Human Melanoma Cell Line A375 Research Articles

Designing JAK2 Inhibitors Beyond Myeloproliferative Neoplasms ‐ Theoretical and Experimental Analysis for Solid Cancers

AbstractSomatic mutations in JAK2 have been reported to cause myeloproliferative neoplasms, autoimmune disorders, rheumatoid arthritis, and inflammation. JAK2 protein with PDB id 3FUP was selected for docking analysis via cross‐docking. Molecular docking of JAK2 with ATP and crystal ligand revealed their binding affinities and interactions. 3D QSAR models with R2=0.997 and Q2=0.984 having high stability and predictivity were generated. The biological activity of the hit compound obtained from virtual screening, LAS 26879206, was predicted to be 4.21 using the 3D QSAR model. Modification of LAS 26879206 utilizing 3D QSAR contours for modulating its JAK2 inhibitory potential yielded two novel inhibitors with a 2‐aminopyrimidine core – ACP1 and ACPM1. Molecular docking, molecular dynamics simulations, and ADMET analysis indicated improvements in the efficiency of these compounds compared to the hit compound. Synthesis of these compounds was carried out using a two‐step reaction‐ first Buchwald – Hartwig coupling reaction to yield an intermediate ACB1 which was then coupled with appropriate boronic acids via Suzuki reaction to get ACP1 and ACPM1. The effects of these designed compounds as anticancer agents were analyzed in hCT‐116 (human colorectal carcinoma), HeLa (cervical carcinoma), A549 (human lung adenocarcinoma), and A375 (human melanoma) cell lines. ACPM1 was found to be active in all the cell lines with a pIC50 of 5 which was in concordance with the in silico studies. Western blot analysis validated the JAK2 inhibitory property of ACPM1 which can be further ensured by in vivo studies.

Plant Based Extract Oil-Based Nano emulsions: Impact on Human Melanoma Cell Line.

Cancer is a challenge for either the patient or the healthcare manager. Treatment protocols based on chemotherapy or radiotherapy, or both are interfering with the patient's life making him suffer rather than being alleviated. This burden pushed the scientists to search for new regimens that may help ameliorate patient as well as doctor inconvenience. Benefits of plant extracts as medical substitutes in cancer management have been proved. New nano formulated drug delivery systems may help overcoming remedy regimens barriers and obstacles. The present research topic aims to evaluate the anticancer power of two plant extracts in nano emulsion formulation on human melanoma cell line. Carvacrol and rosemary essential oils were obtained, and nano emulsions were formulated. NE were characterized using TEM for charge and size distribution. The A375 human melanoma cell line was cultured and propagated then IC50 of prepared NE was added. Assessment of cell cytotoxicity, effect on angiogenesis and apoptosis were tested. After synthesis and characterization, both carvacrol nano emulsion (CNE) and rosemary nano emulsion (RNE) were capable of inhibiting melanoma cell line viability, angiogenesis and they enhanced the expression of caspase-3 proapoptotic marker. Rosemary and carvacrol extract nano emulsions could be a new revolutionary agent in human melanoma therapy and these formulations can be applied locally.

