M14 Melanoma Cells Research Articles

Abstract 5936: A novel class of orally bioavailable small molecules induces potent and sustained tumor growth inhibition and T cell infiltration in various solid tumor mouse models

Abstract The introduction of targeted immunomodulating agents has transformed cancer treatment over the last decade by demonstrating unprecedented efficacy in patients who respond. However, limited clinical response rates as well as adverse events underline the need to identify additional novel modalities in cancer immunotherapy. Here we report on the discovery of a novel class of low molecular weight compounds for oral application that selectively enhance tumor-reactive T cell cytotoxicity. Hit-to-lead development of compound hit series A was performed based on medicinal chemistry to investigate structure-activity-relations. A-306, a representative of the A compound class, induced increased T cell proliferation, elevated levels of the activation markers CD25 and CD69 as well as enhanced secretion of IFN-γ, IL-2 and TNF-α upon anti-CD3/CD28 stimulation. A-306 treatment without T cell receptor ligation had no effect on T cell activation, suggesting selective activation potential only in the context of antigen encounter. Furthermore, A-306 strengthened T-cell mediated killing of M21 melanoma cells and enhanced T cell function in response to viral antigens by showing a Th1 signature. Medicinal chemistry efforts resulted in front runner compounds A-481 and A-687 that show a similar profile and potencies down to nanomolar level. In a murine B16-SIY melanoma and EO771 breast cancer model oral single-agent administration with front runner compounds was well tolerated, showed good bioavailability in lymphoid organs and plasma and resulted in significant tumor growth inhibition beyond the end of treatment (D21). T cells from tumor draining lymph nodes of long-term surviving mice in both cancer models showed a distinct activation pattern indicative of anti-tumor immunity. Furthermore, oral application of A-481 in combination with intra peritoneal administration of anti-PD1 antibodies eradicated B16-SIY melanoma cells in a synergistic manner. Taken together, we report on the identification of a novel class of small molecules possessing high potential for selective anti-tumor activation of the immune system upon oral administration. Front runner compounds A-481 and A-687, which significantly inhibit tumor growth in a B16-SIY melanoma and EO771 breast cancer mouse model, lead to prolonged survival beyond the end of treatment and display a good safety profile. The final candidate compound will undergo testing in toxicity studies and enter a first-in-human trial in selected solid tumors in 2025. Citation Format: Bianca Gapp, Gunther Zischinsky, Maria Urban, Nora Birnbacher, Alice Schwarzböck, Sabine Grünaug, Tommaso Gastaldi, Anton Stütz, Peter Nussbaumer, Stefan Stranner, Julia Buchberger, Andreas Birbach, Daniela Schögl, Mario Kuttke, Alexander Dohnal, Romana Gugenberger. A novel class of orally bioavailable small molecules induces potent and sustained tumor growth inhibition and T cell infiltration in various solid tumor mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5936.

Enhancing drug delivery with supramolecular amphiphilic macrocycle nanoparticles: selective targeting of CDK4/6 inhibitor palbociclib to melanoma.

Drug delivery systems based on amphiphilic supramolecular macrocycles have garnered increased attention over the past two decades due to their ability to successfully formulate nanoparticles. Macrocyclic (MC) materials can self-assemble at lower concentrations without the need for surfactants and polymers, but surfactants are required to form and stabilize nanoparticles at higher concentrations. Using MCs to deliver both hydrophilic and hydrophobic guest molecules is advantageous. We developed two novel types of amphiphilic macrocycle nanoparticles (MC NPs) capable of delivering either Nile Red (NR) (a hydrophobic model) or Rhodamine B (RhB) (a hydrophilic model) fluorescent dyes. We extensively characterized the materials using various techniques to determine size, morphology, stability, hemolysis, fluorescence, loading efficiency (LE), and loading capacity (LC). We then loaded the CDK4/6 inhibitor Palbociclib (Palb) into both MC NPs using a solvent diffusion method. This yielded Palb-MC NPs in the size range of 65-90 nm. They exhibited high stability over time and in fetal bovine serum with negligible toxicity against erythrocytes. Cytotoxicity was minimal when tested against RAW macrophages, human fibroblast HDFn, and adipose stromal cells (ASCs) at higher concentrations of MC NPs. Cell viability studies were conducted with different concentrations of MC NPs, Palb-MC NPs, and free Palb against RAW macrophages, human U-87 GBM, and human M14 melanoma cell lines in vitro. Flow cytometry experiments revealed that blank MC NPs and Palb-MC NPs were selectively targeted to melanoma cells, resulting in cell death compared to the other two cell lines. Future work will focus on studying the biological effect of MC NPs including their binding affinity with molecules/receptors expressed on the M14 and other melanoma cell surfaces by molecular docking simulations. Subsequently, we will evaluate the MCs as a component of combination therapy in a murine melanoma model.

