A Novel and Effective Method for Human Primary Skin Melanocytes and Metastatic Melanoma Cell Isolation

The development of an effective method of melanocyte isolation and culture is necessary for basic and clinical studies concerning skin diseases, including skin pigmentation disorders and melanoma. In this paper, we describe a novel, non-enzymatic and effective method of skin melanocyte and metastatic melanoma cell isolation and culture (along with the spontaneous spheroid creation) from skin or lymph node explants. The method is based on the selective harvesting of melanocytes and melanoma cells emigrating from the cultured explants. Thereby, isolated cells retain their natural phenotypical features, such as expression of tyrosinase and Melan-A as well as melanin production and are not contaminated by keratinocytes and fibroblasts. Such melanocyte and melanoma cell cultures may be very useful for medical and cosmetology studies, including studies of antitumor therapies.

A UHPLC-Mass Spectrometry View of Human Melanocytic Cells Uncovers Potential Lipid Biomarkers of Melanoma

Melanoma is the deadliest form of skin cancer due to its ability to colonize distant sites and initiate metastasis. Although these processes largely depend on the lipid-based cell membrane scaffold, our understanding of the melanoma lipid phenotype lags behind most other aspects of this tumor cell. Here, we examined a panel of normal human epidermal and nevus melanocytes and primary and metastatic melanoma cell lines to determine whether distinctive cell-intrinsic lipidomes can discern non-neoplastic from neoplastic melanocytes and define their metastatic potential. Lipidome profiles were obtained by UHPLC-ESI mass-spectrometry, and differences in the signatures were analyzed by multivariate statistical analyses. Significant and highly specific changes in more than 30 lipid species were annotated in the initiation of melanoma, whereas less numerous changes were associated with melanoma progression and the non-malignant transformation of nevus melanocytes. Notably, the “malignancy lipid signature” features marked drops in pivotal membrane lipids, like sphingomyelins, and aberrant elevation of ether-type lipids and phosphatidylglycerol and phosphatidylinositol variants, suggesting a previously undefined remodeling of sphingolipid and glycerophospholipid metabolism. Besides broadening the molecular definition of this neoplasm, the different lipid profiles identified may help improve the clinical diagnosis/prognosis and facilitate therapeutic interventions for cutaneous melanoma.

Deciphering the Functional Role of RIPK4 in Melanoma

The receptor-interacting protein kinase 4 (RIPK4) plays an important role in the development and maintenance of various tissues including skin, but its role in melanoma has not been reported. Using patient-derived cell lines and clinical samples, we show that RIPK4 is expressed in melanomas at different levels. This heterogenous expression, together with very low level of RIPK4 in melanocytes, indicates that the role of this kinase in melanoma is context-dependent. While the analysis of microarray data has revealed no straightforward correlation between the stage of melanoma progression and RIPK4 expression in vivo, relatively high levels of RIPK4 are in metastatic melanoma cell lines. RIPK4 down-regulation by siRNA resulted in the attenuation of invasive potential as assessed by time-lapse video microscopy, wound-healing and transmigration assays. These effects were accompanied by reduced level of pro-invasive proteins such as MMP9, MMP2, and N-cadherin. Incubation of melanoma cells with phorbol ester (PMA) increased PKC-1β level and hyperphosphorylation of RIPK4 resulting in degradation of RIPK4. Interestingly, incubation of cells with PMA for short and long durations revealed that cell migration is controlled by the NF-κB signaling in a RIPK4-dependent (RIPK4high) or independent (RIPK4low) manner depending on cell origin (distant or lymph node metastasis) or phenotype (mesenchymal or epithelial).

