Melanoma Cell Viability Research Articles

Microfluidic development and biological evaluation of targeted therapy-loaded biomimetic nano system to improve the metastatic melanoma treatment

Optimizing current therapies is among next steps in metastatic melanoma (MM) treatment landscape. The innovation of this study is the design of production process by microfluidics of cell membrane (CM)-modified nanoparticles (NPs), as an emerging biomimetic platform that allows for reduced immune clearance, long blood circulation time and improved specific tumor targeting. To achieve melanoma selectivity, direct membrane fusion between synthetic liposomes and CMs extracted from MM cell line was performed by microfluidic sonication approach, then the hybrid liposomes were loaded with cobimetinib (Cob) or lenvatinib (Lenva) targeting agents and challenged against MM cell lines and liver cancer cell line to evaluate homotypic targeting and antitumor efficacy. Characterization studies demonstrated the effective fusion of CM with liposome and the high encapsulation efficiency of both drugs, showing the proficiency of microfluidic-based production. By studying the targeting of melanoma cells by hybrid liposomes versus liposomes, we found that both NPs entered cells through endocytosis, whereas the former showed higher selectivity for MM cells from which CM was extracted, with 8-fold higher cellular uptake than liposomes. Hybrid liposome formulation of Cob and Lenva reduced melanoma cells viability to a greater extent than liposomes and free drug and, notably, showed negligible toxicity as demonstrated by bona fide haemolysis test. The CM-modified NPs presented here have the potential to broaden the choice of therapeutic options in MM treatment.

Comparison of chitosan nanoparticles containing Lippia citriodora essential oil and citral on the induction of apoptosis in A375 melanoma cells

BackgroundUsing nanoparticles containing L. citriodora EO and citral has shown potential in treating skin disorders such as melanoma.MethodsIn this study, GC‒MS was used to analyze the chemical composition of L. citriodora essential oil (EO). The ion gelation method prepared free chitosan nanoparticles and chitosan nanoparticles containing L. citriodora EO and citral. The successful loading of the EO and citral was evaluated using ATR-FTIR. The DPPH assay measured the antioxidant effect of citral, L. citriodora EO, Citral-ChiNPs, L. citriodora-ChiNPs, and Free-ChiNPs. A375 melanoma cell viability was assessed using the MTT assay. The qPCR technique was employed to evaluate the expression of apoptotic genes, and flow cytometry was used to detect apoptosis.ResultsThis study showed that in equal concentrations, the antioxidant properties of chitosan nanoparticles containing citral were greater than those of chitosan nanoparticles containing L. citriodora. The IC50 values of chitosan nanoparticles containing citral, L. citriodora EO, and their nonformulated states were 105.6, 199.9, 136.9, and 240 µg/ml, respectively. The gene expression results showed that the ratio of the expression of the apoptosis gene to the inhibitory gene was higher than 1 in all the samples, indicating that the conditions for apoptosis were present. Flow cytometry confirmed cell apoptosis, with 93.5 ± 0.3% in chitosan nanoparticles containing citral, 80 ± 0.2% in chitosan nanoparticles containing L. citriodora EO, 63 ± 0.3 in citral, and 42.03% in L. citriodora EO-treated cells.ConclusionThe results showed that using the Nano form of L. citriodora and citral increased their efficiency in apoptosis pathways and their toxicity against 375 melanoma cancer cells.

AM1172 (a hydrolysis-resistant endocannabinoid analog that inhibits anandamide cellular uptake) reduces the viability of the various melanoma cells, but it exerts significant cytotoxic effects on healthy cells: an in vitro study based on isobolographic analysis.

Despite great advances in our understanding of the impact of cannabinoids on human organism, many of their properties still remain undetermined, including their potential antineoplastic effects. This study was designed to assess the anti-proliferative and cytotoxic effects of AM1172 (a hydrolysis-resistant endocannabinoid analog that inhibits anandamide cellular uptake) administered alone and in combinations with docetaxel (DOCX), paclitaxel (PACX), mitoxantrone (MTX) and cisplatin (CDDP) on various human malignant melanoma A375, FM55P, SK-MEL 28 and FM55M2 cell lines. In the MTT, LDH, and BrdU assays, the potency and safety of AM1172 when administered alone and in combinations with DOCX, PACX, MTX, and CDDP were determined. The isobolographic analysis revealed that combinations of AM1172 with PACX, DOCX, MTX, and CDDP exerted additive interactions, except for a combination of AM1172 with PACX in primary melanoma A375 cell line, for which synergy was observed (*p<0.05). Nevertheless, AM1172 when administered alone produced cytotoxic effects on healthy human melanocytes (HEMa-LP) and human keratinocytes (HaCaT), which unfortunately limits its potential therapeutic utility. AM1172 cannot be used separately as a chemotherapeutic drug, but it can be combined with PACX, DOCX, MTX, and CDDP, offering additive interactions in terms of the anti-proliferative effects in various malignant melanoma cell lines.

