Human Skin Melanoma Cell Line Research Articles

Clinical significance of downregulated NISCH expression in skin cutaneous melanoma: Modulation of tumor cell invasion, migration, and EMT via PAK1 inhibition

ObjectiveThis study aimed to investigate the expression and functional role of NISCH in skin cutaneous melanoma (SKCM), exploring its association with clinical characteristics and its potential impact on human skin melanoma cell behavior. MethodsThe research assessed differential NISCH expression in SKCM tissues using the GEPIA (Gene Expression Profiling Interactive Analysis) database and validated these findings through immunohistochemical staining of 45 clinical samples. To affirm NISCH expression at the cellular level, three human skin melanoma cell lines (RPMI-7951, A375, MEL-5), and the human normal skin cell line HEMa underwent quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting. Transwell experiments evaluated the migration and invasion capabilities of RPMI-7951 and A375 cells post-transduction with NISCH or PAK1 lentiviral activation particles. Additionally, qRT-PCR analysis of epithelial-mesenchymal transition (EMT)-related gene expression (Vimentin, E-cadherin, N-cadherin) was conducted in A375 and RPMI-7951 cells. ResultsSKCM tissues exhibited significantly reduced NISCH expression compared to normal tissues. Immunohistochemical analysis revealed predominant nuclear localization of NISCH in melanoma cells, with reduced expression significantly correlating with sex, advanced stage, and lymph node metastasis. Melanoma cell lines displayed lower NISCH expression levels compared to normal skin cells. Functional experiments showcased that NISCH overexpression suppressed p-PAK1/PAK1, while PAK1 upregulation notably increased melanoma cell migration, invasion, and induced EMT. Remarkably, NISCH overexpression counteracted PAK1-induced effects on EMT, migration, and invasion in melanoma cells. ConclusionNISCH may significantly influence the aggressive behavior of SKCM cells via the PAK1 pathway, making it a potential therapeutic target for managing melanoma metastasis.

Synergistic anticancer effect of Pistacia lentiscus essential oils and 5-Fluorouracil co-loaded onto biodegradable nanofibers against melanoma and breast cancer

Chemoresistance and severe toxicities represent major drawbacks of chemotherapy. Natural extracts, including the essential oils of Pistacia lentiscus (PLEO), exhibit substantial anticancer and anti-inflammatory activities where different cancers are reported to dramatically recess following targeting with PLEO. PLEO has promising antimicrobial, anticancer, and anti-inflammatory properties. However, the therapeutic properties of PLEO are restricted by limited stability, bioavailability, and targeting ability. PLEO nanoformulation can maximize their physicochemical and therapeutic properties, overcoming their shortcomings. Hence, PLEO was extracted and its chemical composition was determined by GC–MS. PLEO and 5-Fluorouracil (5FU) were electrospun into poly-ε-caprolactone nanofibers (PCL-NFs), of 290.71 nm to 680.95 nm diameter, to investigate their anticancer and potential synergistic activities against triple-negative breast cancer cells (MDA-MB-231), human adenocarcinoma breast cancer cells (MCF-7), and human skin melanoma cell line (A375). The prepared nanofibers (NFs) showed enhanced thermal stability and remarkable physical integrity and tensile strength. Biodegradability studies showed prolonged stability over 42 days, supporting the NFs use as a localized therapy of breast tissues (postmastectomy) or melanoma. Release studies revealed sustainable release behaviors over 168 h, with higher released amounts of 5FU and PLEO at pH 5.4, indicating higher targeting abilities towards cancer tissues. NFs loaded with PLEO showed strong antioxidant properties. Finally, NFs loaded with either PLEO or 5FU depicted greater anticancer activities compared to free compounds. The highest anticancer activities were observed with NFs co-loaded with PLEO and 5FU. The developed 5FU-PLEO-PCL-NFs hold potential as a local treatment of breast cancer tissues (post-mastectomy) and melanoma to minimize their possible recurrence.Graphical

Synthesis of N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines and their photodynamic properties in the human skin melanoma cell line G361

A small series of N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines was synthesized from easily accessible 1-phenyl-1H-pyrazol-3-ol via 7-iodo-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine and 7-iodo-4-methyl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine intermediates and their subsequent use in palladium catalyzed Buchwald-Hartwig cross-coupling reaction with various anilines. Majority of the compounds were not significantly cytotoxic to melanoma G361 cells in the dark up to 10 µM concentration, but their activity could be increased by irradiation with visible blue light (414 nm). The most active compound 10 possessed EC50 values of 3.5, 1.6 and 0.9 µM in cells irradiated with 1, 5 and 10 J/cm2, respectively. The treatment caused generation of reactive oxygen species in cells and extensive DNA damage, documented by the comet assay and by detection of phosphorylated histone H2A.X, followed by apoptotic cell death. Our results suggest that N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines could serve as a potential source of photosensitizing compounds with anticancer activities.