Abstract 5647: Investigating the role of AP-2α in melanomagenesis

Abstract Introduction: Transcription factor AP-2α (encoded by the TFAP2A gene) is a protein involved in the early development of multiple tissues, particularly the neural crest. Melanocytes are derived from the neural crest and their development appears to rely on AP-2α and its paralogues, along with master regulator MITF. Prior studies have indicated that AP-2α is a crucial regulator of melanoma metastasis. However, it is unclear if AP-2α plays a role in melanomagenesis. We sought to understand the role of AP-2α in melanomagenesis, hypothesizing that knock out (KO) of TFAP2A would suppress malignant potential in vitro and downregulate genetic pathways linked to malignant transformation. Methods: TFAP2A KO was generated with a single-guide RNA CRISPR/Cas9 system targeting exon 3 of the gene in the human melanoma cell line A375. Tumorigenesis phenotype was assessed by evaluating contact-independent colony formation with the in vitro soft agar assay. To identify downstream regulatory programs controlled by TFAP2A, we then utilized RNA-seq and Gene Set Enrichment Analysis (GSEA, San Diego). RNA-seq was performed on four clones each for wildtype and TFAP2A KO samples and the resulting data was processed using GSEA to identify gene sets modified after TFAP2A KO. Results: Targeting of TFAP2A with CRISPR/Cas9 effectively knocked out AP-2α protein expression in the A375 melanoma cell line. Colony formation on soft agar assay was significantly decreased after TFAP2A KO (P < 0.01). RNA-seq revealed significant differential expression of 11,195 genes after TFAP2A KO. Gene sets with negative enrichment after TFAP2A KO included those overall higher in tumors versus normal tissue (FWER P = 0.003) and multiple signaling pathways related to malignant transformation such as TGF-β (FWER P = 0.003), MYC (FWER P = 0.009), KRAS (FWER P = 0.03), and Notch (FWER P = 0.043). Conclusions: Our data suggests that TFAP2A plays a role in cutaneous melanomagenesis. TFAP2A may represent a therapeutic target for melanoma, though further pre-clinical studies are needed. We plan to further test this hypothesis in a melanoma mouse model with conditional TFAP2A KO under the control of inducible Cre recombinase driven by the mouse tyrosinase gene promoter. Citation Format: Nicholas Bartschat, Jeremy Chang, Mohammed Suraju, Ryan Nagel, Zhijie Li, Jeffrey White, Colin Kenny, Ronald Weigel. Investigating the role of AP-2α in melanomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5647.

Tyrosinase regulates the motility of human melanoma cell line A375 through its hydroxylase enzymatic activity

Melanoma, originating from melanocytes, is a highly aggressive tumor. Tyrosinase is involved in melanin production in melanocytes, and its overexpression is noted in malignant melanomas. However, the role of tyrosinase in melanomas remains unclear. Therefore, this study aimed to evaluate the potential functions of tyrosinase in the human melanoma cell line A375. The expression level of tyrosinase in A375 cells was undetectable. However, markedly increased expression level was observed in the mouse melanoma cell line B16F10 and the human melanoma cell line WM266-4. Subsequently, we investigated the effect of ectopic tyrosinase expression on A375 cell motility using wound-healing assay. The overexpression of tyrosinase resulted in enhanced cell migration in both stable and transient tyrosinase expression cells. The levels of filamentous actin were decreased in tyrosinase-expressing A375 cells, suggesting that tyrosinase regulates cell motility by modulating actin polymerization. Histidine residues in tyrosinase are important for its enzymatic activity for synthesizing melanin. Substitution of these histidine residues to alanine residues mitigated the promotion of tyrosinase-induced A375 cell metastasis. Furthermore, melanin treatment enhanced A375 cell metastasis and phosphorylation of Cofilin. Thus, our findings suggest that tyrosinase increases the migration of A375 cells by regulating actin polymerization through its enzymatic activity.

Fucoxanthin induces human melanoma cytotoxicity by thwarting the JAK2/STAT3/BCL-xL signaling axis.

