High expression of macrophage migration inhibitory factor in human melanoma cells and its role in tumor cell growth and angiogenesis.

Macrophage migration inhibitory factor (MIF) is known to function as a cytokine, hormone, and glucocorticoid-induced immunoregulator. In this study, we reported for the first time that human melanocytes and melanoma cells express MIF mRNA and produce MIF protein. Immunohistochemical analysis demonstrated that MIF was mostly localized in the cytoplasm of melanocytes and G361 cells, a widely available human melanoma cell line. In particular, strong positive staining was observed at the dendrites of these cells. Expression of MIF mRNA and production of MIF protein were much higher in human melanoma cells such as G361, A375, and L32 than in normal cultured melanocytes. To assess the role of MIF overexpression in melanoma cells, G361 cells were transfected with an antisense human MIF plasmid. The results demonstrated that the cell growth rate of the transfected cells was markedly suppressed, suggesting that MIF participates in the mechanism of proliferation of melanoma cells. To further evaluate the function of MIF, we employed the Boyden chamber method to examine the effect on tumor cell migration and found that MIF enhanced the migration of G361 cells in a dose-dependent manner. Furthermore, we administered anti-MIF antibody into tumor (G361 cells in a Millipore chamber)-bearing mice to assess the effect on tumor-associated angiogenesis. The anti-MIF antibody significantly suppressed tumor-induced angiogenesis. Taken together, these results indicated that it is likely that MIF may function as a novel growth factor that stimulates incessant growth and invasion of melanoma concomitant with neovascularization.     

Involvement of macrophage migration inhibitory factor (MIF) in the mechanism of tumor cell growth.

BACKGROUND: Macrophage migration inhibitory factor (MIF) was recently rediscovered as a cytokine, pituitary hormone, and glucocorticoid-induced immunomodulator. MIF is constitutively expressed in various cells and enhances production of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1, and interferon gamma. Recently, it was reported that MIF mRNA was overexpressed in prostatic tumors, which suggests that MIF is a protein involved in tumor cell growth beyond inflammatory and immune responses. MATERIALS AND METHODS: We examined the expression of MIF in the murine colon carcinoma cell line colon 26 by Western and Northern blot analyses and immunohistochemistry. Next, we investigated the effects of transforming growth factor (TGF) beta, basic fibroblast growth factor (b-FGF), and platelet-derived growth factor (PDGF) on the expression of MIF mRNA. Furthermore, we examined whether MIF is involved in tumor cell proliferation, using an MIF anti-sense plasmid transfection technique. RESULTS: We demonstrated that MIF protein and its mRNA were highly expressed in colon 26 cells, using Western and Northern blot analyses, respectively. By immunohistochemical analysis, we found that MIF was localized largely in the cytoplasm of the tumor cells. In response to TGF-beta, b-FGF, and PDGF, MIF mRNA expression was significantly up-regulated. Following this, we transfected the cells with an anti-sense MIF plasmid, which revealed that this treatment induced significant suppression of cell proliferation. CONCLUSION: Although MIF plays multifunctional roles in a broad spectrum of pathophysiological states, little has been done to investigate the role of this protein in association with tumor growth. The current results suggest the possibility that MIF induces tumor cell growth in concert with other growth factors, which encouraged us to investigate a novel approach for tumor therapy using an anti-MIF antibody and an MIF anti-sense plasmid transfection technique.     

Restoration of thrombospondin 1 expression in tumor cells harbouring mutant ras oncogene by treatment with low doses of doxycycline

Oncogenes act as inducers of tumor neovascularization, at least in part through suppression of endogenous angiogenesis inhibitors, e.g., thrombospondin 1 (TSP-1/THSP1). Therefore, restoration of TSP-1 levels can be viewed as a possible means to inhibit tumor angiogenesis. We observed that low concentrations (0.1-10 microg/ml) of doxycycline (but not those of related tetracycline) restore TSP-1 expression in H-ras oncogene-expressing tumor cell lines (528ras1, MT-Ras). Interestingly, this effect was relatively ras-specific, as doxycycline did not alter TSP-1 expression in several cell lines (e.g., 528neu2 fibrosarcoma, B16F1 melanoma, and Lewis lung carcinoma) harbouring other types of transforming alterations. Doxycycline-induced reversal of TSP down-regulation was abrogated under hypoxic conditions. Therefore, we conclude that, in vivo, TSP-1 is likely under dual and/or synergistic control of oncogenes and hypoxia-related pathways. Disruption of both components may be necessary for the 'rescue' of TSP-1 expression in ras-driven cancers.     

