A Preclinical Model of Cutaneous Melanoma Based on Reconstructed Human Epidermis

Malignant melanoma is among the tumor entities with the highest increase of incidence worldwide. To elucidate melanoma progression and develop new effective therapies, rodent models are commonly used. While these do not adequately reflect human physiology, two-dimensional cell cultures lack crucial elements of the tumor microenvironment. To address this shortcoming, we have developed a melanoma skin equivalent based on an open-source epidermal model. Melanoma cell lines with different driver mutations were incorporated into these models forming distinguishable tumor aggregates within a stratified epidermis. Although barrier properties of the skin equivalents were not affected by incorporation of melanoma cells, their presence resulted in a higher metabolic activity indicated by an increased glucose consumption. Furthermore, we re-isolated single cells from the models to characterize the proliferation state within the respective model. The applicability of our model for tumor therapeutics was demonstrated by treatment with a commonly used v-raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor vemurafenib. This selective BRAF inhibitor successfully reduced tumor growth in the models harboring BRAF-mutated melanoma cells. Hence, our model is a promising tool to investigate melanoma development and as a preclinical model for drug discovery.

Suppressing Effect of Na+/Ca2+ Exchanger (NCX) Inhibitors on the Growth of Melanoma Cells

The role of calcium ion (Ca2+) signaling in tumorigenicity has received increasing attention in melanoma research. Previous Ca2+ signaling studies focused on Ca2+ entry routes, but rarely explored the role of Ca2+ extrusion. Functioning of the Na+/Ca2+ exchanger (NCX) on the plasma membrane is the major way of Ca2+ extrusion, but very few associations between NCX and melanoma have been reported. Here, we explored whether pharmacological modulation of the NCX could suppress melanoma and promise new therapeutic strategies. Methods included cell viability assay, Ca2+ imaging, immunoblotting, and cell death analysis. The NCX inhibitors SN-6 and YM-244769 were used to selectively block reverse operation of the NCX. Bepridil, KB-R7943, and CB-DMB blocked either reverse or forward NCX operation. We found that blocking the reverse NCX with SN-6 or YM-244769 (5–100 μM) did not affect melanoma cells or increase cytosolic Ca2+. Bepridil, KB-R7943, and CB-DMB all significantly suppressed melanoma cells with IC50 values of 3–20 μM. Bepridil and KB-R7943 elevated intracellular Ca2+ level of melanoma. Bepridil-induced melanoma cell death came from cell cycle arrest and enhanced apoptosis, which were all attenuated by the Ca2+ chelator BAPTA-AM. As compared with melanoma, normal melanocytes had lower NCX1 expression and were less sensitive to the cytotoxicity of bepridil. In conclusion, blockade of the forward but not the reverse NCX leads to Ca2+-related cell death in melanoma and the NCX is a potential drug target for cancer therapy.

HDAC2 Is Involved in the Regulation of BRN3A in Melanocytes and Melanoma

The neural crest transcription factor BRN3A is essential for the proliferation and survival of melanoma cells. It is frequently expressed in melanoma but not in normal melanocytes or benign nevi. The mechanisms underlying the aberrant expression of BRN3A are unknown. Here, we investigated the epigenetic regulation of BRN3A in melanocytes and melanoma cell lines treated with DNA methyltransferase (DNMT), histone acetyltransferase (HAT), and histone deacetylase (HDAC) inhibitors. DNMT and HAT inhibition did not significantly alter BRN3A expression levels, whereas panHDAC inhibition by trichostatin A led to increased expression. Treatment with the isoform-specific HDAC inhibitor mocetinostat, but not with PCI-34051, also increased BRN3A expression levels, suggesting that class I HDACs HDAC1, HDAC2, and HDAC3, and class IV HDAC11, were involved in the regulation of BRN3A expression. Transient silencing of HDACs 1, 2, 3, and 11 by siRNAs revealed that, specifically, HDAC2 inhibition was able to increase BRN3A expression. ChIP-Seq analysis uncovered that HDAC2 inhibition specifically increased H3K27ac levels at a distal enhancer region of the BRN3A gene. Altogether, our data suggest that HDAC2 is a key epigenetic regulator of BRN3A in melanocytes and melanoma cells. These results highlight the importance of epigenetic mechanisms in regulating melanoma oncogenes.

The Anticancer Potential of Doxycycline and Minocycline—A Comparative Study on Amelanotic Melanoma Cell Lines

Malignant melanoma is still a serious medical problem. Relatively high mortality, a still-growing number of newly diagnosed cases, and insufficiently effective methods of therapy necessitate melanoma research. Tetracyclines are compounds with pleiotropic pharmacological properties. Previously published studies on melanotic melanoma cells ascertained that minocycline and doxycycline exerted an anti-melanoma effect. The purpose of the study was to assess the anti-melanoma potential and mechanisms of action of minocycline and doxycycline using A375 and C32 human amelanotic melanoma cell lines. The obtained results indicate that the tested drugs inhibited proliferation, decreased cell viability, and induced apoptosis in amelanotic melanoma cells. The treatment caused changes in the cell cycle profile and decreased the intracellular level of reduced thiols and mitochondrial membrane potential. The exposure of A375 and C32 cells to minocycline and doxycycline triggered the release of cytochrome c and activated initiator and effector caspases. The anti-melanoma effect of analyzed drugs appeared to be related to the up-regulation of ERK1/2 and MITF. Moreover, it was noticed that minocycline and doxycycline increased the level of LC3A/B, an autophagy marker, in A375 cells. In summary, the study showed the pleiotropic anti-cancer action of minocycline and doxycycline against amelanotic melanoma cells. Considering all results, it could be concluded that doxycycline was a more potent drug than minocycline.