Abstract 2753: Interleukin-10 and transforming growth factor-beta do not suppress tumor killing activity of PBMC-derived SUPLEXA cells

Abstract Introduction: SUPLEXA cells are peripheral blood mononuclear cells (PBMCs) being developed by Alloplex Biotherapeutics as an autologous adoptive therapy for cancer. The novel nature of SUPLEXA cells is that they are developed from peripheral blood mononuclear cells (PBMCs) that have been activated and expanded by engineered leukocyte stimulator cell lines (ENLIST cells), which are melanoma cell lines that express immunomodulatory proteins to program the tumor killing phenotype of SUPLEXA cells. We find that the SUPLEXA cell manufacturing process reproducibly results in a mixture of NK cells, CD8 T cells, and γδ T cells with broad tumor cell killing activity. In this study, we evaluated the effects of two major immunosuppressive cytokines found in the tumor microenvironment (IL-10 and TGF-β) on the tumor cytolytic function and cytokine release by SUPLEXA cells. Results: SUPLEXA cells were prepared by a 14-day process of PBMC induction by ENLIST cells for 5 days followed by 9 days expansion in cytokines. SUPLEXA cell mediated tumor cytolytic assays using red fluorescent protein (RFP) expressing M14 melanoma cells as targets were performed in the absence or presence of IL-10 or TGF-β (1.25 - 10 ng/ml). Cytokine levels in 48-hour culture supernatants from cytolytic reactions and SUPLEXA or M14 target cells alone were measured using a 36-plex Luminex assay. Without cytokine addition, SUPLEXA mediated cytolysis was 47.3% at 8:1 and 30.9% at 4:1 effector to target (E:T) ratios. Significant increases in SUPLEXA cell tumoricidal activity was observed with addition of IL-10 (74.3% at 8:1 and 51.7% at 4:1 E:T ratios at 2.5 ng/ml IL-10 added, P &It 0.001 by two-way ANOVA). Adding TGF-β caused a reduction in cytolytic activity that was not significant (36.5% at 8:1 and 20.8% at 4:1 E:T ratios with 2.5 ng/ml TGF-β added). Supernatants from cytolysis assays showed that SUPLEXA cells produced IFNγ, MIP-1α, RANTES, IL-8, Gro-α, MIG, and IP-10 in response to tumor cells. IL-10 significantly augmented IFNγ, RANTES, and MIP-1α production by SUPLEXA cells in a dose-dependent fashion. In contrast, TGF-β reduced IFNγ, MIP-1α, Gro-α, RANTES, and IP-10 production, but increased IL-8 production by SUPLEXA cells. Conclusions: An in vitro approach was used as a surrogate for what might occur in patients with solid tumors to test whether potent immune suppressive cytokines that are present in the tumor microenvironment might inhibit SUPLEXA cell function. Our findings indicate that IL-10 and TGF-β do not significantly reduce the tumor killing activity of SUPLEXA cells. Instead, we discovered that IL-10 significantly enhances SUPLEXA cell function and cytokine production, which suggests that SUPLEXA cells may acquire heightened tumoricidal activity and promote immune surveillance by augmenting cytokine and chemokine levels within the tumor microenvironment when given therapeutically to cancer patients. Citation Format: Frank Borriello, Joshua W. Keegan, James A. Lederer. Interleukin-10 and transforming growth factor-beta do not suppress tumor killing activity of PBMC-derived SUPLEXA cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2753.