Up-regulation of thrombospondin-1 gene by epidermal growth factor and transforming growth factor beta in human cancer cells--transcriptional activation and messenger RNA stabilization

Thrombospondin-1 (TSP-1), a multifunctional matrix protein, affects tumor growth through modulation of angiogenesis and other stromal biological functions. In several of nine human cancer cell lines derived from liver, brain, pancreas, and bone, expression of TSP-1 was up-regulated in response to the two most representative growth factors, epidermal growth factor (EGF) and transforming growth factor beta1 (TGFbeta1). Expression of TSP-1 was markedly enhanced in hepatic HuH-7 cells by EGF but not by TGFbeta1. In contrast, expression of TSP-1 was markedly enhanced by TGFbeta1, but not by EGF, in osteosarcoma MG63 cells. EGF induced activation of TSP-1 promoter-driven luciferase activity in HuH-7 cells, and the elements between -267 and -71 on the 5' region of TSP-1 gene containing two GC boxes to which Sp1 bound, were found to be responsible for the promoter activation by EGF. However, EGF did not alter TSP-1 mRNA stability in hepatic cells. On the other hand, no such enhancement of the TSP-1 promoter activity by TGFbeta1 appeared in MG63 cells. Enhanced expression of TSP-1 by TGFbeta1 in MG63 cells was partially blocked by exogenous addition of SB203580, an inhibitor of p38 mitogen-activated protein kinase. TGFbeta was found to induce marked elongation of TSP-1 mRNA longevity in osteosarcoma cells when mRNA degradation was assayed in the presence of alpha-amanitin. The up-regulation of TSP-1 by EGF and TGFbeta might play a critical role in modulation of angiogenesis and formation of matrices in tumor stroma.     

IL-18 enhances thrombospondin-1 production in human gastric cancer via JNK pathway

IL-18 is a pleiotropic cytokine that is produced by many cancer cells. A recent report suggested that IL-18 plays a key role in regulating the immune escape of melanoma and gastric cancer cells. Thrombospondin (TSP-1) is known to inhibit angiogenesis in several cancers but some studies have reported that it stimulates angiogenesis in some cancers such as gastric cancer. IL-18 and TSP-1 are related to tumor proliferation and metastasis. This study investigated the relationship between IL-18 and TSP-1 in gastric cancer. RT-PCR and ELISA showed that after the cells had been treated with IL-18, the level of TSP-1 mRNA expression and TSP-1 protein production by IL-18 increased in both a dose- and time-dependent manner. The cells were next treated with specific inhibitors in order to determine the signal pathway involved in IL-18-enhanced TSP-1 production. IL-18-enhanced TSP-1 expression was blocked by SP600125, a c-Jun N-terminal kinase (JNK) specific inhibitor. In addition, Western blot showed that IL-18 enhanced the expression of phosphorylated JNK. Overall, these results suggest that IL-18 plays a key role in TSP-1 expression involving JNK.     

Decrease of Let-7f in Low-Dose Metronomic Paclitaxel Chemotherapy Contributed to Upregulation of Thrombospondin-1 in Breast Cancer

