Metastasis Of B16F10 Melanoma Cells Research Articles

Indoloquinazoline alkaloids suppress angiogenesis and inhibit metastasis of melanoma cells

Metastasis is the leading cause of cancer-related mortality, targeting angiogenesis emerges as a therapeutic strategy for the treatment of melanoma metastasis. Discovery of new antiangiogenic compounds with specific mechanism of action is still desired. In present study, a bioassay-guidance uncovers the EtOAc extract of a marine-derived fungus Aspergillus clavutus LZD32-24 with significant inhibitory activity against the angiogenesis in Tg (fli1a: EGFP) zebrafish model. Extensive chromatographic fractionation led to the isolation of 48 indoloquinazoline alkaloids, including 21 new analogues namely clavutoines A-U (1–21). Their structures were determined by the spectroscopic data, including the ECD, single crystal X-ray diffraction and quantum chemical calculation for the configurational assignments. Among the bioactive analogues, quinadoline B (QB) showed the most efficacy to suppress the zebrafish vascular outgrowth in zebrafish embryos. QB markedly inhibited the migration, invasion and tube formation with weak cytotoxicity in human umbilical vein endothelial cells (HUVECs). Investigation of the mode of action revealed QB suppressed the ROCK/MYPT1/MLC2/coffin and FAK /Src signaling pathways, and subsequently disrupted actin cytoskeletal organization. In addition, QB reduced the number of new vessels sprouting from the ex vivo chick chorioallantoic membrane (CAM), and inhibited the metastasis of B16F10 melanoma cells in lung of C57BL/6 mice through suppressing angiogenesis. These findings suggest that QB is a potential lead for the development of new antiangiogenic agent to inhibit melanoma metastasis.

Src-family kinase inhibitors block early steps of caveolin-1-enhanced lung metastasis by melanoma cells

In advanced stages of cancer disease, caveolin-1 (CAV1) expression increases and correlates with increased migratory and invasive capacity of the respective tumor cells. Previous findings from our laboratory revealed that specific ECM-integrin interactions and tyrosine-14 phosphorylation of CAV1 are required for CAV1-enhanced melanoma cell migration, invasion and metastasis in vivo. In this context, CAV1 phosphorylation on tyrosine-14 mediated by non-receptor Src-family tyrosine kinases seems to be important; however, the effect of Src-family kinase inhibitors on CAV1-enhanced metastasis in vivo has not been studied. Here, we evaluated the effect of CAV1 and c-Abl overexpression, as well as the use of the Src-family kinase inhibitors, PP2 and dasatinib (more specific for Src/Abl) in lung metastasis of B16F10 melanoma cells. Overexpression of CAV1 and c-Abl enhanced CAV1 phosphorylation and the metastatic potential of the B16F10 murine melanoma cells. Alternatively, treatment with PP2 or dasatinib for 2 h reduced CAV1 tyrosine-14 phosphorylation and levels recovered fully within 12 h of removing the inhibitors. Nonetheless, pre-treatment of cells with these inhibitors for 2 h sufficed to prevent migration, invasion and trans-endothelial migration in vitro. Importantly, the transient decrease in CAV1 phosphorylation by these kinase inhibitors prevented early steps of CAV1-enhanced lung metastasis by B16F10 melanoma cells injected into the tail vein of mice. In conclusion, this study underscores the relevance of CAV1 tyrosine-14 phosphorylation by Src-family kinases during the first steps of the metastatic sequence promoted by CAV1. These findings open up potential options for treatment of metastatic tumors in patients in which Src-family kinase activation and CAV1 overexpression favor dissemination of cancer cells to secondary sites.

