Melanoma Cell Proliferation Research Articles

Phytochemical and biological characterization of aqueous extract of Vassobia breviflora on proliferation and viability of melanoma cells: involvement of purinergic pathway

ABSTRACT Vassobia breviflora belongs to the Solanaceae family, possessing biological activity against tumor cells and is a promising alternative for therapy. The aim of this investigation was to determine the phytochemical properties V. breviflora using ESI-ToF-MS. The cytotoxic effects of this extract were examined in B16-F10 melanoma cells and the relationship if any to purinergic signaling was involved. The antioxidant activity of total phenols, (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was analyzed, as well as production of reactive oxygen species (ROS) and nitric oxide (NO) was determined. Genotoxicity was assessed by DNA damage assay. Subsequently, the structural bioactive compounds were docked against purinoceptors P2X7 and P2Y1 receptors. The bioactive compounds found in V. breviflora were N-methyl-(2S,4 R)-trans-4-hydroxy-L-proline, calystegine B, 12-O-benzoyl- tenacigenin A and bungoside B. In vitro cytotoxicity was demonstrated at concentration ranges of 0.1–10 mg/ml, and plasmid DNA breaks only at the concentration of 10 mg/ml. V. breviflora extracts affected hydrolysis by ectoenzymes, such as ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) and ectoadenosine deaminase (E-ADA) which control levels of degradation and formation of nucleosides and nucleotides. In the presence of substrates ATP, ADP, AMP and adenosine, the activities of E-NTPDase, 5´-NT or E-ADA were significantly modulated by V. breviflora. N-methyl-(2S,4 R)-trans-4-hydroxy-L-proline presented higher binding affinity (according to receptor-ligand complex estimated binding affinity as evidenced by ∆G values) to bind to both P2X7 and P2Y1purinergic receptors. Our results suggest a putative interaction of V. breviflora bioactive compounds with growth inhibitory potential in B16-F10 melanoma and suggest that may be considered as promising compounds in melanoma and cancer treatment

WITHDRAWN: Induced PSIG expression by Herbacetin contributes to suppressing the proliferation, migration, and invasion of melanoma cells

Melanoma is a very common malignant tumor with poor prognosis. Herbacetin is a flavonol compound with outstanding anti-tumor effects. Our work investigated the biological effects and mechanism of Herbacetin in melanoma. In our study, the mRNA and protein expressions were assessed using qRT-PCR, Western blot and IHC. MSP was performed to evaluated PGIS promoter methylation level. Cell viability, migration and invasion were examined by MTT assay, transwell migration and invasion assay, respectively. Our results revealed that DNMT3B was markedly upregulated in melanoma, while PGIS was lowly expressed. Herbacetin treatment could not only inhibit the proliferation, migration, invasion of melanoma cells and inhibit the growth of melanoma in vivo. Herbacetin could also restore the abnormal expressions of DNMT3B and PGIS in melanoma cells and tumor tissues. PGIS silencing neutralized the inhibitory effects of Herbacetin on the malignant behaviors of melanoma cells. Besides, DNMT3B knockdown promoted PGIS expression via reducing PGIS promoter methylation level in melanoma cells, thereby inhibiting malignant behaviors of melanoma cells. And as expected, the inhibitory effects of Herbacetin on malignant behaviors of melanoma cells were all abolished by DNMT3B overexpression. Collectively, Herbacetin reduced DNMT3B expression to upregulate PGIS in melanoma cells and participated in suppressing the proliferation, migration, and invasion of melanoma cells.

Hyperforin Enhances Heme Oxygenase-1 Expression Triggering Lipid Peroxidation in BRAF-Mutated Melanoma Cells and Hampers the Expression of Pro-Metastatic Markers.