Low-dose metronomic (LDM) paclitaxel therapy displayed a stronger anti-angiogenic activity on breast tumors with fewer side effects. Upregulation of anti-angiogenic factor Thrombospondin-1 (TSP-1) accords for therapeutic potency of LDM paclitaxel, but its molecular mechanism has not been elucidated yet. microRNAs (miRNAs) have emerged as new important regulators of tumor growth and metastasis. Here, we hypothesize that miRNAs are involved in TSP-1 overexpression in paclitaxel LDM therapy of breast tumors. The miRNA profile of tumor tissues from control, LDM and MTD groups in 4T1 mouse breast cancer model was detected by microarray, and then verified by quantitative real-time PCR (qRT-PCR). Luciferase assay and western blot were employed to explore the mechanisms of miRNAs involved in this process. We found that let-7f, let-7a, miR-19b and miR-340-5p were reduced by >2 fold, and miR-543* and miR-684 were upregulated by at least 50% in paclitaxel LDM therapy. qRT-PCR verification revealed that let-7f level was reduced most significantly in LDM therapy. Computational prediction using TargetScan and miRanda suggested THBS1 which encodes TSP-1 as a potential target for let-7f. Luciferase activity assay further confirmed that let-7f may bind to 3''UTR of THBS1 gene and inhibit its activity. Moreover, forced expression of let-7f led to a decrease of TSP-1 at both mRNA and protein levels in MCF-7 cells. Contrastly, let-7f inhibition induced an increased expression of THBS1 mRNA and TSP-1 protein, but did not affect the proliferation and apoptosis of MCF-7 cells. Paclitaxel LDM therapy led to a decrease of let-7f and the elevation of TSP-1 protein expression in MCF-7 cells, while overexpression of let-7f may abolish LDM-induced the upregulation of TSP-1 in MCF-7 cells. In summary, let-7f inhibition contributed to the upregulation of TSP-1 in paclitaxel LDM therapy, independently of proliferation, cell cycle arrest and apoptosis of breast cancer. This study indicates let-7f as a potential therapeutic target for breast tumor.     

Regulation of thrombospondin-1 expression in alternatively activated macrophages and adipocytes: role of cellular cross talk and omega-3 fatty acids

Thrombospondin-1 (TSP-1) expression in human adipose positively correlates with body mass index and may contribute to adipose dysfunction by activating transforming growth factor-β and/or inhibiting angiogenesis. Our objective was to determine how TSP-1 is regulated in adipocytes and polarized macrophages using a coculture system and to determine whether fatty acids, including the ω-3 fatty acid docosahexaenoic acid (DHA), regulate TSP-1 expression. Coculture of M1, M2a or M2c macrophages with adipocytes induced TSP-1 gene expression in adipocytes (from 2.4- to 4.2-fold, P<.05), and adipocyte coculture induced TSP-1 gene expression in M1 and M2c macrophages (M1: 8.6-fold, M2c: 26-fold; P<.05). TSP-1 protein levels in the shared media of adipocytes and M2c cells were also strongly induced by coculture (>10-fold, P<.05). DHA treatment during the coculture of adipocytes and M2c macrophages potently inhibited the M2c macrophage TSP-1 mRNA level (97% inhibition, P<.05). Adipocyte coculture induced interleukin (IL)-10 expression in M2c macrophages (10.1-fold, P<.05), and this increase in IL-10 mRNA expression was almost completely blocked with DHA treatment (96% inhibition, P<.05); thus, IL-10 expression closely paralleled TSP-1 expression. Since IL-10 has been shown to regulate TSP-1 in other cell types, we reduced IL-10 expression with siRNA in the M2c cells and found that this caused TSP-1 to be reduced in response to adipocyte coculture by 60% (P<.05), suggesting that IL-10 regulates TSP-1 expression in M2c macrophages. These results suggest that supplementation with dietary ω-3 fatty acids could potentially be beneficial to adipose tissue in obesity by reducing TSP-1 and fibrosis.     

Differential expression of thrombospondin and cellular fibronectin during remodeling in proliferative glomerulonephritis.