Angiopoietin-1 deficiency increases tumor metastasis in mice

BackgroundAngipoietin-1 activation of the tyrosine kinase receptor Tek expressed mainly on endothelial cells leads to survival and stabilization of endothelial cells. Studies have shown that Angiopoietin-1 counteracts permeability induced by a number of stimuli. Here, we test the hypothesis that loss of Angiopoietin-1/Tek signaling in the vasculature would increase metastasis.MethodsAngiopoietin-1 was deleted in mice just before birth using floxed Angiopoietin-1 and Tek mice crossed to doxycycline-inducible bitransgenic ROSA-rtTA/tetO-Cre mice. By crossing Angiopoietin-1 knockout mice to the MMTV-PyMT autochthonous mouse breast cancer model, we investigated primary tumor growth and metastasis to the lung. Furthermore, we utilized B16F10 melanoma cells subcutaneous and experimental lung metastasis models in Angiopoietin-1 and Tek knockout mice.ResultsWe found that primary tumor growth in MMTV-PyMT mice was unaffected, while metastasis to the lung was significantly increased in Angiopoietin-1 knockout MMTV-PyMT mice. In addition, angiopoietin-1 deficient mice exhibited a significant increase in lung metastasis of B16F10 melanoma cells, compared to wild type mice 3 weeks after injection. Additional experiments showed that this was likely an early event due to increased attachment or extravasation of tumor cells, since seeding of tumor cells was significantly increased 4 and 24 h post tail vein injection. Finally, using inducible Tek knockout mice, we showed a significant increase in tumor cell seeding to the lung, suggesting that Angiopoietin-1/Tek signaling is important for vascular integrity to limit metastasis.ConclusionsThis study show that loss of the Angiopoietin-1/Tek vascular growth factor system leads to increased metastasis without affecting primary tumor growth.

Curculigoside augments cell-mediated immune responses in metastatic tumor-bearing animals

A positive modulation of immune system is necessary for preparing the body to fight against malignant tumor cells. In the present study, the stimulatory effect of Curculigoside on cell-mediated immune response against the metastasis of B16F10 melanoma cells was analyzed in C57BL/6 mice. Curculigoside is a phenolic glucoside present in the plant Curculigo orchioides Gaertn. (Family – Amaryllidaceae). Administration of Curculigoside enhanced the natural killer (NK) cell activity, antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity in metastatic tumor-bearing animals, when compared to the untreated control animals. The compound was also found to be effective in reducing the levels of proinflammatory cytokines such as TNF-α, IL-1β, IL-6 and GM-CSF during metastasis. Besides these, levels of TH1 cytokines, such as IL-2 and IFN-γ, were significantly enhanced (p < 0.001) by Curculigoside administration and thereby reduces the metastatic lung colony formation along with an increased lifespan of the experimental animals. These studies provide an evidence for the stimulation of cell-mediated immune responses by Curculigoside against B16F10-induced metastatic tumor progression in experimental animals.

BFD-22 a new potential inhibitor of BRAF inhibits the metastasis of B16F10 melanoma cells and simultaneously increased the tumor immunogenicity

Benzofuroxan is an interesting ring system, which has shown a wide spectrum of biological responses against tumor cell lines. We investigated, herein, the antitumor effects of benzofuroxan derivatives (BFDs) in vitro and in a melanoma mouse model. Cytotoxic effects of twenty-two BFDs were determined by MTT assay. Effects of BFD-22 in apoptosis and cell proliferation were evaluated using Annexin V-FITC/PI and CFSE staining. In addition, the effects in the cell cycle were assessed. Flow cytometry, western blot, and fluorescence microscopy analysis were employed to investigate the apoptosis-related proteins and the BRAF signaling. Cell motility was also exploited through cell invasion and migration assays. Molecular docking approach was performed in order to verify the BFD-22 binding mode into the ATP catalytic site of BRAF kinase. Moreover, the BFD-22 antitumor effects were evaluated in a melanoma murine model using B16F10. BFD-22 was identified as a potential hit against melanoma cells. BFD-22 induced apoptosis and inhibited cell proliferation of B16F10 cells. BFD-22 has suppressed, indeed, the migratory and invasive behavior of B16F10 cells. Cyclin D1 and CDK4 expression were reduced leading to cell cycle arrest at G0/G1 phase. Of note, phosphorylation of BRAF at Ser338 was strongly down-regulated by BFD-22 in B16F10 cells. The accommodation/orientation into the binding site of BRAF was similar of BAY43-9006 (co-crystallized inhibitor of BRAF, sorafenib). Importantly, BFD-22 presented in vivo antimetastatic effects and showed better therapeutic efficacy than sorafenib and taxol. BFD-22 can be considered as a new lead compound and, then, can be helpful for the designing of novel drug candidates to treat melanoma.