Hyperforin (HPF) is an acylphloroglucinol compound found abundantly in Hypericum perforatum extract which exhibits antidepressant, anti-inflammatory, antimicrobial, and antitumor activities. Our recent study revealed a potent antimelanoma effect of HPF, which hinders melanoma cell proliferation, motility, colony formation, and induces apoptosis. Furthermore, we have identified glutathione peroxidase-4 (GPX-4), a key enzyme involved in cellular protection against iron-induced lipid peroxidation, as one of the molecular targets of HPF. Thus, in three BRAF-mutated melanoma cell lines, we investigated whether iron unbalance and lipid peroxidation may be a part of the molecular mechanisms underlying the antimelanoma activity of HPF. Initially, we focused on heme oxygenase-1 (HO-1), which catalyzes the heme group into CO, biliverdin, and free iron, and observed that HPF treatment triggered the expression of this inducible enzyme. In order to investigate the mechanism involved in HO-1 induction, we verified that HPF downregulates the BTB and CNC homology 1 (BACH-1) transcription factor, an inhibitor of the heme oxygenase 1 (HMOX-1) gene transcription. Remarkably, we observed a partial recovery of cell viability and an increase in the expression of the phosphorylated and active form of retinoblastoma protein when we suppressed the HMOX-1 gene using HMOX-1 siRNA while HPF was present. This suggests that the HO-1 pathway is involved in the cytostatic effect of HPF in melanoma cells. To explore whether lipid peroxidation is induced, we conducted cytofluorimetric analysis and observed a significant increase in the fluorescence of the BODIPY C-11 probe 48 h after HPF administration in all tested melanoma cell lines. To discover the mechanism by which HPF triggers lipid peroxidation, along with the induction of HO-1, we examined the expression of additional proteins associated with iron homeostasis and lipid peroxidation. After HPF administration, we confirmed the downregulation of GPX-4 and observed low expression levels of SLC7A11, a cystine transporter crucial for the glutathione production, and ferritin, able to sequester free iron. A decreased expression level of these proteins can sensitize cells to lipid peroxidation. On the other hand, HPF treatment resulted in increased expression levels of transferrin, which facilitates iron uptake, and LC3B proteins, a molecular marker of autophagy induction. Indeed, ferritin and GPX-4 have been reported to be digested during autophagy. Altogether, these findings suggest that HPF induced lipid peroxidation likely through iron overloading and decreasing the expression of proteins that protect cells from lipid peroxidation. Finally, we examined the expression levels of proteins associated with melanoma cell invasion and metastatic potential. We observed the decreased expression of CD133, octamer-4, tyrosine-kinase receptor AXL, urokinase plasminogen activator receptor, and metalloproteinase-2 following HPF treatment. These findings provide further support for our previous observations, demonstrating the inhibitory effects of HPF on cell motility and colony formation in soft agar, which are both metastasis-related processes in tumor cells.

Exosome-delivered circRPS5 inhibits the progression of melanoma via regulating the miR-151a/NPTX1 axis.

Circular RNAs (circRNAs) have been reported to exert critical functions in tumorigenesis and development. However, the underlying mechanism by which circRNAs regulate melanoma progression remain to be elucidated. The differentially expressed circRNAs were first identified by circRNA-seq, and circRNAs were validated via qRT-PCR and Sanger sequencing. Then, the impact of circRPS5, miR-151a and NPTX1 expression on the progression of melanoma cell were determined by gain- and loss-of-function assays. The relationship between circRPS5, miR-151a, and NPTX1 was predicted by StarBase website and authenticated by luciferase reporter assay. The melanoma cells-derived exosomes were characterized using nanoparticle tracking analysis (NTA) and western blot. CircRPS5 was significantly downregulated in melanoma tissues and cell lines. Functionally, circRPS5 suppressed the proliferation, migration, and invasion of melanoma cells, and induced cell cycle arrest and apoptosis in vitro. Mechanistically, circRPS5 harbor miR-151a, acting as miRNA sponge, and then miR-151a targeted the 3'-UTR of NPTX1. Finally, circRPS5 was mainly incorporated into exosomes to inhibit the progression of melanoma cells. This finding reveal circRPS5 suppressed the progression of melanoma through miR-151a/NPTX1 pathway, and may provide a promising therapeutic strategies for melanoma.

