Effect of miRNA-193b-5p-mediated decreased expression of transcriptional regulator CITED2 on melanogenesis / 中华皮肤科杂志

Objective:To investigate the effect of miRNA (miR) -193b-5p on melanogenesis and its possible mechanisms.Methods:Human primary melanocytes were isolated from discarded normal foreskin tissues of healthy males after circumcision, and cultured in vitro. miR-NC mimics (miR-NC mimic group) and miR-193b-5p mimics (miR-193b-5p mimic group) were transfected into human primary melanocytes and human MNT1 melanoma cells, separately. After transfection, real-time quantitative PCR (RT-qPCR) was performed to determine the overexpression efficiency of miR-193b-5p at 48 hours, Western blot analysis to determine the expression of melanogenesis-related proteins tyrosinase (TYR) and microphthalmia-associated transcription factor (MITF) in human primary melanocytes and human MNT1 melanoma cells at 72 hours, and the melanin content in the above cells was determined by a sodium hydroxide solubilization method at 1 week. The target gene of miR-193b-5p was predicted by using Targetscan algorithms and verified by dual-luciferase reporter assay, and RT-qPCR and Western blot analysis were performed to analyze changes in mRNA and protein expression of the target gene respectively after the overexpression of miR-193b-5p. Two-independent-samples t test was used for comparisons between two groups. Results:In human primary melanocytes and human MNT1 melanoma cells, the miR-193b-5p expression levels were significantly higher in the miR-193b-5p mimic groups than in the miR-NC mimic groups ( t = 65.57, 22.49, respectively, both P < 0.001) , and the melanin content was significantly lower in the miR-193b-5p mimic groups (0.091 ± 0.007, 0.130 ± 0.004, respectively) than in the miR-NC mimic groups (0.117 ± 0.002, 0.188 ± 0.032, t = 5.98, 3.24, P < 0.01, < 0.05, respectively) . Western blot analysis showed that the expression of melanogenesis-related proteins TYR and MITF in both human primary melanocytes and human MNT1 melanoma cells was significantly lower in the miR-193b-5p mimic groups than in the miR-NC mimic groups (all P < 0.01) . TargetScan analysis and dual-luciferase reporter assay revealed a binding site for miR-193b-5p in the 3′ untranslated region of the transcriptional regulator CITED2. After up-regulation of miR-193b-5p expression in human primary melanocytes and human MNT1 melanoma cells, the CITED2 mRNA and protein expression levels significantly decreased compared with the miR-NC mimic groups (all P < 0.05) . Conclusion:miR-193b-5p overexpression can down-regulate the expression of melanogenesis-related proteins TYR and MITF, and then inhibit melanogenesis, which may be related to the targeted inhibition of CITED2 expression.

Knockdown of NSUN2 Inhibits Melanoma Cells Proliferation by Regulating Cyclin Expression / 中国生物化学与分子生物学报

In order to investigate functions of NOP2/Sun RNA methyltransferase 2 (Nsun2) in melanoma cells, shRNA lentiviral vectors were constructed to target Nsun2 in mouse melanoma B16 cells. After treated B16 cells with recombinant virus, the mRNA and protein levels of NSUN2 in the interference group were significantly reduced, and the knock down efficiency reached 80%. EdU staining assay showed significant inhibition of DNA synthesis in Nsun2 knock-down B16 cells. RNA-seq was used to systematically analyze the gene expression of the Nsun2 knock-down and the control cells. A total of 1062 differentially expressed genes (DEGs) were screened, of which 678 were up-regulated and 384 were down-regulated. DEGs were mainly enriched in chromosome, centromere region, and protein binding GO terms. KEGG analysis showed that DEGs were significantly enriched in cell cycle, DNA replication, cell senescence and other pathways. RT-qPCR and RNA-seq data demonstrated that Cdk2, Ccnal, Cdc25b and other genes related to enhance cell division were significantly down-regulated, while Gadd45g and Gadd45a and other genes arresting cell growth were significantly up-regulated. Collectively, this study indicated that NSUN2 affects melanoma cell proliferation by regulating cell cycle and DNA replication, and provided fundamental data for exploring the molecular mechanism of melanoma genesis and development.