Comparison effects of Ferula gummosa essential oil and Beta-pinene Alginate nanoparticles on human melanoma and breast cancer cells proliferation and apoptotic index in short term normobaric hyperoxic model

BackgroundBreast cancer is the most common cancer among women, and melanoma is the most dreadful type of skin cancer. Due to the side effects of chemotherapy drugs, the development of new herbal nano-medicines has been considered.MethodsThis study first investigated the chemical composition of Ferula gummosa essential oil using GC-MS analysis; β-pinene, with 61.57%, was the major compound. Next, alginate nanoparticles containing β-pinene and the essential oil with particle sizes of 174 ± 7 and 137 ± 6 nm were prepared. Meanwhile, their zeta potentials were 12.4 ± 0.7 and 28.1 ± 1 mV. Besides, the successful loading of β-pinene and the essential oil in nanoparticles was confirmed using ATR-FTIR analysis. After that, their effects on viability and apoptotic index of human melanoma and breast cancer cells were investigated in normoxia and normobaric hyperoxia (NBO) conditions.ResultsThe best efficacy on A-375 and MDA-MB-231 cells was achieved by alginate nanoparticles containing the EO at hyperoxic and normoxia conditions; IC50 76 and 104 µg/mL. Besides, it affected apoptosis-involved genes; as Bax/Bcl-2 ratio was higher than 1, conditions for induction of apoptosis were obtained. Higher sensitivity was observed in the A-375 cell line treated with Alg-EO in the NBO model.ConclusionsAlginate nanoparticles containing F. gummosa EO could be considered for further investigation in anticancer studies. Also, it may be expected that NBO can be a new strategy for delaying cancer progression and improving nanotherapy efficacy.

Synthesis and characterization of 4-nitro benzaldehyde with ZnO-based nanoparticles for biomedical applications.

Globally, cancer is the leading cause of death and morbidity, and skin cancer is the most common cancer diagnosis. Skin problems can be treated with nanoparticles (NPs), particularly with zinc oxide (ZnO) NPs, which have antioxidant, antibacterial, anti-inflammatory, and anticancer properties. An antibacterial activity of zinc oxide nanoparticles prepared in the presence of 4-nitrobenzaldehyde (4NB) was also tested in the present study. In addition, the influence of synthesized NPs on cell apoptosis, cell viability, mitochondrial membrane potential (MMP), endogenous reactive oxygen species (ROS) production, apoptosis, and cell adhesion was also examined. The synthesized 4-nitro benzaldehyde with ZnO (4NBZnO) NPs were confirmed via characterization techniques. 4NBZnO NPs showed superior antibacterial properties against the pathogens tested in antibacterial investigations. As a result of dose-based treatment with 4NBZnO NPs, cell viability, and MMP activity of melanoma cells (SK-MEL-3) cells were suppressed. A dose-dependent accumulation of ROS was observed in cells exposed to 4NBZnO NPs. As a result of exposure to 4NBZnO NPs in a dose-dependent manner, viable cells declined and apoptotic cells increased. This indicates that apoptotic cell death was higher. The cell adhesion test revealed that 4NBZnO NPs reduced cell adhesion and may promote apoptosis of cancer cells because of enhanced ROS levels.