Fibroblasts Influence Metastatic Melanoma Cell Sensitivity to Combined BRAF and MEK Inhibition

The sensitivity of melanoma cells to targeted therapy compounds depends on the tumor microenvironment. Three-dimensional (3D) in vitro coculture systems better reflect the native structural architecture of tissues and are ideal for investigating cellular interactions modulating cell sensitivity to drugs. Metastatic melanoma (MM) cells (SK-MEL-28 BRAF V600E mutant and SK-MEL-2 BRAF wt) were cultured as a monolayer (2D) or cocultured on 3D dermal equivalents (with fibroblasts) and treated with a BRAFi (vemurafenib) combined with a MEK inhibitor (MEKi, cobimetinib). The drug combination efficiently inhibited 2D and 3D MM cell proliferation and survival regardless of their BRAF status. Two-dimensional and three-dimensional cancer-associated fibroblasts (CAFs), isolated from a cutaneous MM biopsy, were also sensitive to the targeted therapy. Conditioned media obtained from healthy dermal fibroblasts or CAFs modulated the MM cell’s response differently to the treatment: while supernatants from healthy fibroblasts potentialized the efficiency of drugs on MM, those from CAFs tended to increase cell survival. Our data indicate that the secretory profiles of fibroblasts influence MM sensitivity to the combined vemurafenib and cobimetinib treatment and highlight the need for 3D in vitro cocultures representing the complex crosstalk between melanoma and CAFs during preclinical studies of drugs.

Chromomycin A5 induces bonafide immunogenic cell death in metastatic melanoma

Some first-line cytotoxic chemotherapics, e.g. doxorubicin, paclitaxel and oxaliplatin, induce activation of the immune system through immunogenic cell death (ICD). Tumor cells undergoing ICD function as a vaccine, releasing damage-associated molecular patterns (DAMPs), which act as adjuvants, and neoantigens of the tumor are recognized as antigens. ICD induction is rare, however it yields better and long-lasting antitumor responses to chemotherapy. Advanced metastatic melanoma (AMM) is incurable for more than half of patients. The discovery of ICD inducers against AMM is an interesting drug discovery strategy with high translational potential. Here we evaluated ICD induction of four highly cytotoxic chromomycins A (CA5-8). B16-F10, a metastatic melanoma cell line, treated with CA5-8 and doxorubicin exhibited ICD features such as autophagy and apoptosis, externalization of calreticulin, and releasing of HMGB1. However, CA5-treated cells had the best profile, also inducing ATP release, ERp57 externalization, phosphorylation of eIF2α and altering expression of transcription of genes related to autophagy, endoplasmic reticulum stress, and apoptosis. Bonafide ICD induction by CA5 was confirmed by a C57BL/6 mice vaccination assay with CA5-treated cells. These findings support a high potential of CA5 as an anticancer candidate against AMM.

Conjugation with Phospholipids as a Modification Increasing Anticancer Activity of Phenolic Acids in Metastatic Melanoma—In Vitro and In Silico Studies

Phenolic acids possess many beneficial biological activities, including antioxidant and anti-inflammatory properties. Unfortunately, their low bioavailability restricts their potential medical uses, as it limits the concentration of phenolic acids achievable in the organism. The conjugation with phospholipids constitutes one of the most effective strategies to enhance compounds bioavailability in biological systems. In the present study, the conjugates of anisic (ANISA) and veratric acid (VA) with phosphatidylcholine (PC) were investigated. Since both ANISA and VA are inhibitors of tyrosinase, a melanocyte enzyme, the expression of which increases during tumorigenesis, anticancer potential of the conjugates was tested in several metastatic melanoma cell lines. The conjugates proved to be antiproliferative, apoptosis-inducing and cell-cycle-affecting agents, selective for cancerous cells and not affecting normal fibroblasts. The conjugates substituted by ANISA and VA, respectively, at both the sn-1 and sn-2 positions of PC, appeared the most promising, since they were effective against the vast majority of metastatic melanoma cell lines. Additionally, the conjugation of phenolic acids to PC increased their antioxidant activity. Molecular modeling was employed for the first time to estimate the features of the investigated conjugates relevant to their anticancer properties and membrane permeation. Again, the conjugates substituted by phenolic acid at both the sn-1 and sn-2 positions of PC seemed to be presumably most bioavailable.

