Inhibited Melanoma Tumor Growth Research Articles

Development of novel naphthalimide derivatives and their evaluation as potential melanoma therapeutics

Synthesis and anti-melanoma activity of various naphthalimide analogs, rationally modified by introducing isothiocyanate (ITC) and thiourea (TU) functionalities, found in well-known anti-cancer agents, is described. The structure–activity relationship comparison of the novel agents in inhibiting cancer cell growth was evaluated in various melanoma cell lines. Both ITC and TU analogs effectively inhibited cell viability and induced apoptosis in various human melanoma cells. Nitro substitution and increase in alkyl chain length, in general, enhanced the apoptotic activity of ITC derivatives. All the new compounds were well tolerated when injected intraperitoneal (i.p.) in mice at effective doses at which both the ITC and TU derivatives inhibited melanoma tumor growth in mice following i.p. xenograft. The nitro substituted naphthalimide–ITC derivative 3d was found to be the most effective in inducing apoptosis, and in inhibiting melanoma cell and tumor growth.

Abstract 600: The HSV-2 oncolytic virus ΔPK induces multiple death and inflammatory programs associated with inhibition of melanoma tumor growth

Abstract Metastatic melanoma is a highly aggressive and drug resistant cancer. Oncolytic virotherapy is a novel therapeutic strategy designed to circumvent cancer resistance through virus replication-induced cancer cell lysis. Unfortunately, its clinical efficacy is modest, apparently related to poor virus replication within the tumors and the failure to stimulate inflammatory/immunotherapeutic pathways. The growth compromised herpes simplex virus type 2 (HSV-2) mutant ΔPK causes metastatic melanoma cell death through activation of multiple non-redundant death pathways. Activation of calpain and caspases-3 and -7 provide the bulk of the death programs, as evidenced by the finding of 80% protection in cells treated with the combination of calpain (PD150606) and pancaspase (zVAD-fmk) inhibitors. In addition, ΔPK upregulates the autophagy proteins Beclin-1 and LC-II and the autophagy inhibitor 3-methyladenine (3-MA) reduces ΔPK-induced cell death by 15-17%. Cell death is further decreased (to a total of 28-34%) by treatment with 3-MA together with the JNK inhibitor SP600125, indicating that JNK signaling and autophagy contribute independently to ΔPK-induced cell death. Inflammatory-related pathways contribute to ΔPK-induced melanoma cell death, as evidenced by caspase-1 activation, interleukin (IL)-1β release into the conditioned medium and upregulation of pro-apoptotic/stress signaling molecules and pro-inflammatory cytokines and chemokines identified by microarray analysis. These include BAD, BIK, BOK, NLRC4, CIDEB, IL-1β, IL-6, IL-8, IL-12α, IL-12β, LTα, CXCL-10, c-JUN and caspases-4, -5, -6, -8, and -10. Tumor burden is significantly reduced by intratumoral injection of ΔPK and xenografts generated from a fresh clinical isolate, were completely eliminated for a period of 1 year following treatment. Growth inhibition is associated with activation of calpain and caspases-3, -7 and -1 and expression of the inflammatory cytokine TNF-α. Collectively, the data indicate that ΔPK eliminates tumor burden through the simultaneous activation of multiple non-redundant death programs and pro-inflammatory signaling, providing a distinct and promising virotherapeutic approach. 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 600. doi:10.1158/1538-7445.AM2011-600

Abstract 1350: Novel ABI analogs target the colchicine site in tubulin and overcome multidrug resistance in metastatic melanoma