Melanoma is the most lethal skin malignancy. Fucoxanthin is a marine carotenoid with significant anticancer activities. Intriguingly, Fucoxanthin's impact on human melanoma remains elusive. Signal Transducer and Activator of Transcription 3 (STAT3) represents a promising target in cancer therapy due to its persistent activation in various cancers, including melanoma. Herein, we revealed that Fucoxanthin is cytotoxic to human melanoma cell lines A2758 and A375 while showing limited cytotoxicity to normal human melanocytes. Apoptosis is a primary reason for Fucoxanthin's melanoma cytotoxicity, as the pan-caspase inhibitor z-VAD-fmk drastically abrogated Fucoxanthin-elicited clonogenicity blockage. Besides, Fucoxanthin downregulated tyrosine 705-phosphorylated STAT3 (p-STAT3 (Y705)), either inherently present in melanoma cells or inducible by interleukin 6 (IL-6) stimulation. Notably, ectopic expression of STAT3-C, a dominant-active STAT3 mutant, abolished Fucoxanthin-elicited melanoma cell apoptosis and clonogenicity inhibition, supporting the pivotal role of STAT3 blockage in Fucoxanthin's melanoma cytotoxicity. Moreover, Fucoxanthin lowered BCL-xL levels by blocking STAT3 activation, while ectopic BCL-xL expression rescued melanoma cells from Fucoxanthin-induced killing. Lastly, Fucoxanthin was found to diminish the levels of JAK2 with dual phosphorylation at tyrosine residues 1007 and 1008 in melanoma cells, suggesting that Fucoxanthin impairs STAT3 signaling by blocking JAK2 activation. Collectively, we present the first evidence that Fucoxanthin is cytotoxic selectively against human melanoma cells while sparing normal melanocytes. Mechanistically, Fucoxanthin targets the JAK2/STAT3/BCL-xL antiapoptotic axis to provoke melanoma cell death. This discovery implicates the potential application of Fucoxanthin as a chemopreventive or therapeutic strategy for melanoma management.

Recent Updates Regarding the Antiproliferative Activity of Galium verum Extracts on A375 Human Malignant Melanoma Cell Line.

The biological activity of Galium verum herba was exerted on various tumor cell lines with incredible results, but their potential effect on malignant melanoma has not been established yet. Therefore, the current study was structured in two directions: (i) the investigation of the phytochemical profile of diethyl ether (GvDEE) and butanol (GvBuOH) extracts of G. verum L. and (ii) the evaluation of their biological profile on A375 human malignant melanoma cell line. The GvDEE extract showed an FT-IR profile different from the butanol one, with high antioxidant capacity (EC50 of GvDEE = 0.12 ± 0.03 mg/mL > EC50 of GvBuOH = 0.18 ± 0.05 mg/mL). The GvDEE extract also showed antimicrobial potential, especially against Gram-positive bacteria strains, compared to the butanol extract, which has no antimicrobial activity against any bacterial strain tested. The results regarding the antitumor potential showed that both extracts decreased A375 cell viability largely (69% at a dose of 55 µg/mL of the GvDEE extract). Moreover, both extracts induce nuclear fragmentation by forming apoptotic bodies and slight chromatin condensation, which is more intense for GvDEE. Considering the results, one can state that the Galium verum herba possesses antitumor effects on the A375 human malignant melanoma cell line, a promising phytocompound for the antitumor approach to skin cancer.

Pharmacological Property and Cytotoxic Effect Showing Antiproliferative Potency in Human Melanoma Cell Lines (A375) of Combretum racemosum P. Beauv. Leaf and Root Extracts Used in Benin.

Combretum racemosum, a plant from the Combretaceae family, is traditionally used in Benin for various health problems. However, scientific research on Beninese samples of this plant is limited. The aim of this study was to identify and assess the bioactive compounds in the plant's leaves and roots. Initial screening involved analyzing powders derived from these parts for total polyphenols, flavonoids, and both condensed and hydrolyzable tannins. The polyphenolic compounds were analyzed using HPLC-DAD-ESI-MS. To evaluate the plant's antimicrobial properties, the agar diffusion method was employed, while FRAP and DPPH assays were used to determine its antioxidant capacity. For anti-inflammatory activity, the study utilized tests for in vitro protein denaturation inhibition and in vivo acute edema induced by carrageenan. Additionally, an antiproliferative assay was conducted using the human melanoma cell line A375. The analysis revealed the presence of significant polyphenolic compounds in both the leaf and root extracts of C. racemosum. Notably, compounds like Pedunculagin, Vescalagin, Casuarictin, and Digalloyl-glucoside were abundant in the leaves, with Vescalagin being especially predominant in the roots. The study also found that the dichloromethane extracts from the leaves and roots exhibited bactericidal effects on a substantial percentage of meat-isolated strains. Moreover, the antioxidant activities of these extracts were confirmed through FRAP and DPPH methods. Interestingly, the dichloromethane root extract showed strong activity in inhibiting thermal albumin denaturation, while the water-ethanol leaf extract demonstrated significant edema inhibition. Finally, the study observed that C. racemosum extracts reduced cell viability in a dose-dependent manner, with leaf extracts showing more pronounced antiproliferative effects than root extracts. These findings highlight the potential of C. racemosum leaves and roots as sources of compounds with diverse and significant biological activities. In conclusion, C. racemosum's leaves and roots exhibit promising biological activities, highlighting their potential medicinal value.