Nrf2 Deficiency Promotes Melanoma Growth and Lung Metastasis.

The role of Nrf2, a key regulator of antioxidant and cytoprotective genes, in tumorigenesis remains controversial. Here we showed that Nrf2 deficiency led to increased local tumor growth in mice following subcutaneous injection of B16-F10 melanoma cells, as indicated by increased proportion of animals with locally palpable tumor mass and time-dependent increases in tumor volume at the injection site. In vivo bioluminescence imaging also revealed increased growth of melanoma in Nrf2-null mice as compared with wild-type mice. By using a highly sensitive bioluminometric assay, we further found that Nrf2 deficiency resulted in a remarkable increase in lung metastasis of B16-F10 melanoma cells as compared with wild-type mice. Taken together, the results of this short communication for the first time demonstrated that Nrf2 deficiency promoted melanoma growth and lung metastasis following subcutaneous inoculation of B16-F10 cells in 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.

Lipid-soluble ginseng extract inhibits invasion and metastasis of B16F10 melanoma cells.

This study was performed to elucidate the effect of a lipid-soluble ginseng extract (LSGE) on cancer invasion and metastasis. The LSGE, even at noncytotoxic concentrations, potently inhibited invasion and migration of B16F10 mouse melanoma cells in a dose-dependent manner. In the presence of 3 μg/mL of LSGE, the invasion and migration of B16F10 cells were significantly inhibited by 98.1% and 71.4%, respectively. Furthermore, the LSGE decreased mRNA and protein levels of matrix metalloproteinase (MMP)-2 in B16F10 cells, leading to a decrease in MMP-2 activity. After B16F10 cells were intravenously injected in the tail vein of C57BL/6 mice, 1000 mg/kg/day of LSGE was orally administered for 13 days, after which lung metastasis of cancer cells was inhibited by 59.3%. These findings indicate that LSGE inhibits cancer cell invasion and migration in vitro and lung metastasis of melanoma cells in vivo by inhibiting MMP-2 expression.

Growth and metastasis of B16-F10 melanoma cells is not critically dependent on host CD73 expression in mice.

BackgroundRecent studies have suggested that adenosine generated by ecto-5′-nucleotidase (CD73) in the tumor microenvironment plays a major role in promoting tumor growth by suppressing the immune response and stimulating angiogenesis via A2A and A2B receptors. However, adenosine has also been reported to inhibit tumor growth acting via A1 and A3 receptors. Therefore the aim of this study was to clarify the role of host CD73, which catalyzes the extracellular hydrolysis of AMP to adenosine, on tumor growth and metastasis of B16-F10 melanoma cells.MethodsCD73 and alkaline phosphatase (AP) activity of B16-F10 melanoma cells were measured by HPLC. Tumor cells were injected either subcutaneously or intradermally in WT and CD73−/− mice and tumor growth was monitored by MRI at 9.4 T. Immune cell subpopulations within tumors were assessed by FACS after enzymatic digestion. An endothelium specific CD73−/− was created using Tie2-Cre+ mice and CD73flox/flox (loxP) mice. Chimeric mice lacking CD73−/− on hematopoietic cells was generated by bone marrow transplantation. Lung metastatic spread was measured after intravenous B16-F10 application.ResultsB16-F10 cells showed very little CD73 and negligible AP activity. Neither complete loss of host CD73 nor specific knockout of CD73 on endothelial cells or hematopoietic cells affected tumor growth after subcutaneous or intradermal tumor cell application. Only peritumoral edema formation was significantly attenuated in global CD73−/− mice in the intradermal model. Immune cell composition revealed no differences in the different transgenic mice models. Also lung metastasis after intravenous B16-F10 injection was not altered in CD73−/− mice.ConclusionsCD73 expression on host cells, particularly on endothelial and hematopoietic cells, does not modulate tumor growth and metastatic spread of B16-F10 melanoma cells most likely because of insufficient adenosine formation by the tumor itself.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-898) contains supplementary material, which is available to authorized users.