Abstract With rapidly rising incidences of metastatic melanoma and its high resistance to current therapeutic agents, developing more effective drugs is urgently needed. We recently discovered a novel class of 2-aryl-4-benzoyl-imidazoles (ABI) analogs. These compounds showed great antiproliferative activity on melanoma cells and the IC50 is as low as 8 nM. ABIs showed equal potency against multidrug resistant melanoma cells and the sensitive parent cells, suggesting that they can effectively overcome P-gp meditated multidrug resistance. ABIs inhibited capillary network formation which indicates high potential for anti-angiogenesis. Cell cycle analysis showed ABIs arrested cells in the G2/M phase and inhibited tubulin polymerization. ABIs bind to the colchicine binding site in the α/β-tubulin heterodimer as indicated by a number of evidences including competitive binding and molecular modeling studies. Immunofluorescence experiments revealed that microtubule modality was dramatically changed with ABIs treatment. In vivo studies using both B16-F1/C57BL model and A375/SHO model showed significant inhibition of melanoma tumor growth by ABIs. These novel analogs hold great promise as new selective agents for the improved treatment of malignant melanoma. 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 1350. doi:10.1158/1538-7445.AM2011-1350

Abstract 2834: Anti-DLL4 treatment inhibits melanoma tumor growth, recurrence, metastases, and reduces frequency of cancer stem cells in a clinically relevant tumor model in NOD/SCID mice

Abstract Metastatic burden at the time of diagnosis is often the cause of death in patients suffering from malignant melanoma. More predictive in vivo animal tumor models are needed to elucidate both the efficacy and mechanism of activity for newly developed anti-cancer therapeutic agents. We have developed an orthotopic melanoma model in NOD/SCID mice with intradermal implant of human melanoma cells that are derived directly from human cancer patient specimens. These tumors retain much of the original human cancer heterogeneity, phenotype, and the ability to form metastases in the lungs, lymph nodes, liver, intestines, and brain _ recapitulating the metastatic invasiveness seen in the clinic. We have developed antibodies against the human and the murine Delta-like Notch ligand DLL4, which target both tumor cells and vasculature, and reduce the frequency of cancer stem cells (CSCs) in human tumors implanted into NOD/SCID mice (Hoey et al., Cell Stem Cell 5:1680177, 2009). In our studies with a human melanoma model, we evaluated the anti-tumor and anti-metastatic activities of anti-DLL4 and found a significant inhibition of both primary tumor and metastases in the lungs, liver, intestine and brain. Moreover, in combination with the standard-of-care Paclitaxel, anti-DLL4 treatment caused a greater reduction of tumor growth than either agent alone and an increase in apoptosis of tumor cells compared to control antibody or paclitaxel alone These data were consistent with the results from the limiting dilution assay experiments that demonstrated a significant reduction of tumor initiating cells by the anti-DLL4 alone (1:15) and a further decrease in combination with paclitaxel (1:33) compared to the control antibody (1:5), but no effect by paclitaxel (1:5). In other studies aimed to examine tumor recurrence after excision of the primary tumors, treatment with anti-DLL4, alone and with paclitaxel, prevented recurrence of the primary tumors. In contrast, paclitaxel alone increased the frequency of tumor relapse when compared to the control and other treatments. In addition, anti-DLL4 alone inhibited metastases to lungs, lymph nodes, liver, brain and intestines and this effect was further enhanced by the combination with paclitaxel in the liver and intestines. The results from these studies demonstrate for the first time a significant inhibition of tumor recurrence and metastases in human melanoma by anti-DLL4 and establish a link between reduction of CSC frequency and the concurrent inhibition of metastases. Therefore our findings reveal great potential of our anti-DLL4 antibody as an effective anti-cancer therapy for melanoma patients. 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 2834. doi:10.1158/1538-7445.AM2011-2834

Phytochemical potential of Eruca sativa for inhibition of melanoma tumor growth

Solvent extracts from the aerial and root parts and seed oil from E. sativa (rocket salad) were assayed for anticancer activity against melanoma cells. The seed oil (isothiocyanates rich) significantly (p < 0.01) reduced the tumor growth comparable to the control. Remarkably, the seed oil inhibited melanoma growth and angiogenesis in mice without any major toxicity. The findings qualify seed oil for further investigations in the real of cancer prevention and treatment.