Tectorigenin functions as a potential drug for melanoma treatment via inhibition of cell growth, migration and aerobic glycolysis

Purpose: To investigate the role of tectorigenin (TG) in melanoma cells.&#x0D; Methods: Viability of A375 and A2058 melanoma cells was determined by cell counting kit (CCK-8) assay. Cell cycle progression was analyzed by 5-ethynyl-2’-deoxyuridine (EdU) staining. Migration and invasion of melanoma cells were determined by Transwell assay. The epithelial-mesenchymal transition (EMT) of melanoma cells was investigated by determining expression of E-Cadherin and Snail. Expression of glucose transporter 1 (GLUT1) and lactose dehydrogenase A (LDHA) was assessed via western blotting. Glucose and lactate production was evaluated by corresponding assay kit. The NOX4 and FOXM1 expressions were determined using western blotting. &#x0D; Results: Tectorigenin inhibited proliferation, migration and invasion of A375 and A2058 melanoma cells. Tectorigenin regulated cell cycle progression by decreasing the number of cells in S-phase and significantly inhibited snail expression and increased expression of E-Cadherin (p &lt; 0.05), thus inhibiting the EMT of A375 and A2058 cells. Tectorigenin inhibited expression of GLUT1 and LDHA, thereby leading to reduction in glucose consumption and lactate production. It also significantly inhibited expressions of NOX4 and FOXM1 (p &lt; 0.05), indicating an inhibitory effect on the activity of NOX4/FOXM1 pathway.&#x0D; Conclusion: Tectorigenin inhibits aerobic glycolysis, growth and migration in melanoma cells by suppressing the activity of NOX4/FOXM1 pathway, suggesting its potential in melanoma treatment.

Three-dimensional, PEG-based hydrogels induce spheroid formation and enhance viability of A2058 melanoma cells.

Traditional drug screening methods use monolayer (2D) tumor cell cultures, which lack basic features of tumor complexity. As an alternative, 3D hydrogels have begun to emerge as simple, time-, and cost-saving systems. One of the most promising candidates, synthetic alkoxysilane-PEG (polyethylene glycol)-based hydrogels, are formed by "sol-gel" polymerization in an aqueous medium, which allows control over the incorporated elements. Our aims were to optimize siloxane-PEG hydrogels for three different cell lines of skin origin and utilize these 3D hydrogels as a feasible drug (e.g., daunorubicin) screening assay. A drastic increase in survival and the formation of cellular aggregates (spheroids) could be observed in A2058 melanoma cells, but not in keratinocyte and endothelial cell lines. A deep-learning neural network was trained to recognize and distinguish between the cellular formations and allowed the fast processing of hundreds of microscopic images. We developed an artificial intelligence (AI)-assisted application (https://github.com/enyecz/CancerDetector2), which indicated that, in terms of average area of the spheroids treated with daunorubicin, A2058 melanoma cell 3D aggregates have better survival in a hydrogel containing 15% bis-mono-ethoxysilane-PEG.

Development of a novel NiCu nanoparticle-loaded polysaccharide-based hydrogel for 3D printing of customizable dressings with promising cytotoxicity against melanoma cells.

Polysaccharide hydrogels and metal alloy nanoparticles have already found use in a range of biomedical applications. Nickel-copper nanoparticles (NiCu NPs) are particularly promising due to their tunable properties, such as ferromagnetism, biocompatibility, and antimicrobial activity. At the same time, polysaccharide hydrogels made of polymer mixtures such as alginate and methylcellulose with incorporated metal alloy nanoparticles are reported in the scientific literature. In view of this, in this work, NiCu NPs are combined with polysaccharide hydrogels and 3D printed to construct geometrically customizable dressings with tailorable properties for melanoma treatment. This novel combination exploits the intrinsic magnetic properties of NiCu NPs and the same time builds on their less known properties to improve the mechanic stability of 3D printed materials, both contributing to a previously not reported application as potent cytotoxic dressing against melanoma cells. The dressings were evaluated in terms of their physico-chemical characteristics, and their potential application, namely melanoma cell cytotoxicity. While all dressings exhibited similar degradation profiles regardless of composition, the addition of NiCu NPs had an effect on the hydrophilicity, swelling rates, and topographical properties of the dressings. Compression results showed that the presence of NPs increased the stiffness of the dressings, while the ultimate tensile strength was highest at 0.31MPa for the dressings with 0.5wt% NPs. We show that although the base formulation of the dressings is biocompatible with skin-derived cells, dressings loaded with NPs exhibit promising antimelanoma activity. Extracts obtained from dressings containing 0.5wt% NPs reduced melanoma cell viability to 61%±11% and 40%±2% after 24h and 72h of soaking, respectively. Furthermore, extracts of dressings with 1wt% NPs reduced melanoma cell viability to less than 15% within the first 24h. By adjusting the NP content, the mechanical properties, surface roughness, and wettability can be tuned so that the dressings can be functionally customized. In addition, by using 3D printing as a fabrication process, the shape and composition of the dressings can be tailored to the patient's needs. The dressings also remained intact after soaking in simulated physiological solution for 14 days, indicating their suitability for long-term topical application.