INAM Plays a Critical Role in IFN-γ Production by NK Cells Interacting with Polyinosinic-Polycytidylic Acid–Stimulated Accessory Cells

Polyinosinic-polycytidylic acid strongly promotes the antitumor activity of NK cells via TLR3/Toll/IL-1R domain-containing adaptor molecule 1 and melanoma differentiation-associated protein-5/mitochondrial antiviral signaling protein pathways. Polyinosinic-polycytidylic acid acts on accessory cells such as dendritic cells (DCs) and macrophages (Mφs) to secondarily activate NK cells. In a previous study in this context, we identified a novel NK-activating molecule, named IFN regulatory factor 3-dependent NK-activating molecule (INAM), a tetraspanin-like membrane glycoprotein (also called Fam26F). In the current study, we generated INAM-deficient mice and investigated the in vivo function of INAM. We found that cytotoxicity against NK cell-sensitive tumor cell lines was barely decreased in Inam(-/-) mice, whereas the number of IFN-γ-producing cells was markedly decreased in the early phase. Notably, deficiency of INAM in NK and accessory cells, such as CD8α(+) conventional DCs and Mφs, led to a robust decrease in IFN-γ production. In conformity with this phenotype, INAM effectively suppressed lung metastasis of B16F10 melanoma cells, which is controlled by NK1.1(+) cells and IFN-γ. These results suggest that INAM plays a critical role in NK-CD8α(+) conventional DC (and Mφ) interaction leading to IFN-γ production from NK cells in vivo. INAM could therefore be a novel target molecule for cancer immunotherapy against IFN-γ-suppressible metastasis.

Abstract 249: Carnosic acid effectively inhibits metastasis of B16F10 melanoma cells

Abstract Carnosic acid is a natural benzenediol abietane diterpene found in rosemary and common sage and reported to exhibit anti-inflammatory, antioxidant, and anticarcinogenic activities. The present study examined the effects of carnosic acid on the metastatic characteristics of B16F10 cells. Results from transwell migration assays revealed that carnosic acid inhibited B16F10 migration in a dose-dependent manner. Western blot analysis of cell conditioned media showed that carnosic acid significantly suppressed the secretion of MMP-9, TIMP-1, uPA, and VCAM-1. However, the secretion of TIMP-2 was significantly increased in cells treated with 10 μmol/L of carnosic acid. Moreover, carnosic acid suppressed the expression of mesenchymal markers such as snail, Slug, vimentin, and N-cadherine and increased that of E-cadherin, an epithelial marker. We previously noted that chronic consumption of a high-fat diet (HFD) increased solid tumor growth and lymph node (LN) metastasis in C57BL/6 mice injected with B16F10. To assess the effects of dietary carnosic acid on HFD-stimulated LN metastasis, male C57BL/6 mice were fed a control diet (CD, 10 kcal% fat) or a HFD (60 kcal% fat) containing 0, 0.02, or 0.04% of carnosic acid for 21 weeks. B16F10 was subcutaneously injected 16 weeks after the initiation of feeding. The body weight and solid tumor growth were significantly increased in the HFD group as compared to the CD group, and these increases were suppressed by dietary carnosic acid. Lean body mass and body fat mass were estimated by dual energy X-ray absorptiometry. The body fat mass was tremendously increased in HFD-fed mice, and this increase was substantially decreased in a carnosic acid-dose dependent manner. A small but significant increase in lean body mass was noted in HFD-fed mice, which was not altered by dietary carnosic acid. Fasting blood glucose was markedly increased in HFD-fed mice, which was suppressed by 0.04% dietary carnosic acid. The tumor was resected 3 weeks after the B16F10 injection. Immunohistochemical staining of tumor tissues revealed that the expression of Ki67, platelet endothelial cell adhesion molecule-1, lymphatic vessel endothelial hyaluronan receptor-1, phospho-tyrosine phosphatase (a leukocyte marker), and vimentin was markedly increased in the HFD group as compared to the CD group, and these increases were blocked by carnosic acid. Two weeks after the tumor resection, LN metastasis was increased in mice fed HFD, and this increase was markedly suppressed by carnosic acid. In the present study, we demonstrate that dietary carnosic acid potently inhibits HFD-induced solid tumor growth and LN metastasis of B16F10 as well as tumor angiogenesis and lymphangiogenesis. Carnosic acid effectively suppresses HFD-induced body fat gains and fasting hyperglycemia, which may be parts of the mechanisms by which carnosic acid suppresses HFD-stimulated tumor progression. Citation Format: So Young Park, YooJin Jung, Ki Won Lee, Mi-Kyung Sung, Jung Han Yoon Park. Carnosic acid effectively inhibits metastasis of B16F10 melanoma 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 249. doi:10.1158/1538-7445.AM2014-249