Thrombospondin-1 (TSP-1) and an alternatively spliced fibronectin (Fn)-EIIIA isoform are adhesive proteins associated with embryogenesis and tissue remodeling. We compared, by immunohistochemistry and in situ hybridization, the course of TSP-1 and Fn-EIIIA expression in a model of glomerulonephritis induced by Habu snake venom (HV) and characterized by mesangial cell migration, proliferation, and extracellular matrix (ECM) synthesis. At 24 hr after HV, TSP-1 and Fn-EIIIA proteins localized in the central aspects of lesions associated with platelets and macrophages and at the margins of lesions coinciding with mesangial cell migration (determined by Thy-1 staining). Mesangial cells at this time expressed TSP-1 but not Fn-EIIIA mRNA. TSP-1 protein and mRNA peaked in lesions at 48 hr and were associated with cell proliferation (determined by PCNA, alpha-smooth muscle actin phenotype, and expression of beta-PDGF receptor mRNA). TSP-1 expression declined at 72 hr when expression of ECM synthesis peaked, as determined by increased expression of collagen Type IV, laminin, and TGF-beta1 protein and mRNA. Mesangial cell expression of Fn-EIIIA was first observed at 48 hr and was most abundant at 72 hr after HV. Therefore, platelet- and macrophage-derived Fn-EIIIA and TSP-1 in early lesions are associated with mesangial cell migration. Mesangial cell upregulation of TSP-1 is associated with migration and proliferation but not maximal ECM accumulation, whereas mesangial cell expression of Fn-EIIIA is associated with proliferation and ECM accumulation. These results suggest distinctive temporal and spatial roles for TSP-1 and Fn-EIIIA in remodeling during glomerular disease. (J Histochem Cytochem 47:533-543, 1999)     

The tumor suppressor PTEN inhibits EGF-induced TSP-1 and TIMP-1 expression in FTC-133 thyroid carcinoma cells

Thrombospondin-1 (TSP-1) is a multidomain extracellular macromolecule that was first identified as natural modulator of angiogenesis and tumor growth. In the present study, we found that epidermal growth factor (EGF) up-regulated TSP-1 expression in FTC-133 (primary tumor) but not in FTC-238 (lung metastasis) thyroid cancer cells. Both EGF and TSP-1 induced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In FTC-133 cells, EGF induced proliferation in a TSP-1- and TIMP-1-dependent manner. In addition, we determined that re-expression of the tumor suppressor protein PTEN induced cell death, an effect that correlated with a block of Akt kinase phosphorylation. EGF-induced TSP-1 and TIMP-1 promoter activity and protein expression were inhibited in FTC-133 cells stably expressing wtPTEN but not in cells expressing mutant PTEN. Furthermore, we found that wtPTEN inhibited EGF--but not TSP-1--stimulated FTC-133 cell migration and also inhibited invasion induced by EGF and by TSP-1. Finally, an antibody against TSP-1 reversed EGF-stimulated FTC-133 cell invasion as well as the constitutive invasive potential of FTC-238 cells. Overall, our results suggest that PTEN can function as an important modulator of extracellular matrix proteins in thyroid cancer. Therefore, analyzing differential regulation of TSP-1 by growth factors such as EGF can be helpful in understanding thyroid cancer development.     

Expression and localization of thrombospondin-1 and -2 and their cell-surface receptor,CD36, during rat follicular development and formation of the corpus luteum

Thrombospondin (TSP)-1 and -2 are extracellular matrix glycoproteins that are both antiangiogenic and important in regulating cellular development, differentiation, and function. To evaluate the expression of TSP in follicular and luteal development, ovarian cycles of Sprague-Dawley rats were synchronized and tissues collected daily at stages corresponding to the early antral, ovulatory, early luteal, and late luteal phases of the ovarian cycle. Immunohistochemistry and Western blot analyses demonstrated that TSP-1 protein and its receptor, CD36, were present in the early antral phase and were localized primarily to the granulosa cells of antral follicles. Both proteins were also present immediately after ovulation and were localized to the developing corpus luteum. Messenger RNA for TSP-1 showed a similar pattern, with expression at the early antral and ovulatory phases. Protein and mRNA expression for TSP-2 was relatively delayed compared to TSP-1, although TSP-2 also was expressed in granulosa cells. Both TSP-1 and -2 were increased in response to LH stimulation in vitro, whereas TSP-2 was suppressed by FSH. The temporal pattern of expression of TSP-1, -2, and CD36, which mirrors the active phases of angiogenesis in this experimental model, is compatible with a role for these proteins in the control of ovarian vascularization.     