PLA2G6 Silencing Suppresses Melanoma Progression and Affects Ferroptosis Revealed by Quantitative Proteomics.

Although phospholipase A2 group VI (PLA2G6) is involved in oncogenesis in several human tumors, its expression and role in cutaneous malignant melanoma (CMM) pathogenesis remains unclear. Here, by using the Oncomine and CCLE online database, immunohistochemistry, RT-qPCR, and western blotting analysis, we revealed that PLA2G6 was markedly up-regulated in CMM tissues compared to nevus tissues, as well as remarkably increased in vitro in SK-MEL-28 and M14 melanoma cell lines compared to human melanocytes. In vivo, PLA2G6 was also elevated in nine melanoma tissues compared to adjacent tissues. To investigate the malignant behaviors of PLA2G6 in CMM, SK-MEL-28 and M14 cell lines with PLA2G6 stable knockdown by RNAi strategy were constructed. Through CCK8 and colony formation assays in vitro and xenograft tumor experiment in vivo, we found that knockdown of PLA2G6 dramatically inhibited cell proliferation. The results of scratch-wound and transwell assays suggested that the migration and invasion of melanoma cells were prominently suppressed after silencing PLA2G6. In addition, flow cytometry showed that the knockdown of PLA2G6 promoted the apoptosis rate of melanoma cells. To further explore the potential molecular mechanism, we used liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) proteomic and bioinformatics analysis. The GO and KEGG analysis suggested that the underlying mechanism of PLA2G6 in CMM might be associated with the ferroptosis pathway, and ferroptosis-related proteins were validated to be differentially expressed in PLA2G6 knockdown SK-MEL-28 and M14 cells. Together, these results suggested that PLA2G6 knockdown significantly inhibited cell proliferation, metastasis, and promoted apoptosis in melanoma. Our findings on the biological function of PLA2G6 and the underlying association between PLA2G6 and ferroptosis in melanoma may contribute to developing a potential therapeutic strategy for melanoma.

Advanced Nanosystems for Clinical Translation

Advanced Nanosystems for Clinical Translation

QSAR and Docking Studies on Some Potential Anti-Cancer Agents to Predict their Effect on M14 Melanoma Cell Line

The global incidence of melanoma cancer is increasing rapidly and its metastatic form causes high mortality rates. The research report illustrates the development of a quantitative structure–activity relationship (QSAR) to predict the activities (pGI50) of some anti-cancer agents on melanoma cancer cell line. Subsequently, most potent compounds that showed better pGI50 activities were selected and screened via Lipinski’s rule of five for drug likeness, ADMET risk parameters and lastly docking simulation studies was performed to elucidate their binding mode. Kennard-Stone algorithm was adopted for the data division, the genetic function algorithm was employed for variable selection and multiple linear regressions method was utilized for model development. The derived QSAR model showed, respectively acceptable [( $$R^{2}$$ (0.805), $$R_{adjusted}^{2}$$ (0.773), Qcv2 (0.754) and $$R_{pred}^{2}$$ (0.703)]. The obtained cRP2 for Y-randomization is 0.649, and applicability-domain (A-D) was assessed via leveraged method. Among the screened compounds via Lipinski’s rule of five for oral bioavailability, ADMET risk filter for drug-like features and Docking simulation studies, compound 18 and 25 were identified as the best ligands having the better interactions energies (− 150.679 kcal mol−1 and − 176.246 kcal mol−1) and showed better interactions with the receptor than vemurafenib (− 147.245 kcal mol−1). Thus, the results of this research would be helpful in identification of lead molecule and optimization of novel drug.