Caffeine reduces viability, induces apoptosis, inhibits migration and modulates the CD39/CD73 axis in metastatic cutaneous melanoma cells.

We aimed to evaluate the effect of caffeine on viability, apoptosis, migration, redox profile and modulatory effect of the purinergic system of cutaneous melanoma cells. The melanoma cells SK-MEL-28 and non-tumoural CCD-1059sk cells were treated for 24 h with different concentrations of caffeine. Cell viability was evaluated by a biochemical assay and fluorescence microscopy, and flow cytometry assessed apoptosis induction. A wound-healing assay assessed cell migration. The redox profile was evaluated by the levels of markers of reactive oxygen species (ROS), nitric oxide (NOx), total thiols (PSH) and non-protein thiols (NPSH). RT-qPCR and flow cytometry assessed the expression of CD39 and CD73. ATPase/ADPase and AMPase enzyme activities were evaluated by hydrolysis of ATP, ADP and AMP nucleotides. A bioluminescent assay assessed extracellular ATP levels. Caffeine significantly reduced melanoma cell viability and migration and did not affect non-tumoural cells. Caffeine increased ROS levels and improved PSH levels in melanoma cells. Furthermore, caffeine reduced CD39 and CD73 expression, decreased ATP, ADP and AMP nucleotide hydrolysis and increased extracellular ATP levels. We have shown that caffeine reduces metastatic cutaneous melanoma cell viability and migration, induces ROS generation and improves PSH levels. In an unprecedented manner, we also showed that caffeine reduces the expression of CD39 and CD73 and, consequently, ATPase/ADPase/AMPase hydrolytic activity of ectonucleotidases, thus displacing the CD39/CD73 axis and increasing extracellular ATP levels. Therefore, caffeine may be an interesting compound for clinical trials with the CD39/CD73 axis as a therapeutic target.

T-Lymphocytes Activated by Dendritic Cells Loaded by Tumor-Derived Vesicles Decrease Viability of Melanoma Cells In Vitro.

Immunotherapy represents an innovative approach to cancer treatment, based on activating the body's own immune system to combat tumor cells. Among various immunotherapy strategies, dendritic cell vaccines hold a special place due to their ability to activate T-lymphocytes, key players in cellular immunity, and direct them to tumor cells. In this study, the influence of dendritic cells processed with tumor-derived vesicles on the viability of melanoma cells in vitro was investigated. Dendritic cells were loaded with tumor-derived vesicles, after which they were used to activate T-cells. The study demonstrated that such modified T-cells exhibit high activity against melanoma cells, leading to a decrease in their viability. Our analysis highlights the potential efficacy of this approach in developing immunotherapy against melanoma. These results provide new prospects for further research and the development of antitumor strategies based on the mechanisms of T-lymphocyte activation using tumor-derived vesicles.

Abstract 27 Secretome of Umbilical Cord Mesenchymal Stem Cells: Potential Effects on Melanoma