Quantitative Autofluorescence Imaging of A375 Human Melanoma Cell Samples: A Pilot Study.

Introduction: Skin cancer is one of the most common types of malignancy worldwide. Human skin naturally contains several endogenous fluorophores, as potential sources can emit inherent fluorescence, called intrinsic autofluorescence (AF). The melanin endogenous fluorophore in the basal cell layer of the epidermis seems to have a strong autofluorescence signal among other ones in the skin. This pilot study aimed to investigate the feasibility of the detection of autofluorescence signals in the A375 human melanoma cell line in the cell culture stage using the FluoVision optical imaging system. Methods: The human skin melanoma cell line (A375) donated as a gift from Switzerland (University Hospital Basel) was cultured. For the imaging of the A375 human melanoma cell sample in this pilot study, the FluoVision optical imaging device (Tajhiz Afarinan Noori Parseh Co) was applied. The proposed clustering image processing code was developed based on the K-mean segmentation method, using MATLAB software (version 16). Results: The quantification of color pixels in the color bar along with the intensity score of the autofluorescence signal ranged between 0 and 70 was written in the image processing code execution and a threshold higher than 40%, proportional to the ratio of autofluorescent cells. The percentage of the signal of A375 autofluorescent melanoma cells in the 3 studied cell samples was calculated as 3.11%±0.6. Conclusion: This imaging method has the advantage of no need for fluorophore labels over the existing fluorescence imaging methods, and it can be regarded as one of the important choices of label-free imaging for this A375 melanoma cell line containing the intrinsic endogenous fluorophore in cell studies.

Cyclic RGD and isoDGR Integrin Ligands Containing cis-2-amino-1-cyclopentanecarboxylic (cis-β-ACPC) Scaffolds.

Integrin ligands containing the tripeptide sequences Arg-Gly-Asp (RGD) and iso-Asp-Gly- Arg (isoDGR) were actively investigated as inhibitors of tumor angiogenesis and directing unit in tumor-targeting drug conjugates. Reported herein is the synthesis, of two RGD and one isoDGR cyclic peptidomimetics containing (1S,2R) and (1R,2S) cis-2-amino-1-cyclopentanecarboxylic acid (cis-β-ACPC), using a mixed solid phase/solution phase synthetic protocol. The three ligands were examined in vitro in competitive binding assays to the purified αvβ3 and α5β1 receptors using biotinylated vitronectin (αvβ3) and fibronectin (α5β1) as natural displaced ligands. The IC50 values of the ligands ranged from nanomolar (the two RGD ligands) to micromolar (the isoDGR ligand) with a pronounced selectivity for αvβ3 over α5β1. In vitro cell adhesion assays were also performed using the human skin melanoma cell line WM115 (rich in integrin αvβ3). The two RGD ligands showed IC50 values in the same micromolar range as the reference compound (cyclo[RGDfV]), while for the isoDGR derivative an IC50 value could not be measured for the cell adhesion assay. A conformational analysis of the free RGD and isoDGR ligands by NMR (VT-NMR and NOESY experiments) and computational studies (MC/EM and MD), followed by docking simulations performed in the αVβ3 integrin active site, provided a rationale for the behavior of these ligands toward the receptor.

Synthesis and photodynamic properties of pyrazole-indole hybrids in the human skin melanoma cell line G361

Three conjugated pyrazole-indole hybrids, 5, 7 and 10, were synthesized from easily accessible 3-(hexyloxy)-1-phenyl-1H-pyrazole-4-carbaldehyde 1 and 5-iodo-3,3-dimethyl-2-phenyl-3H-indole 4 employing olefination reactions (Wittig, Ramirez), Vilsmeier-Haack formylation and palladium-catalysed cross coupling reactions (ligand-free Heck, Sonogashira). The compounds displayed no cytotoxicity in the melanoma cell line G361 unless irradiated with visible blue light (414 nm), and the potency of the compounds increased with light intensity. Lead compound 7 displayed EC50 values of 3.08, 0.26 and 0.05 μM in cells irradiated with 1, 10 and 50 J/cm2, respectively. The following experiments revealed increased levels of reactive oxygen species in treated cells and a decrease in JC-1 polymer aggregation within the mitochondria, suggesting mitochondrial membrane depolarization. In addition, extensive DNA fragmentation was detected using the comet assay and by detection of phosphorylated histone H2A.X, the level of which increased with both increased compound concentration and irradiation energy. In control experiments, cells kept either in the dark or without compound 7 did not show any DNA damage. Overall, the results demonstrated that fluorescent pyrazole-indole hybrids may serve as an interesting source of photosensitizing compounds with anticancer activity.