Silver Nanocarriers Targeted with a CendR Peptide Potentiate the Cytotoxic Activity of an Anticancer Drug

AbstractSilver nanoparticles (AgNP) can be tracked in cells and tissues by optical imaging and isotopic fingerprinting. AgNPs are particularly useful as endocytosis probes as they can be rapidly dissolved with a biocompatible etching solution to eliminate extracellular NPs.Here, affinity‐targeted AgNPs are evaluated as therapeutic carriers of a potent cytotoxic compound, monomethyl auristatin E (MMAE). AgNPs are coated with MMAE via a lysosomal protease cathepsin B sensitive linker and functionalized with a prototypic CendR peptide (RPARPAR) that targets neuropilin‐1 (NRP‐1). This gives the AgNPs dual tumor specificity as both cathepsin B and NRP‐1 are overexpressed in many types of solid tumors. Cellular imaging, flow cytometry, viability assays, and high‐performance liquid chromatography‐mass spectrometry (HPLC‐MS) analysis show that the RPARPAR–MMAE–AgNPs are internalized and induce apoptotic cell death in NRP‐1‐positive PPC‐1 prostate cancer cells while sparing NRP‐1‐negative M21 melanoma cells. Furthermore, in a mixed culture of PPC‐1 and M21 cells, RPARPAR–MMAE–AgNP treatment selectively eliminates NRP‐1‐positive PPC‐1 cells. The study demonstrates that affinity‐targeted AgNPs can be used as carriers to selectively deliver and potentiate the activity of cytotoxic compounds in vitro, and that treatment with a biocompatible etching solution can be used to control the internalization and, thus, the cytotoxicity of these AgNPs.

Disintegrin Tablysin-15 Suppresses Cancer Hallmarks in Melanoma Cells by Blocking FAK/Akt/ERK and NF-κB Signaling.

Integrins are crucial anti-cancer therapy targets. We previously showed that tablysin-15 is an integrin antagonist with its Arg-Gly-Asp motif in a novel structural context. Here we investigated the anti-cancer effects and mechanisms of action of tablysin-15 in melanoma cells. Cell adhesion, competitive binding, cell viability, and ATP chemiluminescence assays were used to analyze the binding of tablysin-15 to αvβ3 integrin and its phenotypic effects. Wound healing, transwells, and zymography were performed to detect motility and matrix metalloproteinase- 2/-9 activities. PARP and caspase-3 cleavage were used as apoptosis assays, while LDH release and flow cytometry were used for necrosis and cell cycle analysis. The expression of mRNAs and proteins of target molecules was measured by qRT-PCR and western blotting, respectively. Tablysin-15 dose-dependently inhibited the proliferation, migration, and invasion of M21 cells through integrin αvβ3. The proliferation inhibition caused by tablysin-15 was attributable to G0/G1 phase arrest rather than apoptosis or necrosis. Furthermore, tablysin-15 suppressed MMP-2/- 9 activities and the mRNA expression of MMP-2/-9 and COX-2 but was upregulated TIMP-1 in M21 cells. Meanwhile, tablysin-15 suppressed the expression of cyclin D1/E and CDK 2/6, the phosphorylation of FAK, Akt, and ERK, and nuclear translocation of NF-κB, while increasing the expression of the CDK inhibitor p21waf1/C1. Taken together, tablysin-15 might inhibit melanoma cell metastasis and proliferation by competing with αvβ3 integrin, thereby blocking FAK-associated signaling pathways and nuclear translocation of NF-κB. Tablysin-15 has reliable anti-cancer effects against M21 melanoma cells, suggesting tablysin-15 is a promising anti-tumor drug.

Abnormal overexpression of G9a in melanoma cells promotes cancer progression via upregulation of the Notch1 signaling pathway.