Low-dose imiquimod induces melanogenesis in melanoma cells through an ROS-mediated pathway

BackgroundMelanogenesis is the process of melanin maturation which not only protects skin from UV radiation but also plays an important role in antigenicity of melanomas. Imiquimod (IMQ) is a toll-like receptor 7 (TLR7) agonist that exhibits antiviral and anticancer activity. ObjectiveTo explore whether IMQ could induce melanogenesis in melanoma cells. MethodsThe mouse melanoma cell line B16F10, the mouse immortalized melanocyte Melan-A, and human melanoma cell lines MNT-1, C32 and A375 were utilized in this study. The pigmented level was observed by the centrifuged cell pellet. The intracellular and extracellular melanin levels were examined in the absorbance in NaOH-extracted cell lysate and cell-cultured medium, respectively. The expression of melanogenesis related proteins was examined by immunoblotting. The intracellular cyclic AMP amount was evaluated by the cAMP Glo assay kit. The activity of phosphodiesterase 4B (PDE4B) was investigated by CREB reporter assay with overexpressed PDE4B or not. ResultsWe demonstrated that a low dose of IMQ could trigger melanogenesis in B16F10 cells. IMQ induced microphthalmia-associated transcription factor (MITF) nuclear translocation, upregulated the expression of melanogenesis-related proteins, increased tyrosinase (TYR) activity, and led to pigmentation in B16F10 cells. Next, we found that IMQ-induced melanogenesis was activated by excessive intracellular cAMP accumulation, which was regulated through IMQ-mediated PDE4B inhibition. Finally, IMQ-induced ROS production was found to be involved in melanogenesis by its control of PDE4B activity. ConclusionsLow dose of IMQ could activate melanogenesis through the ROS/PDE4B/PKA pathway in melanoma cells.

In vitro cytotoxic potential of cow dung and expired tomato sauces-derived carbon nanodots against A-375 human melanoma cell line

Converting biowaste into a functional product is put to the test by the growing amount of biowaste in the world and the environmental problems it causes. In this research study, we synthesized, characterized, and evaluated bluish-green luminescent carbon nanodots (CNDs) from cow dung and expired tomato sauces via a hydrothermal synthesis method at 160 °C for 8 h. The carbon nanodots were fabricated without additional passivating agents and exhibited good physicochemical and optical properties. The intrinsic properties of carbon nanodots were characterized using various spectral techniques. First, we evaluated the cytotoxic potential of carbon nanodots against A-375 human melanoma cell lines. This study revealed that carbon nanodots exhibited potent cytotoxicity and significantly inhibited the proliferation of A-375 cells in a dose-dependent manner. Next, we demonstrated these carbon nanodot's free radical scavenging potential by employing 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The bluish-green fluorescent carbon nanodots fabricated using a green synthesis approach have broad potential for biological applications.

Melanocortin receptor 4 as a new target in melanoma therapy: Anticancer activity of the inhibitor ML00253764 alone and in association with B-raf inhibitor vemurafenib