Exogenous hydrogen sulfide inhibits human melanoma cell development via suppression of the PI3K/AKT/ mTOR pathway

Background: Melanoma is one of the most aggressive, therapy-resistant skin cancers in the world. Hydrogen sulfide (H2S), a newly discovered gasotransmitter, plays a crucial role in the progression and development of many types of cancers. However, the effect of H2S on human skin melanoma remains to be elucidated. Objective: We aimed to explore the effect of exogenous H2S on melanoma cells and its underlying mechanisms. Methods: In this study, human skin melanoma cell lines, including A375 and SK-MEL-28, were treated with a donor of H2S (NaHS). CCK-8, scratch assay, flow cytometric analysis, western blotting and transmission electron microscopy (TEM) were performed to explore the effects of H2S on cell behaviors. Results: Treatment with NaHS inhibited cell proliferation, migration and division, while it could induce cell apoptosis and autophagy in melanoma cell lines. Moreover, NaHS significantly decreased the expression of p-PI3K, p-Akt and mTOR proteins. Furthermore, insulin-like growth factor-1 (IGF-1), the activator of PI3K/AKT/mTOR pathway, could reverse the cell behaviors caused by NaHS. Conclusion: Our results demonstrated that exogenous hydrogen sulfide could inhibit human melanoma cell development via suppression of the PI3K/AKT/mTOR pathway. Hydrogen sulfide might serve as a potential therapeutic option for melanoma.

Anti-melanoma and UV-B protective effect of microbial pigment produced by marine Pseudomonas aeruginosa GS-33

Bioactivity of a microbial pigment, extracted from fermented broth of culture marine Pseudomonas aeruginosa was screened for anticancer activity against human skin melanoma cell line SK-MEL-2. Upon characterisation, the pigment was confirmed as Phenazine-1-carboxylic acid (PCA). The PCA was found effective against SK-MEL-2 cell line at low concentration (GI50 value <10 μg/mL). Reduced cell density and cell shrinkage with typical morphological changes such as rounding of cells with loss/breaking of cell membrane were seen in SK-MEL-2 cells treated with PCA and Adriamycin. The pigment exhibited UV-B protecting activity as calculated by in vitro spectrophotometric assay and potentiated sun protection factor of commercial sunscreen lotion. Moreover, the pigment was non-toxic up to concentration of 100 ppm as assessed erythrocyte haemolysis assay. These results suggest that microbial pigment PCA could be effective and promising in the treatment as well as prevention of melanoma skin cancers.

Anticancer properties of phospholipase A2 from Daboia siamensis venom on human skin melanoma cells.

BackgroundPhospholipase A2 (PLA2) is a major component of the Daboia siamensis venom, which is able to hydrolyse the membrane of various cells. For this reason, the activity of PLA2 was investigated regarding its pharmaceutical properties. This study was conducted to explore the pharmacological properties of a PLA2 from Daboia siamensis (dssPLA2) venom on human skin melanoma cell line (SK-MEL-28).MethodsdssPLA2 was isolated by ion exchange and gel filtration columns. Various concentrations of dssPLA2 were investigated for cytotoxic activity and inhibition of migration on SK-MEL-28 cells. Cell death analysis, mRNA expression levels of Notch I-III and BRAF V600E genes were also determined.ResultsdssPLA2 exhibited cytotoxicity on SK-MEL-28 for 24 and 72 h as compared with untreated cells. However, it had no toxic effects on CCD-1064sk cells under the same conditions. dssPLA2 (0.25 and 0.5 μg/mL) induced 17.16 and 30.60 % of apoptosis, while activated 6.53 and 7.05 % of necrotic cells. dssPLA2 at 0.25, 0.5, 1 and 2 μg/mL could inhibit migration on SK-MEL-28 cells for 24 h by 31.06, 41.66, 50 and 68.75 %, respectively. The action of dssPLA2 significantly reduced the levels of Notch I and BRAF V600E genes expression on SK-MEL-28 cells compared with untreated cells at 72 h.ConclusionsThis study indicates that dssPLA2 had potential effects of apoptosis, necrosis, cytotoxicity and inhibition of migration on SK-MEL-28 cells. dssPLA2 could possibly be a selective agent that targets cancer cells without affecting normal cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s40409-016-0061-z) contains supplementary material, which is available to authorized users.