Abstract Introduction Melanoma, a tumor resulting from the malignant transformation of melanocytes, is characterized by its aggressive nature and propensity to metastasize. Current treatment options for advanced melanoma are limited and mostly ineffective, highlighting the need for novel therapeutic approaches. Mesenchymal stem cells (MSCs) have increased considerable attention due to their anti-cancer and immunomodulatory properties. In particular, human umbilical cord MSCs (hUCMSCs) have shown promise in various therapeutic applications. Objectives The objective of this research was to assess and understand the potential therapeutic effects of conditioned media (CM) derived from hUCMSCs on melanoma cells in vitro. Methods hUCMSC, isolated from the umbilical cord of seven healthy neonates by enzymatic digestion followed by direct plastic adherence method, were cultivated and their CM stored for subsequent assays. Then, malignant melanoma B16F10 cells were treated using a final concentration of 100% of hUCMSC-CM for 24 hours. Afterward, their viability, adhesion, and motility were accessed by MTT, crystal violet, and scratch wound healing assays, respectively. Results The MTT assay showed the inhibition of B16F10 cell viability in all treated groups compared to the control group (32%; p&amp;lt;0,05 vs control, n=7). Moreover, crystal violet staining revealed that hUCMSC-CM enhanced the adhesion capacity of melanoma cells by 16% (p&amp;lt;0,05 vs control, n=7). In terms of motility, the hUCMSC-CM reduced migration by 24% (p&amp;lt;0,05 vs control, n=7) after 24 hours. Discussion Our results suggest that hUCMSCs may exhibit dual effects on melanoma cells. On one hand, certain factors produced by hUCMSCs have shown potential in reducing the viability and motility of B16F10 melanoma cells, indicating a potential therapeutic benefit. On the other hand, it has also been observed that CM of hUCMSCs can increase the adhesion capacity of melanoma cells. While hUCMSC-released factors may exhibit beneficial effects on melanoma cells, further research and clinical trials are needed to establish their safety, efficacy, and optimal therapeutic protocols for malignant melanoma.

Schisandrin B Inhibits Cell Viability and Malignant Progression of Melanoma Cells via Wnt/β-catenin Signaling Pathway.

Melanoma is of great interest due to its aggressive behavior and less favorable prognosis. The need for the development of novel drugs for the treatment of melanoma is urgent. Considerable evidence indicated that Schisandrin B (Sch B), a bioactive compound extracted from Schisandra chinensis, has numerous anti-tumor properties in multiple malignant tumors. A few studies have reported the effect of Sch B on melanogenesis in the melanoma B16F10 cell line; however, the specific anti-tumor effects and mechanisms need to be further explored. This study aimed to investigate the effects of Sch B on the cell viability, migration, invasion, and cell cycleblocking of melanoma cells and explore its potential anti-tumor mechanism in vitro and in vivo. Melanoma cells (A375 and B16) were treated with different concentrations of Sch B (0, 20, 40, 60, or 80 μM), with dimethyl sulfoxide (DMSO) as control. The inhibitory effect of Sch B on A375 and B16 melanoma cells was verified by crystal violet assay and CCK8 assay. The flow cytometry was performed to observe cell cycle blocking. The effect of Sch B on the migration and invasion of melanoma cells was detected by wound healing assay and transwell assay, respectively. Western blot analysis was used to determine protein expression levels. The growth of the A375 melanoma xenograft-treated groups and immunohistochemical staining were conducted to assess the anti-tumor effect of Sch B in vivo. The crystal violet assay and CCK8 assay showed that Sch B significantly inhibited melanoma cell viability in a dose-dependent manner. Meanwhile, the flow cytometry analysis revealed that Sch B induced melanoma cell cycleblocking at the G1/S phase. In addition, the wound healing assay and transwell assay showed that Sch B inhibited the migration and invasion of melanoma cells. Furthermore, by establishing an animal model, we found that Sch B significantly inhibited the growth of melanoma in vivo. The potential mechanism could be that Sch B inhibited the activity of the Wnt/β-catenin signaling pathway. These findings indicated that Sch B inhibits the cell viability and malignant progression of melanoma cells via the Wnt/β-catenin pathway and induces cell cycle arrest. Our study suggests that Sch B has potential as a bioactive compound for the development of new drugs for melanoma.

The Antimalarial Drug Artesunate Mediates Selective Cytotoxicity by Upregulating HO-1 in Melanoma Cells.

Despite great efforts to develop new therapeutic strategies to combat melanoma, the prognosis remains rather poor. Artesunate (ART) is an antimalarial drug displaying anti-cancer effects in vitro and in vivo. In this in vitro study, we investigated the selectivity of ART on melanoma cells. Furthermore, we aimed to further elucidate the mechanism of the drug with a focus on the role of iron, the induction of oxidative stress and the implication of the enzyme heme oxygenase 1 (HO-1). ART treatment decreased the cell viability of A375 melanoma cells while it did not affect the viability of normal human dermal fibroblasts, used as a model for normal (healthy) cells. ART's toxicity was shown to be dependent on intracellular iron and the drug induced high levels of oxidative stress as well as upregulation of HO-1. Melanoma cells deficient in HO-1 or treated with a HO-1 inhibitor were less sensitive towards ART. Taken together, our study demonstrates that ART induces oxidative stress resulting in the upregulation of HO-1 in melanoma cells, which subsequently triggers the effect of ART's own toxicity. This new finding that HO-1 is involved in ART-mediated toxicity may open up new perspectives in cancer therapy.