The aim of our study is to investigate in vitro and in vivo MC4R as a novel target in melanoma using the selective antagonist ML00253764 (ML) alone and in combination with vemurafenib, a B-rafV600E inhibitor.The human melanoma B-raf mutated A-2058 and WM 266-4 cell lines were used. An MC4R null A-2058 cell line was generated using a CRISPR/Cas9 system. MC4R protein expression was analysed by western blotting, immunohistochemistry, and immunofluorescence. Proliferation and apoptotic assays were performed with ML00253764, whereas the synergism with vemurafenib was evaluated by the combination index (CI) and Loewe methods. ERK1/2 phosphorylation and BCL-XL expression were quantified by western blot. In vivo experiments were performed in Athymic Nude-Foxn1nu male mice, injecting subcutaneously melanoma cells, and treating animals with ML, vemurafenib and their concomitant combination. Comet and cytome assays were performed.Our results show that human melanoma cell lines A-2058 and WM 266-4, and melanoma human tissue, express functional MC4R receptors on their surface. MC4R receptors on melanoma cells can be inhibited by the selective antagonist ML, causing antiproliferative and proapoptotic activity through the inhibition of phosphorylation of ERK1/2 and a reduction of BCL-XL. The concomitant combination of vemurafenib and ML caused a synergistic effect on melanoma cells in vitro and inhibited in vivo tumor growth in a preclinical model, without causing mouse weight loss or genotoxicity.Our original research contributes to the landscape of pharmacological treatments for melanoma, providing MC4R antagonists as drugs that can be added to established therapies.

Dataset: Impact of β-Galactosylceramidase Overexpression on the Protein Profile of Braf(V600E) Mutated Melanoma Cells

β-Galactosylceramidase (GALC) is a lysosomal enzyme involved in sphingolipid metabolism by removing β-galactosyl moieties from β-galactosyl ceramide and β-galactosyl sphingosine. Previous observations have shown that GALC exerts a pro-oncogenic activity in human melanoma. Here, the impact of GALC overexpression on the proteomic landscape of BRAF-mutated A2058 and A375 human melanoma cell lines was investigated by liquid chromatography–tandem mass spectrometry analysis of the cell extracts. The results indicate that GALC overexpression causes the upregulation/downregulation of 172/99 proteins in GALC-transduced cells when compared to control cells. Gene ontology categorization of up/down-regulated proteins indicates that GALC may modulate the protein landscape in BRAF-mutated melanoma cells by affecting various biological processes, including RNA metabolism, cell organelle fate, and intracellular redox status. Overall, these data provide further insights into the pro-oncogenic functions of the sphingolipid metabolizing enzyme GALC in human melanoma.

Investigating the antitumor effects of a novel ruthenium (II) complex on malignant melanoma cells: An NMR-based metabolomic approach

Metals have been used for many years in medicine, particularly for the treatment of cancer. Cisplatin is one of the most used drugs in the treatment of cancer. Although platinum-containing therapeutics have unparalleled efficacy in cancer treatment, they are coupled with adverse effects and the development of tumour resistance. This has led to the exploration of other metal-based modalities including ruthenium-based compounds. Thus, in our previous study, we synthesized and characterized a novel ruthenium (II) complex (referred to herein as GA113) containing a bis-amino-phosphine ligand. The complex was subsequently screened for its anti-cancerous potential against a human malignant melanoma A375 cell line and findings revealed favourable cytotoxicity. In the current study, a nuclear magnetic resonance (NMR)-based cellular metabolomics approach was applied to probe the possible mechanism of GA113 in A375 cells. In addition, other biological assays including light microscopy, Hoechst-33258 and MitoTracker Orange CM-H2TMRos stain were used to assess cellular viability and apoptosis in GA113-treated cells. Consequently, multivariate statistical data analysis was applied to the metabolomic data to identify potential biomarkers. Six signatory metabolites were altered after treatment. Changes in these metabolites were linked to two metabolic pathways, which include the alanine, aspartate, and glutamate metabolic pathway as well as the glycine, serine, and threonine pathway. By means of an NMR-based metabolomic approach, we identified the potential mechanism of action of complex GA113 in A375 cancer cells thus providing new insights into the metabolic pathways affected by complex GA113 and establishing a foundation for further development, research, and eventual application in cancer.