Malignant melanoma is a type of very dangerous skin cancer. Histone modifiers usually become dysregulated during the process of carcinoma development, thus there is potential for a histone modifier inhibitor as a useful drug for cancer therapy. There is a multitude of evidence regarding the role of G9a, a histone methyltransferase (HMTase), in tumorigenesis. In this study, we first showed that G9a was significantly upregulated in melanoma patients. Using the TCGA database, we found a significantly higher expression of G9a in primary melanoma samples (n = 461) compared to normal skin samples (n = 551). Next, we knocked down G9a in human M14 and A375 melanoma cell lines in vitro via small interfering RNA (siRNA). This resulted in a significant decrease in cell viability, migration and invasion, and an increase in cell apoptosis. UNC0642 is a small molecule inhibitor of G9a that demonstrates minimal cell toxicity and good in vivo pharmacokinetic characteristics. We investigated the role of UNC0642 in melanoma cells, and detected its anti-cancer effects in vitro and in vivo. Next, we treated cells with UNC0642, and observed a significant decrease in cell viability in M14 and A375 cell lines. Furthermore, treatment with UNC0642 resulted in increased apoptosis. In immunocompetent mice bearing A375 engrafts, treatment with UNC0642 inhibited tumor growth. Results of Western blot analysis revealed that administration of UNC0642 or silencing of G9a expression by siRNA reduced Notch1 expression significantly and decreased the level of Hes1 in A375. All in all, the data from our study demonstrates potential of G9a as a therapeutic target in the treatment of melanoma.

1,2,3-Triazole-Chalcone hybrids: Synthesis, in vitro cytotoxic activity and mechanistic investigation of apoptosis induction in multiple myeloma RPMI-8226

A new series of 1,2,3-triazole-chalcone hybrids has been synthesized and screened in vitro against a panel of 60 human cancer cell lines according to NCI (USA) protocol. Compound 4d having 3, 4-dimethoxyphenyl chalcone moiety, the most potent derivative, inhibited the growth of RPMI-8226 and SR leukemia cell lines by 99.73% and 94.95% at 10 μM, respectively. Also, it inhibited the growth of M14 melanoma, K-562 leukemia, and MCF7 breast cancer cell lines by more than 80% at the same test concentration. 4d showed IC50 values less than 1 μM on six types of tumor cells and high selectivity index reached to 104 fold on MCF7. Compound 4d showed superior activity than methotrexate and gefitinib against the most sensitive leukemia cell lines in addition to higher or comparable activity against the rest sensitive cell lines. Flow cytometry analysis in RPMI-8226 cells revealed that compound 4d caused cell cycle arrest at G2/M phase and induced apoptosis in a dose dependant manner. Mechanistic evaluation referred this apoptosis induction to triggering mitochondrial apoptotic pathway through inducing ROS accumulation, increasing Bax/Bcl-2 ratio and activation of caspases 3, 7 and 9.

Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells.

Malignant melanoma is the most life-threatening neoplasm of the skin. Despite the increase in incidence, melanoma is becoming more resistant to current therapeutic agents. The bioactive compound frugoside has been recently reported to inhibit growth when used in various cancer cells. However, this effect has not been demonstrated in melanoma. Here, we found that frugoside inhibited the rate of reduction of hyperoxidized peroxiredoxins (Prxs) by downregulating sulfiredoxin (Srx) expression. Furthermore, frugoside increased the accumulation of sulfinic Prxs and reactive oxygen species (ROS) and stimulated p-p38 activation, resulting in the mitochondria-mediated death of M14 and A375 human melanoma cells. The mitochondria-mediated cell death induced by frugoside was inhibited by the overexpression of Srx and antioxidants, such as N-acetyl cysteine and diphenyleneiodonium. In addition, we observed that frugoside inhibited tumor growth without toxicity through a M14 xenograft animal model. Taken together, our findings reveal that frugoside exhibits a novel antitumor effect based on a ROS-mediated cell death in melanoma cells, which may have therapeutic implications.

Imaging modification of colon carcinoma cells exposed to lipid based nanovectors for drug delivery: a scanning electron microscopy investigation.

The adsorption at cell surfaces and cell internalization of two drug delivery lipid based nanovectors has been investigated by means of Field Emission Scanning Electron Microscopy (FE-SEM) operating at low beam voltage on two different colon carcinoma cell lines, CaCo-2 and CoLo-205, that were compared with the M14 melanoma cell line, as a reference. The cells were incubated with the investigated multifunctional nanovectors, based on liposomes and magnetic micelles loaded with 5-fluorouracil, as a chemotherapeutic agent, and a FE-SEM systematic investigation was performed, enabling a detailed imaging of any morphological changes of the drug exposed cells as a function of time. The results of the FE-SEM investigation were validated by MTS assay and immunofluorescence staining of the Ki-67 protein performed on the investigated cell lines at different times. The two nanoformulations resulted in a comparable effect on CaCo-2 and M14 cell lines, while for CoLo 205 cells, the liposomes provided an cytotoxic activity higher than that observed in the case of the micelles. The study highlighted the high potential of FE-SEM as a valuable complementary technique for imaging and monitoring in time the drug effects on the selected cells exposed to the two different nanoformulations.