Morusin shows potent antitumor activity for melanoma through apoptosis induction and proliferation inhibition

BackgroundThe discovery of new anti-melanoma drugs with low side effect is urgently required in the clinic. Recent studies showed that morusin, a flavonoid compound isolated from the root bark of Morus Alba, has the potential to treat multiple types of cancers, including breast cancer, gastric cancer, and prostate cancer. However, the anti-cancer effect of morusin on melanoma cells has not been investigated.MethodsWe analyzed the effects of morusin on the proliferation, cell cycle, apoptosis, cell migration and invasion ability of melanoma cells A375 and MV3, and further explored the effects of morusin on tumor formation of melanoma cell. Finally, the effects of morusin on the proliferation, cycle, apoptosis, migration and invasion of A375 cells after knockdown of p53 were detected.ResultsMorusin effectively inhibits the proliferation of melanoma cells and induces cell cycle arrest in the G2/M phase. Consistently, CyclinB1 and CDK1 that involved in the G2/M phase transition were down-regulated upon morusin treatment, which may be caused by the up-regulation of p53 and p21. In addition, morusin induces cell apoptosis and inhibits migration of melanoma cells, which correlated with the changes in the expression of the associated molecules including PARP, Caspase3, E-Cadherin and Vimentin. Moreover, morusin inhibits tumor growth in vivo with little side effect on the tumor-burden mice. Finally, p53 knockdown partially reversed morusin-mediated cell proliferation inhibition, cell cycle arrest, apoptosis, and metastasis.ConclusionCollectively, our study expanded the spectrum of the anti-cancer activity of morusin and guaranteed the clinical use of the drug for melanoma treatment.

Ircinia ramosa Sponge Extract (iSP) Induces Apoptosis in Human Melanoma Cells and Inhibits Melanoma Cell Migration and Invasiveness.

Marine compounds represent a varied source of new drugs with potential anticancer effects. Among these, sponges, including those belonging to the Irciniidae family, have been demonstrated to exert cytotoxic effects on different human cancer cells. Here, we investigated, for the first time, the therapeutic effect of an extract (referred as iSP) from the sponge, Ircinia ramosa (Porifera, Dictyoceratida, and Irciniidae), on A375 human melanoma cells. We found that iSP impaired A375 melanoma cells proliferation, induced cell death through caspase-dependent apoptosis and arrested cells in the G1 phase of the cell cycle, as demonstrated via both flow cytometry and qPCR analysis. The proapoptotic effect of iSP is associated with increased ROS production and mitochondrial modulation, as observed by using DCF-DHA and mitochondrial probes. In addition, we performed wound healing, invasion and clonogenic assays and found that iSP was able to restrain A375 migration, invasion and clonogenicity. Importantly, we observed that an iSP treatment modulated the expression of the EMT-associated epithelial markers, E-CAD and N-CAD, unveiling the mechanism underlying the effect of iSP in modulating A375 migration and invasion. Collectively, this study provides the first evidence to support the role of Ircinia ramosa sponge extracts as a potential therapeutic resource for the treatment of human melanoma.

A novel cell-wall polysaccharide derived from the stipe of Agaricus bisporus inhibits mouse melanoma proliferation and metastasis