β-Elemene inhibits the metastasis of B16F10 melanoma cells by downregulation of the expression of uPA, uPAR, MMP-2, and MMP-9

β-Elemene has been reported to be effective for the treatment of leukemia and certain solid tumors in basic and clinical studies. However, the mechanism of action of this phytochemical remains unknown. This study aimed to investigate the effect and mechanism of β-elemene in the mouse melanoma cell line B16F10. Cell viability was measured using the MTT assay. β-Elemene inhibited B16F10 melanoma cell metastasis, examined using scratch and Transwell migration/invasion assays. The mRNA and protein expression of urokinase-type plasminogen activator (uPA), the uPA receptor (uPAR), matrix metalloproteinase (MMP)-2, and MMP-9 were assayed using real-time PCR, immunocytochemistry, and western blotting methods. The results indicated that β-elemene inhibited the viability of B16F10 melanoma cells in a dose-dependent and time-dependent manner. The migratory and invasive capacities of B16F10 cells were also inhibited by β-elemene. The expression of uPA, uPAR, MMP-2, and MMP-9 was reduced by β-elemene at both the mRNA and protein level. β-Elemene inhibits the metastasis of B16F10 melanoma cells through downregulation of the expression of uPA, uPAR, MMP-2, and MMP-9. Thus, β-elemene is a natural potential anticancer drug.

CSE1L modulates Ras-induced cancer cell invasion: correlation of K-Ras mutation and CSE1L expression in colorectal cancer progression

BackgroundRas plays an important role in colorectal cancer progression. CSE1L (chromosome segregation 1-like) gene maps to 20q13, a chromosomal region that correlates with colorectal cancer development. We investigated the association of CSE1L with Ras in colorectal cancer progression. MethodsThe effect of CSE1L on metastasis-stimulating activity of Ras was studied in an animal model with tumor cells expressing CSE1L-specific shRNA and v-H-Ras. CSE1L expression was evaluated by the immunohistochemical analysis of 127 surgically resected colorectal tumors. K-Ras mutations were analyzed by direct sequencing. ResultsCSE1L knockdown reduced Ras-induced metastasis of B16F10 melanoma cells in C57BL/6 mice. v-H-Ras expression altered the cellular trafficking of CSE1L and increased CSE1L secretion. Most colorectal tumors were positive for CSE1L staining (98.4%, 125 of 127). Colorectal tumors with K-Ras mutation or high cytoplasmic CSE1L expression were correlated with T status (depth of tumor penetration; P = .004), stage (P = .004), and lymph node metastasis (P = .019). ConclusionsCSE1L may be a target for treating Ras-associated tumors. Analysis of K-Ras mutation and CSE1L expression may provide valuable clinical and pathological information to aid in the determination of treatment options for colorectal cancer.

Abstract 4869: β-caryophyllene inhibits solid tumor growth and metastasis of B16F10 melanoma cells in C57BL/6 mice fed on a high-fat diet.