Viscoelastic parameterization of human skin cells characterize material behavior at multiple timescales

Countless biophysical studies have sought distinct markers in the cellular mechanical response that could be linked to morphogenesis, homeostasis, and disease. Here, an iterative-fitting methodology visualizes the time-dependent viscoelastic behavior of human skin cells under physiologically relevant conditions. Past investigations often involved parameterizing elastic relationships and assuming purely Hertzian contact mechanics, which fails to properly account for the rich temporal information available. We demonstrate the performance superiority of the proposed iterative viscoelastic characterization method over standard open-search approaches. Our viscoelastic measurements revealed that 2D adherent metastatic melanoma cells exhibit reduced elasticity compared to their normal counterparts—melanocytes and fibroblasts, and are significantly less viscous than fibroblasts over timescales spanning three orders of magnitude. The measured loss angle indicates clear differential viscoelastic responses across multiple timescales between the measured cells. This method provides insight into the complex viscoelastic behavior of metastatic melanoma cells relevant to better understanding cancer metastasis and aggression.

Nuclear Localization of BRAFV600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells

Background: Previously, we have demonstrated that nuclear BRAFV600E is associated with melanoma aggressiveness and vemurafenib resistance. However, the underlying mechanisms of how nuclear localization of BRAFV600E promotes cell aggressiveness have not yet been investigated. Despite therapeutic advancements targeting cutaneous melanoma, unknown cellular processes prevent effective treatment for this malignancy, prompting an urgent need to identify new biological targets. This study aims to explore the association of inducible heme oxygenase 1 (HMOX-1) with nuclear BRAFV600E in promoting melanoma aggressiveness. Methods: Proteomics analysis was performed to identify the interacting partner(s) of nuclear BRAFV600E. Immunohistochemistry was applied to evaluate the levels of HMOX-1 and nuclear BRAFV600E expression in melanoma and adjacent healthy tissues. Immunofluorescence assessed the nuclear localization of BRAFV600E in vemurafenib-resistant A375R melanoma cells. Further study of HMOX-1 knockdown or BRAFV600E overexpression in melanoma cells suggested a role for HMOX-1 in the regulation of cell proliferation in vivo and in vitro. Finally, Western blot analysis was performed to confirm the pathway by which HMOX-1 mediates Akt signaling. Results: Proteomics results showed that HMOX-1 protein expression was 10-fold higher in resistant A375R cells compared to parental counterpart cells. In vitro and in vivo results illustrate that nuclear BRAFV600E promotes HMOX-1 overexpression, whereas HMOX-1 reduction represses melanoma cell proliferation and tumor growth. Mechanistic studies revealed that HMOX-1 was associated with nuclear BRAFV600E localization, thus promoting melanoma proliferation via a persistent activation of the AKT pathway. Conclusions: Our results highlight a previously unknown mechanism in which the nuclear BRAFV600E/HMOX-1/AKT axis plays an essential role in melanoma cell proliferation. Targeting HMOX-1 could be a novel method for treating melanoma patients who develop BRAF inhibitor resistance.

The Effects of Hedgehog Signaling Pathway on the Proliferation and Apoptosis of Melanoma Cells

Background. Studies have found that the abnormality of the Hedgehog signaling pathway is related to the occurrence and development of a variety of tumors, but the effect of this signaling pathway on melanoma cells is still unclear. Methods. This study aimed to discuss the effect of Hedgehog signaling pathway on the proliferation and apoptosis of human malignant melanoma A375 cells and explore its possible mechanism in the proliferation and apoptosis of melanoma cells. Different concentrations of Hedgehog signaling pathway inhibitor cyclopamine (5, 10, 20 and 40 μM) were used to treat human melanoma A375 cells for 24, 48, and 72 h, and set a blank control group (0 μM). Trypan blue cell counting method was used to detect cell viability. MTT method was used to detect the inhibition rate of cell proliferation. Transwell was used to detect cell invasion, and flow cytometry was used to detect cell apoptosis. Results. Through the trypan blue cell counting method and MTT experiment, it was found that the Hedgehog signaling pathway inhibitor cyclopamine has an inhibitory effect on the proliferation and viability of melanoma A375 cells ( P < 0.05 ), and the proliferation inhibitory effect is enhanced with prolonged action time in a dose- and time-dependent manner. Transwell experiment showed that compared with the blank control group, the invasion and migration ability of the treated melanoma A375 cells are significantly reduced, and the difference is statistically significant ( P < 0.05 ). Cell apoptosis experiment showed that compared with the blank control group, the apoptosis rate of A375 cells is significantly higher after treated by 40 μM cyclopamine for 24 h, and the difference is statistically significant ( P < 0.05 ). Gli1 and Bcl-2 protein are highly expressed in melanoma A375 cells, and their expressions show a downward trend ( P < 0.05 ) after being treated by cyclopamine. Conclusion. Cyclopamine inhibits cell proliferation and induces cell apoptosis by downregulating Gli1. Hedgehog signaling pathway can be used as a new target for the treatment of malignant melanoma, and multiple measures can be used to inhibit the signaling pathway to achieve a therapeutic effect.