Exogenous recombinant N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) inhibits progression of B16F10 cutaneous melanomas and modulates cell signaling

In the syngeneic, subcutaneous B16F10 mouse model of malignant melanoma, treatment with exogenous ARSB markedly reduced tumor size and extended survival. In vivo experiments showed that local treatment with exogenous N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) led to reduced tumor growth over time (p < 0.0001) and improved the probability of survival up to 21 days (p = 0.0391). Tumor tissue from the treated mice had lower chondroitin 4-sulfate (C4S) content and lower sulfotransferase activity. The free galectin-3 declined, and the SHP2 activity increased, due to altered binding with chondroitin 4-sulfate. These changes induced effects on transcription, which were mediated by Sp1, phospho-ERK1/2, and phospho-p38 MAPK. Reduced mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4), carbohydrate sulfotransferase 15 (N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase), and matrix metalloproteinases 2 and 9 resulted. Experiments in the human melanoma cell line A375 demonstrated similar responses to exogenous ARSB as in the tumors, and inverse effects followed ARSB siRNA. ARSB, which removes the 4-sulfate group at the non-reducing end of C4S, acts as a tumor suppressor, and treatment with exogenous ARSB impacts on vital cell signaling and reduces the expression of critical genes associated with melanoma progression.

IMP3 Immunohistochemical Expression Is Related with Progression and Metastases in Xenografted and Cutaneous Melanomas

Introduction: Insulin-like growth factor-II messenger RNA-binding protein-3 (IMP3) over-expression is a predictor of tumor recurrence and metastases in some types of human melanoma. Our objective was to evaluate the immunohistochemical expression of IMP3 and other molecules related to tumor prognosis in melanoma-xeno-tumors undergoing treatment. We test the effect of radiotherapy (RT) and mesenchymal stromal cells (MSCs) treatment, analyzing the tumorigenic and metastatsizing capacity in a mice melanoma xenograft model. Materials and Methods: We inoculated A375 and G361 human melanoma cell lines into NOD/SCID gamma mice (n = 64). We established a control group, a group treated with MSCs, a group treated with MSCs plus RT, and a group treated with RT. We assessed the immunohistochemical expression of IMP3, E-cadherin, N-cadherin, PARP1, HIF-1α, and the proliferation marker Ki-67. Additionally, we performed a retrospective study including 114 histological samples of patients diagnosed with malignant cutaneous superficial spreading melanoma (n = 104) and nodular melanoma (n = 10) with at least 5 years of follow-up. Results: Most morphological and immunohistochemical features show statistically significant differences between the 2 cell lines. The A375 cell line induced the formation of metastases, while the G361 cell line provoked tumor formation but not metastases. All three treatments reduced the cell proliferation evaluated by the Ki-67 nuclear antigen (p = 0.000, one-way ANOVA test) and reduced the number of metastases (p = 0.004, one-way ANOVA test). In addition, the tumor volumes reduced in comparison with the control groups, 31.74% for RT + MSCs in the A357 tumor cell line, and 89.84% RT + MSCs in the G361 tumor cell line. We also found that IMP3 expression is associated with greater tumor aggressiveness and was significantly correlated with cell proliferation (measured by the expression of Ki-67), the number of metastases, and reduced expression of adhesion molecules. Conclusions: The combined treatment of RT and MSCs on xenografted melanomas reduces tumor size, metastases frequency, and the epithelial to mesenchymal transition/PARP1 metastatic phenotype. This treatment also reduces the expression of molecules related to cellular proliferation (Ki-67), molecules that facilitate the metastatic process (E-cadherin), and molecules related with prognosis (IMP3).

Effects of temozolomide on tumor mutation burden and microsatellite instability in melanoma cells.

The efficacy of combination therapy with an immune checkpoint inhibitor (ICI) and cytotoxic chemotherapeutic agents has been investigated in cancer, including melanoma. Before ICIs were introduced, dacarbazine or temozolomide (TMZ) were used to treat melanoma. Several studies using glioma or colorectal cancer cells showed that TMZ can increase the tumor mutation burden (TMB) and induce mismatch repair (MMR) deficiency associated with microsatellite instability (MSI). These could increase immunoreactivity to an ICI, but this has not been evaluated in melanoma cells. We investigated the effects of TMZ on MSI status and TMB in melanoma cells. To evaluate the TMB, we performed whole-exome sequencing using genomic DNA from the human melanoma cell lines Mel18, A375, WM266-4, G361, and TXM18 before and after TMZ treatment. Polymerase chain reaction amplification of five mononucleotide repeat markers, BAT25, BAT26, NR21, NR24, and MONO27, was performed, and we analyzed changes in the MSI status. In all cell lines, the TMB was increased after TMZ treatment (the change amount of TMB with ≤ 5% variant allele frequency [VAF] was 18.0-38.3 mutations per megabase) even in the condition without obvious cytological damage. MSI after TMZ treatment was not observed in any cells. TMZ increased TMB but did not change MSI status in melanoma cells.