Malignant melanoma is an invasive and highly aggressive skin cancer that—if diagnosed—poses a serious threat to the patient's health and life. In this work, a novel purified cell-wall polysaccharide (termed Abwp) was obtained from the discarded stipe of Agaricus bisporus (A. bisporus) and characterized to be a novel homogeneous polysaccharide consisted of a β-(1 → 4)- glucosyl backbone with β-(1 → 2) and (1 → 6)-d-glucosyl side-chains. The anti-melanoma effects of Abwp and its associated mechanisms in mice were then explored using in vitro and in vivo approaches. In vitro results showed that Abwp inhibited B16 melanoma cell proliferation and promoted their apoptosis in both time- and dose-dependent manners. In B16 cells induced with tumor necrosis factor (TNF-α), Abwp significantly decreased the protein expression of inflammatory-related signaling pathway (e.g., p38 MAPK and NF-κB) in time-, concentration-, and dose-dependent manners. Moreover, Abwp blocked nuclear entry of NF-κB-p65. In an in vivo mouse model featuring neoplasm transplantation with B16 melanoma cells, Abwp significantly inhibited the growth and proliferation of mouse melanoma. Hematoxylin staining showed that the invasion of melanoma cells into the lung tissue of the Abwp-treated group was significantly reduced. Immunohistochemical analysis showed that the expression of proliferation cell nuclear antigen (PCNA), N-cadherin, MMP-9, and Snail in the lung of mouse was significantly inhibited. Immunofluorescence showed that Abwp significantly interfered with the nuclear transcription of NF-κB-p65 in a dose-dependent manner. Collectively, these results showed that Abwp mediated p38 MAPK and NF-κB signaling pathways to inhibit the inflammatory response and malignant proliferation and metastasis of melanoma in mice.

Histone Deacetylases (HDAC) Inhibitor-Valproic Acid Sensitizes Human Melanoma Cells to Dacarbazine and PARP Inhibitor.

The inhibition of histone deacetylases (HDACs) holds promise as a potential anti-cancer therapy as histone and non-histone protein acetylation is frequently disrupted in cancer, leading to cancer initiation and progression. Additionally, the use of a histone deacetylase inhibitor (HDACi) such as the class I HDAC inhibitor-valproic acid (VPA) has been shown to enhance the effectiveness of DNA-damaging factors, such as cisplatin or radiation. In this study, we found that the use of VPA in combination with talazoparib (BMN-673-PARP1 inhibitor-PARPi) and/or Dacarbazine (DTIC-alkylating agent) resulted in an increased rate of DNA double strand breaks (DSBs) and reduced survival (while not affecting primary melanocytes) and the proliferation of melanoma cells. Furthermore, the pharmacological inhibition of class I HDACs sensitizes melanoma cells to apoptosis following exposure to DTIC and BMN-673. In addition, the inhibition of HDACs causes the sensitization of melanoma cells to DTIV and BMN-673 in melanoma xenografts in vivo. At the mRNA and protein level, the histone deacetylase inhibitor downregulated RAD51 and FANCD2. This study aims to demonstrate that combining an HDACi, alkylating agent and PARPi could potentially enhance the treatment of melanoma, which is commonly recognized as being among the most aggressive malignant tumors. The findings presented here point to a scenario in which HDACs, via enhancing the HR-dependent repair of DSBs created during the processing of DNA lesions, are essential nodes in the resistance of malignant melanoma cells to methylating agent-based therapies.

Polysaccharide extracted from the Sargassum fusiforme induces cell cycle arrest and apoptosis of B16F10 melanoma cells through the PI3K/AKT pathway.

SARGASSUM FUSIFORME: (S. fusiforme) is a brown alga that has been utilized as a medicine for a long time. Polysaccharides extracted from S. fusiforme demonstrate antitumor activities. The impact of S. fusiforme polysaccharides (SFPS 191,212) on the proliferation, apoptosis, and cell cycle kinetics of B16F10 murine melanoma cells were thoroughly investigated in this work. The anticancer activities of the SFPS 191,212 compounds were assayed in the B16F10 cells at both transcriptional and translational levels. The compound exhibited concentration-dependent effects. Moreover, SPFS 191,212 increased the numbers of apoptotic cells and arrested the cell cycle in the S phase of the quantitative real-time PCR. From western blotting, it was verified that the SFPS 191,212 treatment improved the expression of Bax, Caspase-9, and Caspase-3 genes and proteins, while it reduced phosphatidylinositol 3 kinase and Bcl-2 genes and proteins, suggesting the involvement of mitochondria. Overall, SFPS 191,212 can be further explored as a potential functional food or adjuvant agent for the prevention or treatment of melanoma.