Direct early growth response-1 knockdown decreases melanoma viability independent of mitogen-activated extracellular signal-related kinase inhibition.

To investigate downstream molecular changes caused by mitogen-activated protein kinase (MEK) inhibitor treatment and further explore the impact of direct knockdown of early growth response-1 ( EGR1 ) in melanoma cell culture. RNA-sequencing (RNA-Seq) was performed to determine gene expression changes with MEK inhibitor treatment. Treatment with MEK inhibitor (trametinib) was then assessed in two cutaneous (MEL888, MEL624) and one conjunctival (YUARGE 13-3064) melanoma cell line. Direct knockdown of EGR1 was accomplished using lentiviral vectors containing shRNA. Cell viability was measured using PrestoBlueHS Cell Viability Reagent. Total RNA and protein were assessed by qPCR and SimpleWestern. RNA-Seq demonstrated a profound reduction in EGR1 with MEK inhibitor treatment, prompting further study of melanoma cell lines. Following trametinib treatment of melanoma cells, viability was reduced in both cutaneous (MEL888 26%, P < 0.01; MEL624 27%, P < 0.001) and conjunctival (YUARGE 13-3064 33%, P < 0.01) melanoma compared with DMSO control, with confirmed EGR1 knockdown to 0.04-, 0.01-, and 0.16-fold DMSO-treated levels (all P < 0.05) in MEL888, MEL624, and YUARGE 13-3064, respectively. Targeted EGR1 knockdown using shRNA reduced viability in both cutaneous (MEL624 78%, P = 0.05) and conjunctival melanoma (YUARGE-13-3064 67%, P = 0.02). RNA-Sequencing in MEK inhibitor-treated cells identified EGR1 as a candidate effector molecule of interest. In a malignant melanoma cell population, MEK inhibition reduced viability in both cutaneous and conjunctival melanoma with a profound downstream reduction in EGR1 expression. Targeted knockdown of EGR1 reduced both cutaneous and conjunctival melanoma cell viability independent of MEK inhibition, suggesting a key role for EGR1 in melanoma pathobiology.

Anti-melanoma effects of S-allyl-L-cystine sulfoxide (ACSO/alliin) from garlic in C57BL/6J mice

Certain foods modify the risk of developing melanoma. Various garlic compounds, including odor precursors and their derivatives, inhibit tumor growth; thus, garlic could protect against melanoma. However, it is unclear if S-allyl-L-cystine sulfoxide (ACSO/alliin), which is the most abundant (>10 mg/g) odor precursor in fresh raw garlic, has anti-melanoma effect. We investigated if ACSO and the related compounds suppress tumor growth in vivo and in vitro. We orally administered ACSO, S-methyl-L-cystine sulfoxide (MCSO), and S-allyl-L-cystine (ACS) to mice with xenografted melanoma and observed the effect of these administrations on tumor size and lactate dehydrogenase activity. The effect of ACSO, MCSO, and ACS on B16F1 melanoma cells viability was also studied. We found that only ACSO suppressed tumor growth in vivo and in vitro. In addition, the sera collected from rats after oral administration of ACSO suppressed melanoma proliferation in vitro. ACSO may be the key anti-melanoma compound in garlic.

SPI1 involvement in malignant melanoma pathogenesis by regulation of HK2 through the AKT1/mTOR pathway.