Suicide-Gene-Modified Extracellular Vesicles of Human Primary Uveal Melanoma in Future Therapies.

Extracellular vesicles secreted from uveal melanoma (UM) cells are involved in the establishment of the premetastatic niche and display transforming potential for the formation of metastases, preferentially in the liver. In this study, we cultivated human primary UM cells and uveal melanoma-associated fibroblasts in vitro to be transduced by infection with a retrovirus containing the suicide gene-fused yeast cytosine deaminase::uracil phospho-ribosyl transferase (yCD::UPRT). A homogenous population of yCD::UPRT-UM cells with the integrated provirus expressed the gene, and we found it to continuously secrete small extracellular vesicles (sEVs) possessing mRNA of the suicide gene. The yCD::UPRT-UM-sEVs were internalized by tumor cells to the intracellular conversion of the prodrug 5-fluorocytosine (5-FC) to the cytotoxic drug 5-fluorouracil (5-FU). The host range of the yCD::UPRT-UM-sEVs was not limited to UMs only. The yCD::UPRT-UM-sEVs inhibited the growth of the human cutaneous melanoma cell line A375 and uveal melanoma cell line MP38, as well as other primary UMs, to various extents in vitro. The yCD::UPRT-UM-sEVs hold the therapeutic and prophylactic potential to become a therapeutic drug for UM. However, the use of yCD::UPRT-UM-sEVs must first be tested in animal preclinical studies.

Antimicrobial and Apoptotic Efficacy of Plant-Mediated Silver Nanoparticles.

Phytogenically synthesised nanoparticle (NP)-based drug delivery systems have promising potential in the field of biopharmaceuticals. From the point of view of biomedical applications, such systems offer the small size, high surface area, and possible synergistic effects of NPs with embedded biomolecules. This article describes the synthesis of silver nanoparticles (Ag-NPs) using extracts from the flowers and leaves of tansy (Tanacetum vulgare L.), which is known as a remedy for many health problems, including cancer. The reducing power of the extracts was confirmed by total phenolic and flavonoid content and antioxidant tests. The Ag-NPs were characterised by various analytical techniques including UV-vis spectroscopy, scanning electron microscopy (SEM), energy-dispersive spectrometry (EDS), Fourier transform infrared (FT-IR) spectroscopy, and a dynamic light scattering (DLS) system. The obtained Ag-NPs showed higher cytotoxic activity than the initial extracts against both human cervical cancer cell lines HeLa (ATCC CCL-2) and human melanoma cell lines A375 and SK-MEL-3 by MTT assay. However, the high toxicity to Vero cell culture (ATCC CCL-81) and human fibroblast cell line WS-1 rules out the possibility of their use as anticancer agents. The plant-mediated Ag-NPs were mostly bactericidal against tested strains with MBC/MIC index ≤4. Antifungal bioactivity (C. albicans, C. glabrata, and C. parapsilosis) was not observed for aqueous extracts (MIC > 8000 mg L-1), but Ag-NPs synthesised using both the flowers and leaves of tansy were very potent against Candida spp., with MIC 15.6 and 7.8 µg mL-1, respectively.

1249 Research of hiperfect-mediated RNA interference targeting hTERT on the biological behavior of the human malignant melanoma cell line A375

1249 Research of hiperfect-mediated RNA interference targeting hTERT on the biological behavior of the human malignant melanoma cell line A375

Determination of Some Isoquinoline Alkaloids in Extracts Obtained from Selected Plants of the Ranunculaceae, Papaveraceae and Fumarioideae Families by Liquid Chromatography and In Vitro and In Vivo Investigations of Their Cytotoxic Activity.