LSM2 is associated with a poor prognosis and promotes cell proliferation, migration, and invasion in skin cutaneous melanoma

BackgroundSkin cutaneous melanoma (SKCM) is an extremely malignant tumor that is associated with a poor prognosis. LSM2 has been found to be related to different types of tumors; however, its role in SKCM is poorly defined. We aimed to determine the value of LSM2 as a prognostic biomarker for SKCM.MethodsThe expression profile of LSM2 mRNA was compared between tumor and normal tissues in public databases, such as TCGA, GEO, and BioGPS. LSM2 protein expression was explored using immunohistochemistry (IHC) on a tissue microarray containing 44 SKCM tissues and 8 normal samples collected at our center. Kaplan-Meier analysis was performed to assess the prognostic value of LSM2 expression in patients with SKCM. SKCM cell lines with LSM2 knockdown were used to determine the effects of LSM2. Cell counting kit-8 (CCK8) and colony formation assays were conducted to assess cell proliferation, whereas wound healing and transwell assays were carried out to assess the migration and invasion abilities of SKCM cells.ResultsLSM2 was more highly expressed at the mRNA and protein levels in SKCM than that in normal skin. Moreover, elevated expression of LSM2 was associated with shorter survival time and early recurrence in patients with SKCM. The in vitro results revealed that the silencing of LSM2 in SKCM cells significantly inhibited cell proliferation, migration, and invasion.ConclusionOverall, LSM2 contributes to malignant status and poor prognosis in patients with SKCM and may be identified as a novel prognostic biomarker and therapeutic target.

Pirin is a prognostic marker of human melanoma that dampens the proliferation of malignant cells by downregulating JARID1B/KDM5B expression

Originally considered to act as a transcriptional co-factor, Pirin has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. Here, we have analyzed the diagnostic and prognostic value of Pirin expression in the early stages of melanoma, and its role in the biology of melanocytic cells. Pirin expression was analyzed in a total of 314 melanoma biopsies, correlating this feature with the patient’s clinical course. Moreover, PIR downregulated primary melanocytes were analyzed by RNA sequencing, and the data obtained were validated in human melanoma cell lines overexpressing PIR by functional assays. The immunohistochemistry multivariate analysis revealed that early melanomas with stronger Pirin expression were more than twice as likely to develop metastases during the follow-up. Transcriptome analysis of PIR downregulated melanocytes showed a dampening of genes involved in the G1/S transition, cell proliferation, and cell migration. In addition, an in silico approach predicted that JARID1B as a potential transcriptional regulator that lies between PIR and its downstream modulated genes, which was corroborated by co-transfection experiments and functional analysis. Together, the data obtained indicated that Pirin could be a useful marker for the metastatic progression of melanoma and that it participates in the proliferation of melanoma cells by regulating the slow-cycling JARID1B gene.

BS148 Reduces the Aggressiveness of Metastatic Melanoma via Sigma-2 Receptor Targeting.

The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer.

Exogenous Hydrogen Sulfide Induces A375 Melanoma Cell Apoptosis Through Overactivation of the Unfolded Protein Response.

Melanomas are highly malignant and rapidly develop drug resistance due to dysregulated apoptosis. Therefore, pro-apoptotic agents could be effective for the management of melanoma. Hydrogen sulfide is ubiquitous in the body, and exogenous hydrogen sulfide has been reported to show inhibitory and pro-apoptotic effects on cancer cells. However, whether high concentrations of exogenous hydrogen sulfide have pro-apoptotic effects on melanoma and its mechanisms remain unknown. Hence, this study aimed to explore the pro-apoptotic effects and mechanisms of exogenous hydrogen sulfide on the A375 melanoma cell line treated with a hydrogen sulfide donor (NaHS). The cell proliferation test, flow cytometric analysis, Hoechst 33258 staining, and Western blotting of B-cell lymphoma 2 and cleaved caspase-3 were used to explore the pro-apoptotic effects of hydrogen sulfide on A375 cells. The transcriptional profile of NaHS-treated A375 cells was further explored via high-throughput sequencing. Western blotting of phosphorylated inositol-requiring enzyme 1α (p-IRE1α), phosphorylated protein kinase R-like ER kinase (p-PERK), phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α), C/EBP homologous protein, glucose-regulating protein 78, IRE1α, PERK, and eIF2α was performed to verify the changes in the transcriptional profile. NaHS inhibited A375 melanoma cell proliferation and induced apoptosis. The endoplasmic reticulum stress unfolded protein response and apoptosis-associated gene expression was upregulated in NaHS-treated A375 melanoma cells. The overactivation of the unfolded protein response and increase in endoplasmic reticulum stress was verified at the protein level. Treatment with NaHS increased endoplasmic reticulum stress, which triggered the overactivation of the unfolded protein response and ultimately lead to melanoma cell apoptosis. The pro-apoptotic effect of NaHS suggests that it can be explored as a potential therapeutic agent in melanoma.