JTC-801 Suppresses Melanoma Cells Growth through the PI3K‑Akt‑mTOR Signaling Pathways.

Melanoma is considered as one of the most potentially fatal and aggressive malignancies. Due to the limited efficacy or drug resistance of the current targeted therapies of melanoma, developing new therapeutic drugs against new targets to effectively control tumor growth is greatly needed. In this study, the effect of JTC-801, a selective small-molecule antagonist of nociceptin receptor and analgesic agent, on a melanoma cell line, M14, has been studied. We demonstrate herein that JTC-801 could efficiently suppress the proliferation, migration and invasion capacity of the M14 melanoma cells, and induced a strong apoptosis. Importantly, our results provide the underlying molecular mechanism of these effects. JTC-801 cells regulate M14 cells by inhibiting the PI3K-Akt‑mTOR pathway. These results suggest that JTC-801 should be further studied in preclinical modes to establish whether it represents a potential small anticancer candidate drug against melanoma.

UNBS5162 inhibits proliferation of human melanoma cells by inducing apoptosis via the PI3K/Akt pathway.

UNBS5162, a novel naphthalimide, is generated by UNBS3157 hydrolysis in physiological saline. In the present study, the effects of UNBS5162 on M14 human melanoma cells were evaluated by Cell Counting Kit‑8 and transwell assays, as well as western blotting. The underlying mechanism of apoptosis induced by UNBS5162 was investigated. The results demonstrated that proliferation of UNBS5162‑treated M14 melanoma cells was markedly inhibited in a time‑dependent manner. The flow cytometry results indicated a markedly increased apoptosis rate in the experimental group compared with in the control group (23.8±0.4 vs. 7.62±0.5%). Microscopy analysis revealed that the invasive and migratory abilities of UNBS5162‑treated M14 cells were markedly suppressed. Furthermore, UNBS5162 treatment led to decreased expression of the anti‑apoptotic protein B‑cell lymphoma 2, but increased expression of the pro‑apoptotic proteins Bcl‑2‑associated X protein and caspase‑3. In addition, the expression of several key proteins involved in the phosphatidylinositol‑4,5‑bisphosphate3‑kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway was altered in M14 cells treated with UNBS5162. Based on these results, it may be hypothesized that UNBS5162 suppresses the proliferation of M14 cells by inducing apoptosis via inhibition of key proteins in the PI3K/Akt/mTOR signaling pathway.

The antioxidant, aged garlic extract, exerts cytotoxic effects on wild-type and multidrug-resistant human cancer cells by altering mitochondrial permeability

Aged garlic extract (AGE) has been shown to possess therapeutic properties in cancer; however its mechanisms of action are unclear. In this study, we demonstrate by MTT assay that AGE exerts an anti-proliferative effect on a panel of both sensitive and multidrug-resistant (MDR) human cancer cell lines and enhances the effects of hyperthermia (42˚C) on M14 melanoma cells. The evaluation of the mitochondrial activity in whole cancer cells treated with AGE, performed by cytofluorimetric analysis in the presence of the lipophilic cationic fluorochrome JC-1, revealed the occurrence of dose-dependent mitochondrial membrane depolarization. Membrane potential was measured by the TPP+ selective electrode. In order to shed light on its mechanisms of action, the effects of AGE on isolated rat liver mitochondria were also examined. In this regard, AGE induced a mitochondrial membrane hyperpolarization of approximately 15mV through a mechanism that was similar to that observed with the ionophores, nigericin or salinomycin, by activating an exchange between endogenous K+ with exogenous H+. The prolonged incubation of the mitochondria with AGE induced depolarization and matrix swelling, indicative of mitochondrial permeability transition induction that, however, occurs through a different mechanism from the well-known one. In particular, the transition pore opening induced by AGE was due to the rearrangement of the mitochondrial membranes following the increased activity of the K+/H+ exchanger. On the whole, the findings of this study indicate that AGE exerts cytotoxic effects on cancer cells by altering mitochondrial permeability. In particular, AGE in the mitochondria activates K+/H+ exchanger, causes oxidative stress and induces mitochondrial permeability transition(MPT).