Abstract 730: Targeting colchicine binding site in tubulin for the treatment of advanced melanoma

Abstract Inhibition of tubulin dynamics has been a fruitful drug target for cancer treatment. Out the three ligand binding sites in tubulin, drugs targeting taxane binding site (e.g. taxol and epithilones) and vinca alkaloids binding site (e.g. vinblastine) are clinically very successful drugs. However, no FDA-approved cancer drugs targeting the colchicine binding site in tubulin currently exist. In our ongoing efforts to search for effective drugs for the treatment of advanced melanoma that are highly resistant to all existing cancer treatment, we have discovered a new class of compounds that are not substrates of P-glycoprotein (Pgp) and are highly efficacious against human melanoma tumors in A375 xenograft models. At relatively low doses (5∼15 mg/kg), these compounds inhibit melanoma tumor growth more effectively than DTIC (the gold standard drug for advanced melanoma) at 60 mg/kg. Extensive biological and molecular modeling studies suggest that these compounds work by competitively binding to the colchicine site in tubulin. Further optimization of these compounds hold great promise in providing an effective agent for the treatment of advanced melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 730.

Synthesis, in vitro structure-activity relationship, and in vivo studies of 2-arylthiazolidine-4-carboxylic acid amides as anticancer agents.

A series of (2RS,4R)-2-arylthiazolidine-4-carboxylic acid amide (ATCAA) was synthesized. Antiproliferative activity against melanoma and prostate cancer cells compared with control cells (fibroblast and RH7777, respectively) was evaluated. Compound 3id showed the best selectivity and growth-inhibition activity against three melanoma cell lines (B16-F1, A375, and WM-164). Compounds 15b and 3ac had good selectivity and potency against four prostate cancer cell lines (DU 145, PC-3, LNCaP, and PPC-1). The structure-activity relationship (SAR) of the side chain, the thiazolidine ring, and phenyl substituents is discussed. Cell cycle analysis showed that the percentage of cancer cells undergoing apoptosis (sub-G1 phase) increased after treatment with 1b and 3ad, which also strongly inhibited melanoma colony formation. In vivo studies on nude mice bearing A375 melanoma tumors showed that compound 1b inhibited tumor growth in a dose-dependent manner. At a dose of 10mg/kg, 1b significantly inhibited melanoma tumor growth and showed higher efficacy than did dacarbazine at 60mg/kg.

Small interfering RNA‐mediated CXCR1 or CXCR2 knock‐down inhibits melanoma tumor growth and invasion

CXCR1 and CXCR2 are receptors for CXCL-8 and are differentially expressed on melanoma and endothelial cells. In this study, we determined the functional role of these receptors in melanoma progression. We stably knock-down the expression of CXCR1 and/or CXCR2 in A375-SM (SM; high metastatic) human melanoma cells by short-hairpin RNA transfection. Cell proliferation, migration, invasion, ERK phosphorlyation and cytoskeletal rearrangements were carried out in vitro. In vivo growth was evaluated using murine subcutaneous xenograft model. Our data demonstrate that knock-down of CXCR1 and/or CXCR2 expression, inhibited melanoma cell proliferation, survival, migration and invasive potential in vitro. Moreover, we also observed inhibition of ERK phosphorylation and cytoskeltal rearrangement in SM-shCXCR1, SM-shCXCR2 and SM-shCXCR1/2 cells. Furthermore, when SM-shCXCR1 or SM-shCXCR2 cells implanted in nude mice, tumor growth, proliferation and microvessel density was significantly inhibited as compared to SM-control cells. In addition, we observed a significant increase in melanoma cell apoptosis in SM-shCXCR1 and SM-shCXCR2 tumors compared to SM-control tumors. Together, these data demonstrate that CXCR1 and CXCR2 expression play a critical role in human melanoma tumor progression and, functional blockade of CXCR1 and CXCR2 could be potentially used for future therapeutic intervention in malignant melanoma.

