conducted to evaluate expression and activation of various signaling molecules involved in the IGF-I receptor (IGF-IR) and ErbB3 signaling pathways. Results: IGF-I and HRG-β stimulated HT-29 cell growth but did not abrogate kaempferol-induced growth inhibition and apoptosis. Kaempferol reduced IGF-II secretion, HRG expression and phosphorylation of Akt and extracellular signal-regulated kinase (ERK)-1/2. Kaempferol reduced IGF-I- and HRG-β-induced phosphorylation of the IGF-IR and ErbB3, their association with p85, and phosphatidylinositol 3-kinase (PI3K) activity. Additionally, kaempferol inhibited IGF-I- and HRG-β-induced phosphorylation of Akt and ERK-1/2. Conclusions: The results demonstrate that kaempferol downregulates activation of PI3K/Akt and ERK-1/2 pathways by inhibiting IGF-IR and ErbB3 signaling in HT-29 cells. We suggest that kaempferol could be a useful chemopreventive agent against colon cancer.

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 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