Functional Involvement of ADRA1D in Cutaneous Melanoma Progression and Angiogenesis.

Cutaneous melanoma is a highly aggressive and malignant skin cancer, and its high recurrence rate and drug resistance increase the difficulty of treating advanced-stage patients. Studies have revealed that treatment via stimulation of alpha-1 adrenergic receptor (ADRA1) subtypes inhibits melanoma growth in mice. However, the associations between alpha-1D adrenergic receptor (ADRA1D) and cutaneous melanoma are poorly understood. Tissue specimens from 16 pairs of patients with a pigmented nevus and cutaneous melanoma were analyzed for ADRA1D expression using immunohistochemical staining. Western blotting and RT-qPCR were carried out in order to detect ADRA1D expression levels in melanoma cells and human epidermal melanocytes (HEMs), hypoxia-inducible factor-1α (HIF-1α), and vascular endothelial growth factor (VEGF) levels in HUVECS. A375 cells were transfected with a lentivirus overexpressing ADRA1D. Wound-healing, Transwell, and cell proliferation assays were utilized to identify the ADRA1D effect on the migration, invasion, and proliferation of the two groups of A375 cells in vitro. In order to evaluate the function of ADRA1D in vivo, a melanoma xenograft model was developed in immunodeficient mice. ADRA1D was low expressed in cutaneous melanoma tissues. Overexpression of ADRA1D inhibited the tubulation and migration of HUVECs in vitro. Overexpression of ADRA1D significantly decreased the HIF-1α and VEGF expression. Overexpression of ADRA1D inhibited the invasion and proliferation of A375 melanoma cells in vitro and reduced its angiogenesis in vivo. ADRA1D inhibits cutaneous melanoma growth and angiogenesis. It attenuates melanoma cell proliferation and invasion. Meanwhile, its anti-angiogenic effect is achieved by negatively regulating the HIF-1α/VEGF axis in melanoma tissue, thereby attenuating the growth of cutaneous melanoma and reducing the potential of metastasis.

The Vicious Cycle of Melanoma-Microglia Crosstalk: Inter-Melanoma Variations in the Brain-Metastasis-Promoting IL-6/JAK/STAT3 Signaling Pathway.

Previous studies from our lab demonstrated that the crosstalk between brain-metastasizing melanoma cells and microglia, the macrophage-like cells of the central nervous system, fuels progression to metastasis. In the present study, an in-depth investigation of melanoma-microglia interactions elucidated a pro-metastatic molecular mechanism that drives a vicious melanoma-brain-metastasis cycle. We employed RNA-Sequencing, HTG miRNA whole transcriptome assay, and reverse phase protein arrays (RPPA) to analyze the impact of melanoma-microglia interactions on sustainability and progression of four different human brain-metastasizing melanoma cell lines. Microglia cells exposed to melanoma-derived IL-6 exhibited upregulated levels of STAT3 phosphorylation and SOCS3 expression, which, in turn, promoted melanoma cell viability and metastatic potential. IL-6/STAT3 pathway inhibitors diminished the pro-metastatic functions of microglia and reduced melanoma progression. SOCS3 overexpression in microglia cells evoked microglial support in melanoma brain metastasis by increasing melanoma cell migration and proliferation. Different melanomas exhibited heterogeneity in their microglia-activating capacity as well as in their response to microglia-derived signals. In spite of this reality and based on the results of the present study, we concluded that the activation of the IL-6/STAT3/SOCS3 pathway in microglia is a major mechanism by which reciprocal melanoma-microglia signaling engineers the interacting microglia to reinforce the progression of melanoma brain metastasis. This mechanism may operate differently in different melanomas.