Antiproliferative Activity of Selected Triterpene Acids from Rosemary on Metastatic Melanoma Cell Line WM-266-4

BACKGROUND: Natural products and their derivatives, particularly secondary metabolites, have been recognized for many years as an important source of therapeutic agents. In this context, pentacyclic triterpene acids including betulinic acid (BA), oleanolic acid (OA), and ursolic acid (UA) are highly valuable triterpenic acids because of their wide range of biological activities. AIM: Therefore, the aim of our study was to investigate any potential effect of BA, UA, and OA on human melanoma WM-266-4 cells’ proliferation activity. METHODS: BA, UA, and OA have been prepared in dimethyl sulfoxide in concentration range from 0.002 to 200 μM separately or in selected combination (UA+OA ratio 1:1 or 3.5:1), while cells in cell culture medium served as controls. The rapid colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was used to measure proliferation activity of the metastatic melanoma cell line WM-266-4 after being exposed to selected concentrations of BA, UA, OA, or UA+OA and during different time periods. Student’s t-test was used for single statistical comparisons. Data were analyzed using IBM SPSS 25.0 (IBM Corp., Armonk, NY). To account for multiple comparisons bias, p < 0.001 was considered statistically significant. RESULTS: Our results showed decreased cell proliferation activity after 4 h of incubation of WM-266-4 cells with BA, UA, OA, and UA+OA. The highest inhibitory effect was noted when cells were incubated with selected triterpenic acids and both combinations of UA+OA during the incubation period of 48 h. When compared to control cells, concentration of 2 μM was the lowest concentration of BA that showed a significant decrease of the cells’ proliferation activity regardless the incubation period (4 h, 24 h, and 48 h) (p < 0.001). CONCLUSION: Our encouraging results could be a good starting point for further studies on possible use of BA, UA, and OA in prevention and treatment of metastatic melanoma.

Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential

Peroxidasin, a heme peroxidase, has been shown to play a role in cancer progression. mRNA expression has been reported to be upregulated in metastatic melanoma cell lines and connected to the invasive phenotype, but little is known about how peroxidasin acts in cancer cells. We have analyzed peroxidasin protein expression and activity in eight metastatic melanoma cell lines using an ELISA developed with an in-house peroxidasin binding protein. RNAseq data analysis confirmed high peroxidasin mRNA expression in the five cell lines classified as invasive and low expression in the three non-invasive cell lines. Protein levels of peroxidasin were higher in the cell lines with an invasive phenotype. Active peroxidasin was secreted to the cell culture medium, where it accumulated over time, and peroxidasin protein levels in the medium were also much higher in invasive than non-invasive cell lines. The only well-established physiological role of peroxidasin is in the formation of a sulfilimine bond, which cross-links collagen IV in basement membranes via catalyzed oxidation of bromide to hypobromous acid. We found that peroxidasin secreted from melanoma cells formed sulfilimine bonds in uncross-linked collagen IV, confirming peroxidasin activity and hypobromous acid formation. Moreover, 3-bromotyrosine, a stable product of hypobromous acid reacting with tyrosine residues, was detected in invasive melanoma cells, substantiating that their expression of peroxidasin generates hypobromous acid, and showing that it does not exclusively react with collagen IV, but also with other biomolecules.

Development and Pilot Screen of Novel High Content Assay for Down Regulators of Expression of Heterogenous Nuclear Ribonuclear Protein H2

BACKGROUND/AIMS: Despite recent advances in melanoma drug discovery, the average overall survival of patients with late-stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al., Cell Physiol Biochem 2019;53:656-686). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H2 that can be used for melanoma therapy and research. METHODS: We established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. RESULTS: All plate-based QC parameters were acceptable: %CV = 6.7±0.3, S/B = 21±2.1, Z' = 0.75±0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis. CONCLUSION: We developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNPH1 and 2 in a large compound collection and, therefore, is amenable to a large-scale screening effort.