Abstract We previously observed that chronic consumption of a high-fat diet (HFD) increased the melanoma progression and lymph node metastasis in C57BL/6N mice injected with B16F10 melanoma cells. β-caryophyllene (BCP) is a natural bicyclic sesquiterpene found in many essential oils especially clove and oregano oils. In the present study, we examined whether BCP inhibits HFD-induced melanoma growth and metastasis in the B16F10 allograft model. Four-week old, male C57BL/6 mice were fed on a HFD (60 kcal% fat) or control diet (CD, 10 kcal% fat) with or without BCP (0.15, 0.3% in diet) for a period of 21 weeks. Following 16 weeks of either CD or HFD in the absence or presence of BCP, B16F10 melanoma cells were subcutaneously injected into mice and all mice were continuously fed identical diets. Three weeks post-injection, tumors were resected and mice were sacrificed three weeks post-resection. The body weights and the growth of solid tumors were markedly increased in the HFD group as compared to the CD group, and these increases were suppressed by BCP treatment. The expression of proteins related to cell proliferation (Ki67, CDK4, cyclin A, and cyclin D1), angiogenesis (VEGF-A, VEGFR-2, and CD31) and lymphangiogenesis (VEGF-C, VEGF-D, VEGFR-3, and LYVE-1) was markedly increased in tumor tissues of the HFD group as compared to the CD group, and these increases were blocked by BCP treatment. HFD feeding increased the infiltration of CD45+ leukocytes and F4/80+ macrophages into tumor tissues. The administration of BCP significantly suppressed HFD-induced the infiltration of the immune cells into tumor tissues. HFD feeding reduced TUNEL-positive apoptotic cells in the tumor tissues, which was prevented by BCP treatment. Two weeks after tumor resection, the mice evidenced lung and lymph node metastasis. Lung and lymph node metastasis were increased in the mice fed on HFD, and these increases were markedly suppressed by BCP treatment. The present results demonstrate that BCP suppressed HFD-stimulated melanoma progression and metastasis, which may have been mediated via the suppression of cell cycle progression, angiogenesis and lymph angiogenesis, and induction of apoptosis in tumor tissues. These results suggest that BCP has potential as an anti-cancer agent for the prevention of obesity-stimulated melanoma progression. Citation Format: Jae In Jung, Eun Ji Kim, Yoo Jin Jung, Hyang Sook Chun, Jung Han Yoon Park. β-caryophyllene inhibits solid tumor growth and metastasis of B16F10 melanoma cells in C57BL/6 mice fed on a high-fat diet. [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 4869. doi:10.1158/1538-7445.AM2013-4869

CSE1L, a Novel Microvesicle Membrane Protein, Mediates Ras-Triggered Microvesicle Generation and Metastasis of Tumor Cells

Tumor-derived microvesicles are rich in metastasis-related proteases and play a role in the interactions between tumor cells and tumor microenvironment in tumor metastasis. Because shed microvesicles may remain in the extracellular environment around tumor cells, the microvesicle membrane protein may be the potential target for cancer therapy. Here we report that chromosome segregation 1-like (CSE1L) protein is a microvesicle membrane protein and is a potential target for cancer therapy. v-H-Ras expression induced extracellular signal-regulated kinase (ERK)-dependent CSE1L phosphorylation and microvesicle biogenesis in various cancer cells. CSE1L overexpression also triggered microvesicle generation, and CSE1L knockdown diminished v-H-Ras-induced microvesicle generation, matrix metalloproteinase (MMP)-2 and MMP-9 secretion and metastasis of B16F10 melanoma cells. CSE1L was preferentially accumulated in microvesicles and was located in the microvesicle membrane. Furthermore, anti-CSE1L antibody-conjugated quantum dots could target tumors in animal models. Our findings highlight a novel role of Ras-ERK signaling in tumor progression and suggest that CSE1L may be involved in the "early" and "late" metastasis of tumor cells in tumorigenesis. Furthermore, the novel microvesicle membrane protein, CSE1L, may have clinical utility in cancer diagnosis and targeted cancer therapy.

Inhibition of Stabilin-2 elevates circulating hyaluronic acid levels and prevents tumor metastasis

Hyaluronic acid (HA) has been implicated in the proliferation and metastasis of tumor cells. However, most previous studies were conducted on extracellular matrix or pericellular HA, and the role of circulating HA in vivo has not been studied. HA is rapidly cleared from the bloodstream. The scavenger receptor Stabilin-2 (Stab2) is considered a major clearance receptor for HA. Here we report a dramatic elevation in circulating HA levels in Stab2-deficient mice without any overt phenotype. Surprisingly, the metastasis of B16F10 melanoma cells to the lungs was markedly suppressed in the Stab2-deficient mice, whereas cell proliferation was not affected. Furthermore, administration of an anti-Stab2 antibody in Stab2(+) mice elevated serum HA levels and prevented the metastasis of melanoma to the lung, and also suppressed spontaneous metastasis of mammary tumor and human breast tumor cells inoculated in the mammary gland. Administration of the antibody or high-dose HA in mice blocked the lodging of melanoma cells to the lungs. Furthermore, HA at high concentrations inhibited the rolling/tethering of B16 cells to lung endothelial cells. These results suggest that blocking Stab2 function prevents tumor metastasis by elevating circulating HA levels. Stab2 may be a potential target in antitumor therapy.