ADAM10 Is the Constitutive Functional Sheddase of CD44 in Human Melanoma Cells

CD44 proteins are cell surface receptors for hyaluronic acid (HA), a component of the extracellular matrix that has multiple effects on cell behavior. CD44 can be shed from the cell surface by proteolytic cleavage. The resulting soluble form can interfere with the interaction between HA and membrane-bound CD44. Soluble CD44 can abolish the cell proliferation-promoting effect of HA on melanoma cell lines, suggesting that a better understanding of the shedding process might identify ways of blocking tumor cell proliferation. ADAM10, ADAM17, and MMP14 have previously been implicated in the shedding of CD44 from various tumor cells. Using immunohistochemistry we demonstrate that ADAM10 and ADAM17 but not MMP14 are significantly expressed on melanoma cells in histological sections. In human melanoma cell lines expression of these proteases could be blocked by transfection with appropriate siRNAs. However, only blocking of ADAM10 expression led to decreased shedding of CD44. In parallel, cell proliferation was promoted. Confocal microscopy demonstrated that ADAM10 and CD44 colocalize on the cell surface. We conclude that ADAM10 is the predominant protease involved in the constitutive shedding of endogenous CD44 from melanoma cells, and that enhancement of ADAM10 activity could be an approach to decrease the proliferation of melanoma cells.

Reduction of Human Melanoma Tumor Growth in Severe Combined Immunodeficient Mice by Passive Transfer of Antibodies Induced by a High Molecular Weight Melanoma-Associated Antigen Mimotope Vaccine

The high molecular weight melanoma-associated antigen (HMW-MAA) is an attractive target for immunotherapy of malignant melanoma. We have recently generated a vaccine based on the HMW-MAA mimotope 225D9.2+ that was able to induce anti-HMW-MAA antibodies with antitumor activity in vitro. Here, we investigated the antitumor activity of these antibodies in a human melanoma xenotransplant severe combined immunodeficient (SCID) mouse model. Tumors were established by injecting the human melanoma 518A2 cells into C.B.17 SCID/SCID mice. In tumor prevention experiments, 200 microg purified total IgG antibodies were injected intravenously the same day or on day 5 in therapeutic experiments. Antibody administration was repeated every fourth day and tumor volumes were measured. Antibody specificity and tumor infiltration by macrophages were investigated by immunohistochemistry. Within 35 days after cell inoculation, antibody treatment reduced tumor growth up to 40% in the therapeutic and up to 62% in the tumor prevention experiments compared with the control mice. In tumors of all groups, a similar distribution of the HMW-MAA and no differences in infiltration of macrophages were detected by immunohistochemistry. Here, we showed that antibodies induced by the 225D9.2+ mimotope effectively inhibited melanoma tumor growth. Additional mechanisms besides antibody-dependent cell cytotoxicity like disruption of interactions of melanoma cells mediated by extracellular matrix components seem to be involved in tumor growth inhibition. Based on our findings, we suggest that active immunization with this mimotope might be a promising strategy for treatment of melanoma.

Synthesis and Anticancer Activity Comparison of Phenylalkyl Isoselenocyanates with Corresponding Naturally Occurring and Synthetic Isothiocyanates

Synthesis and identification of novel phenylalkyl isoselenocyanates (ISCs), isosteric selenium analogues of naturally occurring phenylalkyl isothiocyanates (ITCs), as effective cytotoxic and antitumor agents are described. The structure-activity relationship comparison of ISCs with ITCs and effect of the increasing alkyl chain length in inhibiting cancer cell growth were evaluated on melanoma, prostate, breast, glioblastoma, sarcoma, and colon cancer cell lines. IC(50) values for ISC compounds were generally lower than their corresponding ITC analogues. Similarly, in UACC 903 human melanoma cells, the inhibition of cell proliferation and induction of apoptosis were more pronounced with ISCs compared to ITCs. Further, ISCs and ITCs effectively inhibited melanoma tumor growth in mice following intraperitoneal xenograft. A similar reduction in tumor size was observed at 3 times lower doses of ISCs compared to corresponding ITCs.