TNF-alpha downregulates vascular endothelial Flk-1 expression in human melanoma xenograft model

High-dose TNF with melphalan has significant antitumor activity in regional perfusion of the limbs and liver in human malignancies. TNF is believed to target tumor vasculature, but the precise molecular mechanism is unknown. The present study demonstrates that TNF downregulates the VEGF receptor, fetal liver kinase-1 (Flk-1), on tumor endothelium in a human melanoma xenograft model. NIH1286 human melanoma cells were transduced with a 720-bp fragment of the human VEGF(121) gene to develop well-vascularized tumors that served as an amplified system for measuring Flk-1 expression changes. We injected 5 x 10(6) cells subcutaneously, each of two distinct single cell clones (NIH1286/3 and NIH1286/15), into athymic nude mice to produce tumors approximately 10 mm in size. Each animal then received either BSA or TNF in BSA by tail vein. Tumors harvested at different time points post-TNF were analyzed for Flk-1 mRNA and protein expression. Data obtained showed that intravascular TNF downregulated Flk-1 expression in tumor endothelial cells. This effect could contribute to the antitumor activity of TNF known to target tumor vasculature.     

Platelets, thrombospondin-1 and human dermal fibroblasts cooperate for stimulation of endothelial cell tubulogenesis through VEGF and PAI-1 regulation

During cutaneous wound repair, platelets, dermal fibroblasts (DF) and endothelial cells all cooperate. We have presently investigated the regulation of endothelial cell tubulogenesis by human platelet thrombospondin-1 (TSP-1), in comparison to transforming growth factor-beta1 (TGF-beta1) and total platelet lysates (PL), in a fibrin matrix cell culture system incorporating DF. TSP-1, TGF-beta1 and PL all stimulated VEGF expression in DF dose dependently at mRNA and protein level. TSP-1- and PL-treated DF supernatants significantly stimulated capillary-like structure formation (tubulogenesis) by dermal microvascular endothelial cells (HMEC-1 and HDMEC), in part via VEGF, as confirmed with neutralizing anti-VEGF antibodies. In contrast, TGF-beta1-treated DF supernatants did not induce tubulogenesis. This apparent discrepancy could be explained by the differential expression regulation in HMEC-1 of fibrinolysis and metalloproteinase mediators by TSP-1 and TGF-beta1. TSP-1 potently reduced the expression of plasminogen activator inhibitor-1 (PAI-1) (mRNA and protein), whereas TGF-beta1 enhanced it. The crucial role of PAI-1 in tubulogenesis was confirmed via the addition of active recombinant PAI-1, which abrogated tubulogenesis. In contrast, neutralizing PAI-1 antibodies enhanced tubulogenesis. Our results suggest that platelet TSP-1 released in a wound stimulates endothelial cell tubulogenesis through an upregulation of DF VEGF expression and a downregulation of endothelial cell PAI-1 expression.     

Regulation by retinoic acid of ICAM-1 expression on human tumor cell lines.

In a group of four human tumor cell lines comprising one melanoma, one glioma, one teratocarcinoma and one neuroblastoma, the expression of the intercellular adhesion molecule-1 (ICAM-1) was found to be significantly increased following treatment with 10 microM of all-trans retinoic acid. In the melanoma and glioma cell lines HS 294T and HS 683, greater than 90% of the cells reacted with the anti-ICAM-1 monoclonal antibody (mAb) CL203.4 in the absence of treatment. Retinoic acid increased the cell surface expression of the molecule by 2-fold. In the teratocarcinoma and neuroblastoma cell lines, TERA-2 and SK-N-SH, the constitutive expression of ICAM-1 was weak, the percentage of cells stained above the background being less than 25%. Retinoic acid induced ICAM-1 expression in greater than 80% of the cells and increased the levels of expression by 2.5 to 3-fold. Immunoprecipitation studies in biosynthetically labeled cells as well as RNase protection analysis confirmed that retinoic acid treatment increased the amount of ICAM-1 at both the protein and mRNA level. The induction or stimulation occurred within 24 h, was maximal after 4 days and reversible.