Abstract B181: A lentivirus-encoding shRNA targeting a noncoding mitochondrial RNA inhibits melanoma tumor growth and metastasis in a mouse model in vivo.

Abstract We have described a family of non-coding mitochondrial RNAs (ncmtRNA) differentially expressed in normal and tumor cells. Normal proliferating cells express high levels of both sense and antisense ncmtRNAs. In contrast, nondividing cells express low levels of these transcripts. Interestingly, tumor cells express sense ncmtRNA and down-regulate antisense ncmtRNA. Previously, we have shown that transfection with oligonucleotides complementary to any of these ncmtRNAs induce death in different cancer cells type in vitro, but not in normal cells, raising the possibility of using interference to target these transcripts as a potential cancer treatment. The aim of this study was to determine whether in vivo administration of a lentivirus encoding shRNA against both ncmtRNA could inhibit melanoma tumor growth and metastasis in a mouse model. Non-replicative lentiviral vectors encoding shRNA against ncmtRNA (Lv-ncmtRNA) and luciferase (Lv-control) were constructed and their effects were evaluated in vitro by transducing B16F10 melanoma cells. A 6-fold decrease in relative expression of sense and antisense ncmtRNA was observed in cells transduced with Lv-ncmtRNA in comparison to cells transduced with Lv-control as measured by real time quantitative PCR. In addition, Lv-ncmtRNA transduction induces apoptotic cell death determined by cell surface exposure of phosphatidylserine and DNA fragmentation. The ability of this strategy to inhibit growth of solid tumors and metastasis of B16F10 melanoma cells was evaluated in vivo. C57BL/6 mice bearing subcutaneous B16F10 tumors received five intratumoral injections with 4×107 TU of Lv-ncmtRNA or Lv-control every two days. Mice treated with Lv-ncmtRNA showed a significant (p=0.0007) decrease in tumor volume (458 × 83 mm3) compared to mice treated with Lv-control (1167 × 154 mm3) 20 days after cell inoculation. For the metastasis assay, C57BL/6 mice were injected intravenously with 4×105 B16F10 cells and four days later treated with four intravenous administrations of 5×107 TU Lv-ncmtRNA or Lv-control every three days. Mice were sacrificed two weeks after cell injection for analysis of lung metastasis. Mice treated with Lv-ncmtRNA showed a significant (p=0,0005) decrease in the number of metastatic foci (77 × 9) compared to mice treated with Lv-control (286 × 18) as well as in lung weight (393,4 × 26,2 mg v/s 290,2 × 9,2 mg, p = 0,0005). In addition, hematoxylin and eosin-stained tissue sections also showed that the lung treated with Lv-ncmtRNA had only a few small nodules. In contrast, metastasis nodules occupied most of the lung when mice were treated whit Lv-control. These results suggest that intratumoral and systemic administration of shRNA targeting ncmtRNA represents a promising strategy towards the development of potential cancer therapies. Financed by CONICYT PFB-16; UNAB DI-11-11/I. MV is supported by a CONICYT doctoral fellowship. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B181.