Thematic Review Series: Sphingolipids. Ganglioside GM3 suppresses the proangiogenic effects of vascular endothelial growth factor and ganglioside GD1a

Thematic Review Series: Sphingolipids. Ganglioside GM3 suppresses the proangiogenic effects of vascular endothelial growth factor and ganglioside GD1a

GPR56 binds tissue transglutaminase and inhibits melanoma tumor growth and metastasis

GPR56 binds tissue transglutaminase and inhibits melanoma tumor growth and metastasis

GPR56, an atypical G protein-coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metastasis.

The survival and growth of tumor cells in a foreign environment is considered a rate-limiting step during metastasis. To identify genes that may be essential for this process, we isolated highly metastatic variants from a poorly metastatic human melanoma cell line and performed expression analyses of metastases and primary tumors from these cells. GPR56 is among the genes markedly down-regulated in the metastatic variants. We show that overexpression of GPR56 suppresses tumor growth and metastasis, whereas reduced expression of GPR56 enhances tumor progression. Levels of GPR56 do not correlate with growth rate in vitro, suggesting that GPR56 may mediate growth suppression by interaction with a component in the tumor microenvironment in vivo. We show that GPR56 binds specifically to tissue transglutaminase, TG2, a widespread component of tissue and tumor stroma previously implicated as an inhibitor of tumor progression. We discuss the mechanisms whereby GPR56-TG2 interactions may suppress tumor growth and metastasis.

Inhibition of melanoma tumor growth by a pharmacological inhibitor of MetAP-2, PPI-2458

Over the past few decades, melanoma has shown the fastest growing incidence rate of all cancers. This malignancy is clinically defined by its potential to rapidly metastasize, and advanced metastatic melanomas are highly resistant to existing therapeutic regimens. Here, we report that PPI-2458, a novel, orally active agent of the fumagillin class of irreversible methionine aminopeptidase-2 (MetAP-2) inhibitors, potently inhibited the proliferation of B16F10 melanoma cells in vitro, with a growth inhibitory concentration 50% (GI50) of 0.2 nM. B16F10 growth inhibition was correlated with the inhibition of MetAP-2 enzyme, in a dose-dependent fashion, as determined by a pharmacodynamic assay, which measures the amount of uninhibited MetAP-2 following PPI-2458 treatment. Prolonged exposure of B16F10 cells to PPI-2458 at concentrations of up to 1 microM, 5,000-fold above the GI50, did not alter their sensitivity to PPI-2458 growth inhibition and no drug resistance was observed. Moreover, prolonged exposure to this agent induced melanogenesis, concomitant with the elevated expression of the melanocyte-specific enzymes tyrosinase and tyrosinase-related proteins (TRP) 1 and 2, a morphological feature associated with differentiated melanocytes. PPI-2458, when administered orally (p.o.), significantly inhibited B16F10 tumor growth in mice in a dose-dependent fashion, with a maximum inhibition of 62% at 100 mg/kg. This growth inhibition was directly correlated to the amount of irreversibly inhibited MetAP-2 (80% at 100 mg/kg PPI-2458) in tumor tissue. These data demonstrate that PPI-2458 has potent antiproliferative activity against B16F10 cells in vitro and in vivo, and that both activities are directly correlated with levels of MetAP-2 enzyme inhibition. This antiproliferative activity, coupled with additional observations from studies in vitro (absence of detectable resistance to PPI-2458 and induction of morphological features consistent with differentiated melanocytes), provides a rationale for assessing the therapeutic potential of PPI-2458 in the treatment of melanoma.