Spi-1 proto-oncogene (SPI1) plays a vital role in carcinogenesis. Our work aimed to investigate the potential regulatory mechanism of SPI1 in melanoma. The mRNA and protein levels were measured via qRT-PCR and Western blotting. Cell viability was assessed by CCK-8 assay. The target relationship between SPI1 and hexokinase 2 (HK2) was determined using dual-luciferase reporter detection. ChIP was conducted to confirm the targeted relationship between SPI1 and the HK2 promoter. Immunohistochemistry analysis was conducted to measure the positive cell number of SPI1 and HK2 in melanoma tissues. The cell migration abilities were determined using a wound healing assay. Glucose consumption, pyruvate dehydrogenase activity, lactate production and ATP levels were measured to assess glycolysis. SPI1 transcription in melanoma cells and tissues was dramatically higher than that in adjacent normal tissues and epidermal melanocyte HEMa-LP, respectively. Knockdown of SPI1 restrained cell viability, metastasis and glycolysis in melanoma cells. SPI1 directly targeted HK2, and knockdown of SPI1 repressed HK2 expression. Overexpression of HK2 weakened the inhibitory effects of SPI1 knockdown on the viability, metastasis and glycolysis of melanoma cells. The serine-threonine kinase 1 (AKT1)/mammalian target of rapamycin (mTOR) axis is involved in melanoma progression. SPI1 knockdown restrained melanoma cell proliferation, metastasis and glycolysis by regulating the AKT1/mTOR pathway.

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

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.

Dual targeting of melanoma translation by MNK/eIF4E and PI3K/mTOR inhibitors

Melanoma is relatively resistant to chemotherapy, and no targeted therapies are fully effective. The most common mutations in melanoma result in hyperactivation of the mitogen-activated protein kinase (MAPK) and PI3K/AKT/ mTOR pathways responsible for initiating and controlling oncogenic protein translation. This makes both the signaling pathways potentially important therapeutic targets in melanoma. Our studies were carried out on human melanoma cell lines WM793 and 1205 LU with similar genomic alteration (BRAFV600E and PTEN loss). We used a highly specific PI3K/mTOR inhibitor, dactolisib (NVP-BEZ235), and Mnk inhibitor - CGP57380 alone and in combination. Here, we explore the mechanism of action of these drugs alone and in combination, as well as their effect on the viability and invasiveness of melanoma cells. Although when used independently, both drugs suppressed cell proliferation and migration, their combination has additional antitumor effects. We demonstrate that simultaneous inhibition of both pathways may prevent possible drug resistance.

A powerful drug combination strategy targeting BRAF-mutant melanoma.

3152 Background: BRAF inhibitors, such as vemurafenib and dabrafenib, are effective in treating patients with melanoma harboring (V600E) BRAF mutations. These drugs block the activity of the mutated BRAF protein, which impacts the development and progression of melanoma. Despite initial efficacy, drug resistance reliably occurs leading to disease relapse. Here we show how BRAF inhibitors cause metabolic reprogramming in melanoma cells to achieve drug resistance and promote cell survival. Previously we demonstrated that wild-type isocitrate dehydrogenase 1 (wtIDH1) supports mitochondrial function and maintains redox homeostasis in melanoma, and that an FDA-approved mutant IDH1 inhibitor, ivosidenib (AG-120), is actually a potent wtIDH1 inhibitor. Thus, we hypothesized that wtIDH1 inhibition would synergize with BRAF inhibition. Methods: Metabolomic profiling using LC-MS determined the impact of BRAF inhibition on cellular metabolism. Cell viability was assessed by PicoGreen in drug combination assays. Synergism was calculated using the Bliss-Independence dose-response model for drug interaction. In vivo modeling of AG-120 in combination with BRAF inhibition used melanoma cell xenografts in athymic nude mice for tumor volume analyses, and survival studies were performed in both immunocompetent C57BL/6J mice (murine YUMM1.7 cells) and immunocompromised athymic nude mice (human A375 cells). Results: Metabolomics data suggest that BRAF-mutated melanoma cells treated with BRAF inhibitors increased oxidative metabolism and mitochondrial dependence (i.e., survival adaptations). These alterations suggest an opportunity for mitochondrial inhibitors to improve drug efficacy. Targeting wtIDH1 with AG-120 in combination with dabrafenib significantly reduced melanoma cell viability. A positive synergy score and Bliss score greater than 1 indicate that the combined treatment was more effective in reducing cell viability than either treatment alone. Further, the combination of AG-120 and dabrafenib was the most effective in vivo, reducing tumor growth in the BRAF-mutated melanoma xenograft model, and improving mouse survival compared to monotherapy controls. Mice tolerated combination therapy with no reduction in body weight observed. Conclusions: Our findings indicate that dual inhibition of mutated BRAF and wild-type IDH1 represents a novel treatment strategy for BRAF-mutated melanomas, with potential for application to other BRAF-mutated cancers (e.g., colon, hepatobiliary, thyroid, etc.).