Induction of apoptosis by sulforaphane in highly metastatic B16F-10 melanoma cells

Sulforaphane (SFN) is a naturally occurring isothiocyanate found in cruciferous vegetables, such as broccoli, cabbage, cauliflower, etc. SFN has received a great deal of attention because of its ability to inhibit cell proliferation and induce apoptosis in several tumor cell lines. Previously, we have demonstrated that SFN inhibits the metastasis of B16F-10 melanoma cells in both in vivo and in vitro models. Melanomas are among the aggressive tumor types because of their notorious resistance to treatment and their high tendency to metastasize. In this study, we investigated the influence of SFN on the induction of apoptosis in B16F-10 melanoma cells, which was evidenced by morphological changes such as membrane blebbing, presence of apoptotic bodies, DNA condensation, and also by nuclear DNA fragmentation. SFN-induced apoptosis was associated with the activation of caspases 3 and 9, Bax, and p53 and the downregulation of Bcl-2, caspase-8, Bid, and NF-kB. Caspase-3 is a most likely candidate to mediate SFN-induced apoptosis. In addition to the caspase-dependent pathway, our results also showed the involvement of proinflammatory cytokines, namely tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, IL-12p40, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the nuclear translocation of factors kappa B (NF-κB) p65, NF-κB p50, NF-κB c-Rel, c-FOS, ATF-2, and CREB-1 in SFN-induced apoptosis. These results raise the possibility that SFN may be a promising candidate for molecular-targeting chemotherapy against melanoma.

CD73-Deficient Mice Have Increased Antitumor Immunity and Are Resistant to Experimental Metastasis

CD73 is a cell-surface enzyme that suppresses immune responses by producing extracellular adenosine. In this study, we employed CD73 gene-targeted mice to investigate the role of host-derived CD73 on antitumor immunity and tumor cell metastasis. We found that CD73 ablation significantly suppressed the growth of ovalbumin-expressing MC38 colon cancer, EG7 lymphoma, AT-3 mammary tumors, and B16F10 melanoma. The protective effect of CD73 deficiency on primary tumors was dependent on CD8(+) T cells and associated with an increased frequency of antigen-specific CD8(+) T cells in peripheral blood and tumors and increased antigen-specific IFN-γ production. Replicate studies in bone marrow chimeras established that both hematopoietic and nonhematopoietic expression of CD73 was important to promote tumor immune escape. Using adoptive reconstitution of T regulatory cell (Treg)-depleted DEREG (depletion of regulatory T cells) mice, we demonstrated that part of the protumorigenic effect of Tregs was dependent on their expression of CD73. CD73-deficient mice were also protected against pulmonary metastasis of B16F10 melanoma cells after intravenous injection. Unexpectedly, we found that the prometastatic effect of host-derived CD73 was dependent on CD73 expression on nonhematopoietic cells. CD73 expression on nonhematopoietic cells, most likely endothelial cells, was critical for promoting lung metastasis in a manner independent from immunosuppressive effects. Notably, in vivo blockade of CD73 with a selective inhibitor or anti-CD73 monoclonal antibody significantly reduced tumor growth and metastasis of CD73-negative tumors. Taken together, our findings indicate that CD73 may be targeted at multiple levels to induce anticancer effects including at the level of tumor cells, Tregs, and nonhematopoietic cells.

Harmine activates intrinsic and extrinsic pathways of apoptosis in B16F-10 melanoma

BackgroundHarmine is a beta-carboline alkaloid from the plant Peganum harmala. Previous studies found that harmine inhibited metastasis of B16F-10 melanoma cells. This study aims to elucidate the role of harmine in apoptosis of B16F-10 cells.MethodsB16F-10 melanoma cells were treated in the presence and absence of harmine in vitro. Morphological changes, cell cycle and expression of various pro and anti- apoptotic genes were analyzed for the study of apoptosis.ResultsMorphological observation and DNA laddering assay showed that harmine treated cells displayed marked apoptotic characteristics, such as nuclear fragmentation, appearance of apoptotic bodies and DNA laddering fragment. TUNEL assay and flow cytometric analysis also confirmed apoptosis. Furthermore, RT-PCR analysis showed that harmine induced apoptosis in B16F-10 melanoma cells by up-regulating Bax and activating Caspase-3, 9 and p53 and down-regulating Bcl-2. Harmine also up-regulated Caspase-8 and Bid, indicating that harmine affected both extrinsic and intrinsic pathways of apoptosis. This study also showed inhibitory effects of harmine on some transcription factors and pro- inflammatory cytokines that protect cell from apoptosis.ConclusionHarmine activates both intrinsic and extrinsic pathways of apoptosis and regulates some transcription factors and pro-inflammatory cytokines.