Mutant V599EB-Raf Regulates Growth and Vascular Development of Malignant Melanoma Tumors

Activating mutations of the B-RAF gene are observed in >60% of human melanomas. Approximately 90% of these mutations occur in the activation segment of the kinase domain as a single-base substitution that converts a valine to glutamic acid at codon 599 (V599E) in exon 15. This mutation causes activation of the kinase as well as downstream effectors of the mitogen-activated protein kinase-signaling cascade, leading to melanoma tumor development by an as yet unknown mechanism. In this study, we have identified the role of (V599E)B-Raf in melanoma tumor development by characterizing the mechanism by which this mutant protein promotes melanoma tumorigenesis. Small interfering RNA targeted against B-Raf or a Raf kinase inhibitor (BAY 43-9006) was used to reduce expression and/or activity of (V599E)B-Raf in melanoma tumors. This inhibition led to reduced activity of the mitogen-activated protein kinase-signaling cascade and inhibited tumor development in animals. Targeted reduction of mutant (V599E)B-Raf expression (activity) in melanoma cells before tumor formation inhibited tumorigenesis by reducing the growth potential of melanoma cells. In contrast, reduction of mutant (V599E)B-Raf activity in preexisting tumors prevented further vascular development mediated through decreased vascular endothelial growth factor secretion, subsequently increasing apoptosis in tumors. These effects in combination with reduced proliferative capacity halted growth, but did not shrink the size of preexisting melanoma tumors. Thus, these studies identify the mechanistic underpinnings by which mutant (V599E)B-RAF promotes melanoma development and show the effectiveness of targeting this protein to inhibit melanoma tumor growth.

Gene therapy for colon cancer by adeno-associated viral vector-mediated transfer of survivin Cys84Ala mutant

Reactivation of survivin expression is involved in carcinogenesis and angiogenesis in colon cancer. Previous in vitro studies showed that mutation of the cysteine residue at position 84 (Cys84Ala) of survivin generates a dominant-negative mutant that triggers mitotic catastrophe and apoptosis. We investigated the therapeutic effect of the adeno-associated virus (AAV)-mediated survivin mutant (Cys84Ala) on colon cancer. Survivin mutant (Cys84Ala) (Sur-Mut(Cys84Ala)) was cloned into the AAV expression vector pAM/CAG-WPRE.poly(A) to generate recombinant AAV-Sur-Mut(Cys84Ala) virus. Cell proliferation, apoptosis, mitotic catastrophe, and tumor growth were measured in vitro and in vivo. Transduction of colon cancer cells with rAAV-Sur-Mut(Cys84Ala) inhibited cell proliferation and induced apoptosis and mitotic catastrophe in vitro. rAAV-Sur-Mut(Cys84Ala) sensitized colon cancer cells to chemotherapeutic drugs. Furthermore, expression of survivin mutant mediated by AAV inhibited tumorigenesis in colon cancer cells. Intratumoral injection of rAAV-Sur-Mut(Cys84Ala) significantly induced apoptosis and mitotic catastrophe and inhibited angiogenesis and tumor growth in a colon cancer xenograft model in vivo. No obvious cytotoxicity to other tissues was observed. More importantly, rAAV-Sur-Mut(Cys84Ala) expression strongly enhanced the antitumor activity of 5-Fluorouracil (5-FU), resulting in regression of established tumors. Our results showed that rAAV-Sur-Mut(Cys84Ala) induced apoptosis and mitotic catastrophe and inhibited tumor angiogenesis and tumor growth. Thus, use of AAV-mediated survivin mutant (Cys84Ala) is a promising strategy in colon cancer gene therapy.