Targeting the cross-talk between Urokinase receptor and Formyl peptide receptor type 1 to prevent invasion and trans-endothelial migration of melanoma cells

BackgroundAccumulating evidence demonstrates that the Urokinase Receptor (uPAR) regulates tumor cell migration through its assembly in composite regulatory units with transmembrane receptors, and uPAR88–92 is the minimal sequence required to induce cell motility through the Formyl Peptide Receptor type 1 (FPR1). Both uPAR and FPR1 are involved in melanoma tumor progression, suggesting that they may be targeted for therapeutic purposes. In this study, the role of the uPAR-FPR1 cross-talk to sustain melanoma cell ability to invade extracellular matrix and cross endothelial barriers is investigated. Also, the possibility that inhibition of the uPAR mediated FPR1-dependent signaling may prevent matrix invasion and transendothelial migration of melanoma cells was investigated.MethodsExpression levels of uPAR and FPR1 were assessed by immunocytochemistry, Western Blot and qRT-PCR. Cell migration was investigated by Boyden chamber and wound-healing assays. Migration and invasion kinetics, trans-endothelial migration and proliferation of melanoma cells were monitored in real time using the xCELLigence technology. The agonist-triggered FPR1 internalization was visualized by confocal microscope. Cell adhesion to endothelium was determined by fluorometer measurement of cell-associated fluorescence or identified on multiple z-series by laser confocal microscopy. The 3D–organotypic models were set up by seeding melanoma cells onto collagen I matrices embedded dermal fibroblasts. Data were analyzed by one-way ANOVA and post-hoc Dunnett t-test for multiple comparisons.ResultsWe found that the co-expression of uPAR and FPR1 confers to A375 and M14 melanoma cells a clear-cut capability to move towards chemotactic gradients, to cross extracellular matrix and endothelial monolayers. FPR1 activity is required, as cell migration and invasion were abrogated by receptor desensitization. Finally, melanoma cell ability to move toward chemotactic gradients, invade matrigel or fibroblast-embedded collagen matrices and cross endothelial monolayers are prevented by anti-uPAR84–95 antibodies or by the RI-3 peptide which we have previously shown to inhibit the uPAR84–95/FPR1 interaction.ConclusionsCollectively, our findings identify uPAR and FPR1 as relevant effectors of melanoma cell invasiveness and suggest that inhibitors of the uPAR84–95/FPR1 cross-talk may be useful for the treatment of metastatic melanoma.

Authentication of M14 melanoma cell line proves misidentification of MDA-MB-435 breast cancer cell line.

A variety of analytical approaches have indicated that melanoma cell line UCLA‐SO‐M14 (M14) and breast carcinoma cell line MDA‐MB‐435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross‐contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA‐MB‐435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA‐MB‐435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA‐MB‐435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA‐MB‐435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research.

1248P - Tumor-stroma interactions as a determinant of drug resistance in BRAF-mut melanoma