Comparative Anti-Proliferative Studies of Natural Phenolic Monoterpenoids on Human Malignant Tumour Cells

In present study we report prominent effect of carvacrol (5-isopropyl-2-methyl phenol), thymol (2-isopropyl-5- methyl phenol) and eugenol (4 allyl-2-methoxyphenol) on Human melanoma cells. These three compounds were screened for Anti-proliferative test by using Sulforhodamine B (SRB) assay. For this investigation we used human breast melanoma cell line MCF-7, human skin melanoma cell line SK-MEL-2, human colon melanoma cell line HCT-15 and human pancreatic melanoma cell line MIAPaCa-2. Carvacrol was identified as most potent molecule with low GI50 value as 0.1 μL for selected human cancer cell lines; which is comparatively equal to standard drug 14 Adriamycin (ADR). Thymol and Eugenol also exhibited remarkable GI50 for human cancer cell 15 lines.

CHARACTERISTICS OF MULTIDRUG RESISTANCE IN HUMAN SKIN MELANOMA CELL LINES

MDR is the main obstacle to chemotherapy efficiency. MDR can grow in cancer cells even if only the one cytostatic agent will act. The aim of the nowadays work is to characterize MDR in metastatic human skin melanoma cell lines prepared in “N.N. Blokhin Russian Cancer Research Center”. pgpl70 expression was detected by immunofluorescence methods. mRNA of MDR gene was identified by Reverse Transcriptase- PCR( RT-PCR) method. Rhodamine 123 (Rhl23) emission has been evaluated by flow cyto- fluorimetiy, cytotoxic activity was estimated by MTT-tests. The cells sensitivity to Aianoza cytostatic effects has showed that mel Kor cells were sensitive to Aranoza acting, but mel Ibr and mel Mtp X were not. Mel Ibr cells had expressed pgpl70 from 35 to 50 per cent, it was detected by immunofluorescence reaction. Mel Kor and mel Mtp-X cells were not expressed P-glycoprotein. mRNA of genes responsible for multi-drug resistance - MDR1, BCRP, MRP1 and LRP (MVP) - were detected by PCR. mRNA of BCRP and MRP1 genes has low expression, barely visible stripes after 33 cycles in all cell lines samples. LRP (MVP) genes expression of mRNA, unfortunately, never managed to see. YB1 gene mRNA expression is well, it is typically for cancer cells. mRNA of gene was found in mel MtpX and mel Ibr subclones cell lines. Mel Kor cells didn't contain mRNA of MDR1 gene. The study of the Rhl23 emission from cells showed that mel Kor control cells had accumulated Rhl23 and didn't throw it out. Mel Ibr cell line accumulated Rhl23 and threw out the half part of it. Mel MtpX cell tine had accumulated the less part of Rhl23 and almost all were thrown out. Thus, the study shows that mel Kor cell tine that are sensitive to Aranoza doesn't express pgpl70, not contain mRNA of multi-chug resistance genes and does not throw Rhl23. Mel Ibr cells resistant to the Aranoza cytotoxic action express pgpl70 ,contain mRNA of MDR1 gene and throw out Rhl23. However, mel MtpX cell line resistant to Aranoza does not express pgpl70, but contains mRNA of MDR1 gene and actively throws out Rhl23.

The Cytotoxic and Proapoptotic Activities of Hypnophilin are Associated with Calcium Signaling in UACC-62 Cells

Hypnophilin (HNP) is a sesquiterpene that is isolated from Lentinus cf. strigosus and has cytotoxic activities. Here, we studied the calcium signaling and cytotoxic effects of HNP in UACC-62 cells, a human skin melanoma cell line. HNP was able to increase the intracellular calcium concentration in UACC-62 cells, which was blocked in cells stimulated in Ca(2+) -free media. HNP treatment with BAPTA-AM, an intracellular Ca(2+) chelator, caused an increase in calcium signals. HNP showed cytotoxicity against UACC-62 cells in which it induced DNA fragmentation and morphological alterations, including changes in the nuclear chromatin profile and increased cytoplasmatic vacuolization, but it had no effect on the plasma membrane integrity. These data suggest that cytotoxicity in UACC-62 cells, after treatment with HNP, is associated with Ca(2+) influx. Together, these findings suggest that HNP is a relevant tool for the further investigation of new anticancer approaches.