Construction and expression of intracellular anti-ATF-1 single chain Fv fragment: a modality to inhibit melanoma tumor growth and metastasis

The expression of the transcriptional regulators activating transcription factor-1 (ATF-1) and cAMP-responsive element (CRE)-binding protein (CREB) is upregulated in metastatic melanoma cells. However, how overexpression of ATF-1/CREB contributes to the acquisition of the metastatic phenotype is unclear. Monoclonal antibodies against ATF-1 have previously been used to inhibit the transcriptional activity of ATF-1 and its associated proteins. Here, the effect of disrupting ATF-1 activity was investigated using intracellular expression of an inhibitory anti-ATF-1 single chain antibody fragment (ScFv). Intracellular expression of ScFv anti-ATF-1 in human melanoma cells caused significant reduction in CRE-dependent promoter activation. In addition, expression of ScFv anti-ATF-1 in melanoma cells suppressed their tumorigenicity and metastatic potential in nude mice. Furthermore, expression of ScFv anti-ATF-1 rendered the melanoma cells susceptible to thapsigargin-induced apoptosis in vitro and caused massive apoptosis in vivo in tumors transplanted subcutaneously into nude mice. These studies demonstrate the potential usage of ScFv anti-ATF-1 as an inhibitor of tumor growth and metastasis of solid tumors overexpressing ATF-1/CREB.

The angiostatic activity of interferon-inducible protein-10/CXCL10 in human melanoma depends on binding to CXCR3 but not to glycosaminoglycan

Human interferon-inducible protein 10 (IP-10; HGMW-approved gene symbol CXCL10) is an ELR− CXC chemokine that contains binding domains for both the chemokine receptor CXCR3 and glycosaminoglycans. IP-10 has been recently demonstrated to be a potent angiostatic protein in vivo. Whether IP-10 exerts its angiostatic function through binding to CXCR3, glycosaminoglycans, or both, is not clear. To clarify this issue, we created expression constructs for mutants of IP-10 that exhibit partial (IP-10C) or total (IP-10C22) loss of binding to CXCR3 or loss of binding to glycosaminoglycans (IP-10H and IP-10C22H). The A375 human melanoma cell line was transfected with these expression vectors, and stable clones were selected and inoculated subcutaneously into nude mice. As expected, tumor cells secreting wild-type IP-10 showed remarkable reduction in tumor growth compared to control vector-transfected tumor cells. Surprisingly, mutation of IP-10 resulting in partial loss of receptor binding (IP-10C), or loss of GAG binding (IP-10H), did not significantly alter the ability to inhibit tumor growth. This tumor growth inhibition was associated with a reduction in microvessel density, leading to the observed increase in both tumor cell apoptosis and necrosis. In contrast, expression of the IP-10C22 mutant failed to inhibit melanoma tumor growth. These data suggest that CXCR3 receptor binding, but not glycosaminoglycan binding, is essential for the tumor angiostatic activity of IP-10. We conclude that the arginine 22 amino acid residue of IP-10 is essential for both CXCR3 binding and angiostasis.

Transforming growth factor-β1 inhibits tumor growth in a mouse melanoma model by down-regulating the plasminogen activation system

The degradation of basement membranes by tumor cells involves secretion and activation of proteinases, such as matrix metalloproteinases (MMPs) and the plasminogen activation system (uPA, tPA, PAI-1), and results from an imbalance between their inhibitors and activators, controlled by various growth factors or cytokines. Among them, the TGF-β family is one of the most intriguing because it has been reported either to decrease or promote cancer progression. In the present paper, we studied the effect of TGF-β1 in a mouse melanoma model. In vivo, TGF-β1 inhibited tumor growth after subcutaneous injection of B16F1 cells in syngenic mice. In vitro, TGF-β1 did not alter B16F1 cell proliferation, but strongly decreased their migration through Matrigel-coated membranes. The protease production was analyzed by zymography, Western blot, or RT-PCR. MMP-2 and TIMP-2 expression were not altered by TGF-β1. In contrast, TGF-β1 triggered a large decrease of uPA and tPA, as well as a decrease of uPA and uPAR mRNAs. By Western blot and RT-PCR analyses, TGF-β1 was shown to induce a strong increase of PAI-1 synthesis. Collectively, these results suggest that TGF-β1 may inhibit melanoma tumor growth by specifically decreasing plasmin activity of tumor cells and play a protective role during the earliest stages of tumor progression.