Alkaloids are heterocyclic bases with widespread occurrence in nature. Plants are rich and easily accessible sources of them. Most isoquinoline alkaloids have cytotoxic activity for different types of cancer, including malignant melanoma, the most aggressive type of skin cancer. The morbidity of melanoma has increased worldwide every year. For that reason, developing new candidates for anti-melanoma drugs is highly needed. The aim of this study was to investigate the alkaloid compositions of plant extracts obtained from Macleaya cordata root, stem and leaves, Pseudofumaria lutea root and herb, Lamprocapnos spectabilis root and herb, Fumaria officinalis whole plant, Thalictrum foetidum root and herb, and Meconopsis cambrica root and herb by HPLC-DAD and LC-MS/MS. For determination of cytotoxic properties, human malignant melanoma cell line A375, human Caucasian malignant melanoma cell line G-361, and human malignant melanoma cell line SK-MEL-3 were exposed in vitro to the tested plant extracts. Based on the in vitro experiments, Lamprocapnos spectabilis herb extract was selected for further, in vivo research. The toxicity of the extract obtained from Lamprocapnos spectabilis herb was tested using an animal zebrafish model in the fish embryo toxicity test (FET) for determination of the LC50 value and non-toxic doses. Determination of the influence of the investigated extract on the number of cancer cells in a living organism was performed using a zebrafish xenograft model. Determination of the contents of selected alkaloids in different plant extracts was performed using high performance liquid chromatography (HPLC) in a reverse-phase system (RP) on a Polar RP column with a mobile phase containing acetonitrile, water and ionic liquid. The presence of these alkaloids in plant extracts was confirmed by LC-MS/MS. Preliminary cytotoxic activity of all prepared plant extracts and selected alkaloid standards was examined using human skin cancer cell lines A375, G-361, and SK-MEL-3. The cytotoxicity of the investigated extract was determined in vitro by cell viability assays (MTT). For in vivo determination of investigated extract cytotoxicity, a Danio rerio larvae xenograft model was used. All investigated plant extracts in in vitro experiments exhibited high cytotoxic activity against the tested cancer cell lines. The results obtained using the Danio rerio larvae xenograft model confirmed the anticancer activity of the extract obtained from Lamprocapnos spectabilis herb. The conducted research provides a basis for future investigations of these plant extracts for potential use in the treatment of malignant melanoma.

Metastatic Melanoma: A Preclinical Model Standardization and Development of a Chitosan-Coated Nanoemulsion Containing Temozolomide to Treat Brain Metastasis.

Melanoma is the most aggressive type of skin cancer. Brain metastasis is the worst scenario in metastatic melanoma and the treatment options for these patients are limited. Temozolomide (TMZ) is a chemotherapy agent used to treat primary central nervous system tumors. Our objective was to develop chitosan-coated nanoemulsion containing temozolomide (CNE-TMZ) for nasal route administration to melanoma brain metastasis treatment. A preclinical model of metastatic brain melanoma was standardized, and the efficiency of the developed formulation was further determined in vitro and in vivo. The nanoemulsion was done by spontaneous emulsification method and the formulation was characterized by size, pH, polydispersity index, and zeta potential. Culture assessments to determine cell viability were done in the A375 human melanoma cell line. To determine the safety of formulation, healthy C57/BL6 mice were treated with a nanoemulsion without TMZ. The model in vivo used B16-F10 cells implanted by stereotaxic surgery in C57/BL6 mice brains. The results demonstrate that the preclinical model used showed to be useful to analyze the efficiency of new candidate drugs to treat melanoma brain metastasis. The chitosan-coated nanoemulsions with TMZ showed the expected physicochemical characteristics and demonstrated safety and efficacy, reducing around 70% the tumor size compared to control mice, and presenting a tendency in mitotic index reduction, becoming an interesting approach to treat melanoma brain metastasis.