Background: In BRAF-mut melanoma combined BRAF/MEK inhibition increases survival; however, pharmacological effects on the genetically “normal” tumor microenvironment (i.e. paradox MAPK activation) may set the stage for the development of drug resistance. Methods: GFP-labeled cutaneous fibroblasts (HFF) were co-cultured with melanoma cells, in the presence or absence of direct cell-cell contact, and response to Dabrafenib (D) and Trametinib (T), alone or combined, was monitored over time. SEMA6A and AXL were preliminarily evaluated as potential mediators of such interactions. Results: HFF significantly protected (60-100% protection at the lowest two drug concentrations) BRAF-mut M14 melanoma cells from the growth inhibitory activity of D and T, alone or combined; however, combined D+T at the highest concentrations overcame stroma-mediated protection and eliminated both cell populations. Thus, combined BRAF/MEK inhibition resulted in strongly synergistic interactions, as compared to single agent treatments, only under co-culture conditions (CI 0.6 and 0.2 for M14 and HFF cells, respectively). Protective melanoma/stroma interactions were mediated by direct cell-cell contact, as co-cultures in trans-well Boyden chambers or isolated cultures using conditioned medium (HFF-conditioned medium for M14; M14-conditioned medium for HFF), did not affect pharmacological response. As SEMA6A expression is tightly controlled by MAPK and AXL mediates resistance to MAPK inhibition in melanoma, we assessed their potential as mediators of stroma-mediated melanoma protection: interestingly, SEMA6A and AXL expression in a panel of melanoma cell lines were inversely correlated; moreover, in cell lines derived by primary and cutaneous metastases of the same patient, AXL expression was upregulated at the mRNA and protein level in cells derived from metastatic lesions. Conclusions: Tumor-stroma interactions protect BRAF-mut melanoma from MAPK inhibition; such functional protection is mediated by cell-cell contact. SEMA6A and AXL are possible mediators of this interaction and their reciprocal relationships are being studied in melanoma cell line models and clinical series. Legal entity responsible for the study: Regina Elena Cancer Institute- San Gallicano Dermatologic Institute Funding: AIRC (18622-14362-9979) Disclosure: All authors have declared no conflicts of interest.

Mechanisms of CXCR7 induction in malignant melanoma development.

Malignant melanoma (MM) is a highly malignant skin tumor. The mechanism of MM pathogenesis and its signaling pathways are not well characterized. C-X-C chemokine receptor type 7 (CXCR7) has been reported to regulate cancer cell invasion. The present study sought to investigate the effects of CXCR7 on MM development. First, CXCR7 expression levels were assessed in the skin tumor tissue of patients with MM. Then, CXCR7 small hairpin RNA was used in M14 melanoma cells in a Transwell culture model and in a transplanted mouse model to test the effects of CXCR7. In addition, immunohistochemistry staining, reverse transcription-quantitative polymerase chain reaction and western blotting were used. The results revealed that CXCR7 expression levels were significantly higher in MM tissue compared with squamous cell carcinoma or basal cell carcinoma tissue. Knocking down CXCR7 in M14 cells significantly inhibited cell migration and invasion in the Transwell culture model. Furthermore, CXCR7 knockdown also significantly reduced the transplanted tumor size, weight and vascular number in the mouse model. It was concluded that CXCR7 interacts with C-X-C motif chemokine ligand 12 to activate the chemokine receptor signaling pathway, and to increase melanoma cell migration, invasion and development.

Synthesis of caffeic acid ester morpholines and their activation effects on tyrosinase

Abstract Two new caffeic acid ester derivatives, caffeic acid-4-(2-hydroxyethyl) morpholine ester (Z-1) and caffeic acid-4-(3-hydroxypropyl) morpholine ester (Z-2), were synthesized, and their structures were characterized by LC–MS, IR, and 1H NMR. The activation effects of the two compounds on tyrosinase activity were evaluated. Compounds Z-1 and Z-2 showed potent activation effects, and their EC50 values were determined to be 0.075 and 0.0375 mmol/L, respectively. Moreover, the activation mechanism was determined to be of noncompetitive activation type. Interactions of the two compounds with tyrosinase were further analyzed by fluorescence quenching and molecular simulation assays. The results indicated that the effects on the tyrosinase activity were relative to the length of the carbon chain of the compounds. In addition, human M14 melanoma cells showed increased intracellular tyrosinase activity after treatment with the two compounds for 24 h. The expressions of TYR, TRP-1, TRP-2, and α-MSH proteins were upregulated in a dose-dependent manner during a 24-h treatment. These results suggested that compounds Z-1 and Z-2 might represent a novel approach for an effective therapy for diseases associate with tyrosinase dysfunction, such as vitiligo and hair graying.