Isolation and in vitro cytotoxic activity of 11-methylixoside isolated from bark of Randia dumetorum Lamk

Summary Randia dumetorum (family Rubiaceae) is highly reputed ayurvedic medicinal tree commonly known as the Mainphal. A large deciduous thorny shrub grows up to 5 m of height. It occurs almost throughout India up to 1200 m of altitude. It is found in Himalaya from Jammu East ward ascending to 400 m and from Kashmir to East ward up to 1200 m. 11-methylixoside (compound 1), an iridoid glucoside, was isolated from the bark of this plant. The structure was characterized by using spectroscopic methods including 1D-1HNMR,13C-NMR and 2D-NMR (HSQC,HMBC, DQF-COSY) experiments and confirmed by comparison of their NMR data with those from the literature. This compound has been reported for the first time in Randia dumetorum bark. The 11-methylixoside was subjected to cytotoxic activity against MDA-MB-231 (breast cancer cell line) and SK-MEL-2 (human skin melanoma cell line), BE(2)C (neuroblastoma cell line derived from human bone marrow) and U87MG (human neuronale glioblastoma (astrozytom) cell line showed appreciable cytotoxic effect with IC50 value 63.10 µg/ml concentration for SK-MEL-2 (human skin melanoma cell line).

Induction of oxidative stress, DNA damage, and apoptosis in a malignant human skin melanoma cell line after exposure to zinc oxide nanoparticles

The widespread use of zinc oxide (ZnO) nanoparticles worldwide exposes humans to their adverse effects, so it is important to understand their biological effects and any associated risks. This study was designed to investigate the cytotoxicity, oxidative stress, and apoptosis caused by ZnO nanoparticles in human skin melanoma (A375) cells. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] and lactate dehydrogenase-based cell viability assays showed a significant decrease in cell viability after exposure to ZnO nanoparticles, and phase contrast images revealed that cells treated with these nanoparticles had a lower density and a rounded morphology. ZnO nanoparticles were also found to induce oxidative stress, evidenced by generation of reactive oxygen species and depletion of the antioxidant, glutathione. Induction of apoptosis was confirmed by chromosomal condensation assay and caspase-3 activation. Further, more DNA damage was observed in cells exposed to the highest concentration of ZnO nanoparticles. These results demonstrate that ZnO nanoparticles have genotoxic potential in A375 cells, which may be mediated via oxidative stress. Our short-term exposure study showing induction of a genotoxic and apoptotic response to ZnO nanoparticles needs further investigation to determine whether there may be consequences of long-term exposure to ZnO nanoparticles.

Hunchback sequence binding protein suppresses mouse TGF-β3 promoter in vitro

Transforming growth factor-β3 (TGF-β3) has a specific role in vivo in the patterning of embryonic and tissue-specific gene expression. We have cloned and sequenced the mouse TGF-β3 5′-flanking region to study the transcriptional regulation of this gene. Promoter fragments were cloned into a promoterless luciferase reporter plasmid to study functional activity in a human skin melanoma cell line A375 (A375). Sequential 5′-deletion encompassing DNA sequences from −2297 to −1003 bp exhibited high promoter activity in A375 cells, whereas the promoter activity decreased to minimal in the −742 to 104 bp regions, suggesting both positive and negative transcriptional regulation in the TGF-β3 promoter. The fragment containing 1.8 kb had the highest luciferase activity. Characterization of this 1.8 kb 5′-flanking region upstream of the translation start site showed a putative hunchback-binding site consensus sequence. The electrophoretic mobility shift assay (EMSA) and transient transfection experiments showed that the putative hunchback-binding site is functional and regulated TGF-β3 promoter transcriptional activity. The DNA-complex including the hunchback sequence binding protein (HbSBP) was important for suppression of the promoter activity in A375 cells. Mutation of the hunchback consensus sequence resulted in up to 2-fold higher promoter activity than the wild type construct. There was an absence of HbSBP in other cell lines tested including 3T3 fibroblast and B-16 mouse skin melanoma as determined by EMSA and Western blot analysis. HbSBP may function as a TGF-β3 gene transcriptional regulator and may be expressed in a cell type-specific manner.