RITA selectively inhibits proliferation of BAP1-deficient cutaneous melanoma cells in vitro

To screen for small molecular compounds with selective inhibitory activity against cutaneous melanoma cells with BAP1 deletion. Cutaneous melanoma cells expressing wild-type BAP1 were selected to construct a BAP1 knockout cell model using CRISPR-Cas9 system, and small molecules with selective inhibitory activity against BAP1 knockout cells were screened from a compound library using MTT assay. Rescue experiment was carried out to determine whether the sensitivity of BAP1 knockout cells to the candidate compounds was directly related to BAP1 deletion. The effects of the candidate compounds on cell cycle and apoptosis were detected with flow cytometry, and the protein expressions in the cells were analyzed with Western blotting. The p53 activator RITA from the compound library was shown to selectively inhibit the viability of BAP1 knockout cells. Overexpression of wild-type BAP1 reversed the sensitivity of BAP1 knockout cells to RITA, while overexpression of the mutant BAP1 (C91S) with inactivated ubiquitinase did not produce any rescue effect. Compared with the control cells expressing wild-type BAP1, BAP1 knockout cells were more sensitive to RITA-induced cell cycle arrest and apoptosis (P < 0.0001) and showed an increased expression of p53 protein, which was further increased by RITA treatment (P < 0.0001). Loss of BAP1 results in the sensitivity of cutaneous melanoma cells to p53 activator RITA. In melanoma cells, the activity of ubiquitinase in BAP1 is directly related to their sensitivity to RITA. An increased expression of p53 protein induced by BAP1 knockout is probably a key reason for RITA sensitivity of melanoma cells, suggesting the potential of RITA as a targeted therapeutic agent for cutaneous melanoma carrying BAP1-inactivating mutations.

USP2 promotes cell proliferation and metastasis in choroidal melanoma via stabilizing Snail.

Choroidal melanoma (CM) is an intraocular tumor that arises from melanocytes. While ubiquitin-specific protease 2 (USP2) modulates the progression of numerous diseases, its role in CM is not known. This study aimed to determine the role of USP2 in CM and elucidate its molecular mechanisms. MTT, Transwell, and wound-scratch assays were used to investigate the function of USP2 in the proliferation and metastasis of CM. Western blotting and qRT-PCR were used to analyze the expression of USP2, Snail, and factors associated with the epithelial-mesenchymal transition (EMT). The relationship between USP2 and Snail was explored by co-immunoprecipitation and in vitro ubiquitination assays. A nude mouse model of CM was established for verifying the role of USP2 in vivo. USP2 overexpression promoted proliferation and metastasis, and induced the EMT in CM cells in vitro, while specific inhibition of USP2 by ML364 produced the opposite effects. ML364 also suppressed CM tumor growth in vivo. Mechanistically, USP2 is known to deubiquitinate Snail, stabilizing the latter through the removal of its K48 poly-ubiquitin chains. However, a catalytically inactive form of USP2 (C276A) had no effect on Snail ubiquitination and failed to increase Snail protein expression. The C276A mutant was also unable to promote CM cell proliferation, migration, and invasion, as well as EMT progression. Furthermore, Snail overexpression partly counteracted the effects of ML364 on proliferation and migration, while rescuing the effects of the inhibitor on the EMT. The findings demonstrated that USP2 modulated CM development through the stabilization of Snail and suggest that USP2 may be a useful target for the development of novel treatments for CM.

Combination of niclosamide and quinacrine inactivates Akt/HK2/Cyclin D1 axis mediated by glucose deprivation towards the inhibition of melanoma cell proliferation

Malignant melanoma is one of the most aggressive and lethal skin cancer. At present, the treatment methods for melanoma have shortcomings. Glucose is the primary energy source of cancer cells. However, it is unclear whether glucose deprivation can be used to treat melanoma. Herein, we first found glucose played an essential role in melanoma proliferation. We then further found a drug combination of niclosamide and quinacrine could inhibit melanoma proliferation and glucose intake. Thirdly, we revealed the mechanism of anti-melanoma effect of the drug combination, which suppressed the Akt pathway. In addition, the first-rate limiting enzyme HK2 of glucose metabolism was inhibited. This work also disclosed that the decrease of HK2 inhibited cyclin D1 by reducing the activity of transcription factor E2F3, which further suppressed the proliferation of melanoma cells. The drug combination treatment also resulted in significant tumor regression in the absence of obvious morphologic changes in primary organ in vivo. In summary, our study demonstrated that the drug combination treatment created glucose deprivation to inactive the Akt/HK2/cyclin D1 axis, thereby inhibited the proliferation of melanoma cells, providing a potential anti-melanoma strategy.

Synergy, Additivity and Antagonism between Esculetin and Six Commonly Used Chemotherapeutics in Various Malignant Melanoma Cell Lines-An Isobolographic Analysis.

(1) Malignant melanomas are dangerous skin cancers, and the treatment of melanomas with various cytostatic drugs often causes side effects and after their prolonged use resistance to these drugs appears. The aim of this study was to evaluate the anticancer effects of esculetin (a simple coumarin) and to assess pharmacodynamic interactions between esculetin and six commonly used cytostatic drugs (cisplatin, epirubicin, docetaxel, paclitaxel, mitoxantrone and vemurafenib) using an isobolographic analysis. (2) The experiments were carried out on four human malignant melanoma cell lines (FM55P, A375, FM55M2 and SK-MEL28). The effects of esculetin on viability, cell proliferation and cytotoxicity were verified in the range of concentrations of 2-200 μM. (3) Esculetin inhibited, in a dose-dependent manner, malignant melanoma cell viability and proliferation. The IC50 for esculetin ranged from 18.20 ± 2.93 to 120.64 ± 30.39 μM depending on the melanoma cell lines used. The combinations of esculetin with epirubicin and vemurafenib showed antagonistic interactions, the combinations of esculetin with cisplatin, docetaxel and paclitaxel showed additive interactions. For the combinations of esculetin with mitoxantrone, the isobolographic analysis displayed synergy. (4) In the treatment of malignant melanoma, esculetin should not be combined with epirubicin or vemurafenib, due to the reduction of their anticancer effects, while the synergistic interactions (esculetin + mitoxantrone) deserve a preclinical recommendation as a beneficial combination during anticancer therapy.

Circ_0081054 facilitates melanoma development via sponging miR-637 and regulating RAB9A.

Accumulating evidence announces that aberrantly expressed circRNAs were closely related to the development of human cancers. However, the role and mechanism of multiple circRNAs remain unclear. Our work aimed to disclose the functional role and mechanism of circ_0081054 in melanoma. Quantitative real-time polymerase chain reaction assay was utilized to detect circ_0081054, microRNA-637 (miR-637) and RAB9A (member RAS oncogene family) mRNA expression. Cell proliferative ability was evaluated via Cell Counting Kit-8 and colony formation assay. Cell invasion was assessed by using wound healing assay. The significant upregulation of circ_0081054 was detected in melanoma tissues and cells. The proliferation, migration, glycolytic metabolism, and angiogenesis in melanoma cells were suppressed, while apoptosis was promoted following the silence of circ_0081054. In addition, circ_0081054 could target miR-637, and miR-637 inhibitor could reverse the effects of circ_0081054 deficiency. Furthermore, RAB9A was a target gene for miR-637 and RAB9A overexpression could reverse the effects of miR-637 overexpression. In addition, the deficiency of circ_0081054 hampered tumor growth in vivo. Moreover, circ_0081054 could regulate RAB9A expression by sponging miR-637. All results indicated that circ_0081054 promoted the malignant behaviors of melanoma cells partly by regulating the miR-637/RAB9A molecular axis.