Antitumor effect of depsidones from lichens on tumor cell lines and experimental murine melanoma

Abstract Lichens have exhibited numerous biological activities, including growth inhibition of tumor cells. This study evaluated the antiproliferative activity of hypostictic and salazinic acids against tumor cell lines (B16-F10, PC-03, MCF7, HT-29, HEP-G2, K562 and 786-0) by the SRB assay in vitro and antitumor activity in experimental murine melanoma in vivo. Activation of caspase-3 was quantified by flow cytometry. The murine experimental melanoma model B16-F10 was used in BALB/c mice for evaluation of antitumor activity. Hypostictic acid showed significant antiproliferative activity in K562 cells (GI50 2.20 µM), B16-F10 (GI50 13.78 µM) and 786-0 (GI50 14.24 µM), whereas salazinic acid was more active against K562 cells (GI50 64.36 µM), HT-29 (GI50 67.91 µM) and B16-F10 (GI5078.64 µM). Quantification of capase-3 revealed that the test compounds did not increase the expression of that enzyme. In the in vivo antitumor evaluation in B16-F10 melanoma, the isolated compounds inhibited tumor growth in relation to weight and volume. Hypostictic acid (16.7 mg/kg) inhibited 72% and salazinic acid 88% of tumor volume (p < 0.05). The results indicated that, both in the in vitro and in vivo models, the compounds evaluated showed antiproliferative and antitumor activities.

Induction of Melanoma Cell-Selective Apoptosis Using Anti-HER2 Antibody-Conjugated Gold Nanoparticles

PURPOSE: This study was conducted to verify the induction and mechanism of selective apoptosis in G361 melanoma cells using anti-HER2 antibody-conjugated gold nanoparticles (GNP-HER2). MATERIALS AND METHODS: Following GNP-HER2 treatment of G361 cells, cell cycle arrest and apoptosis were measured by WST-1 assay, Hemacolor staining, Hoechst staining, immunofluorescence staining, fluorescence-activated cell sorting analysis, and Western blotting.

Effects of PKM1 gene on the migration of ocular melanoma cells / 眼科新进展

Objective To construct the eukaryotic expression vector of pyruvate kinase M1 (PKM1) gene labeled with pXJ-40-myc and detect its biological activity in ocular B16 melanoma cells.Methods Ocular B16 melanoma ceils were randomly divided into experimental and control group,and the experimental group was transfected with pXJ-40-myc-PKM1 plasmid and the control group was transfected with pXJ-40-myc plasmid.Then PKM1 gene was amplified by PCR with human liver cDNA library as the template.The recombinant plasmid pXJ-40-myc-PKM1 was identified by bacteria PCR and double enzyme digestion,followed by transfection of pXJ40-myc-PKM1 and pXJ-40-myc plasmid into B16 melanoma cells,and finally,the expression of PKM1 protein was verified by the Western blot,while wound healing assay was used to detect the effects of PKM1 on the migration of ocular melanoma ceils.Results The length of PKM1 gene was 1800bp,which was consistent with the expected size.Compared with the control group,the result of bacteria PCR was positive.The length of double enzyme digestion was 4000 bp and 1800 bp respectively.Western blot results showed that recombinant plasmld pXJ-40-myc-PKM1 was successfully expressed in ocular B16 melanoma cells.Compared with the control group,wound healing assay showed that recombinant plasmid could inhibit the migration of ocular B16 melanoma cells.Conclusion The eukaryotic expression vector of pXJ-40-myc-PKM1 is successfully constructed,which can suppress the migration of ocular B16 melanoma cells.

The effects of green tea (Camellia sinensis) flower extracton melanin synthesis in B16-F10 melanoma cells / 대한수의학회지

The present study observed the effects of a green tea (Camellia sinensis) flower extract (GTFE) on melanin synthesis in B16-F10 melanoma cells. GTFE exhibited antioxidant activity on 2,2-diphenyl-1-picrylhydrazyl and inhibited mushroom tyrosinase activity in a dose-dependent manner. Furthermore, GTFE significantly diminished α-melanocyte stimulating hormone (α-MSH) stimulated cellular melanin content and tyrosinase activity throughout the concentration range evaluated. Based on RNA sequencing analysis, differential gene expression patterns observed in α-MSH stimulated B16-F10 melanoma cells were normalized by the addition of GTFE. In particular, the expression levels of melanoregulin and tyrosinase genes which are key regulating genes in melanin synthesis were up-regulated by 3.5 and 3 fold respectively by α-MSH, and were normalized to control levels by the addition of GTFE. The results suggest that GTFE inhibits melanin synthesis in α-MSH stimulated B16-F10 melanoma cells by normalizing expression of genes that are essential for melanin synthesis. Overall, the results suggest that GTFE could be applied in the development of a whitening agent for the treatment of dermal hyperpigmentation.

Effects of human BRE1B gene on the growth of B16 melanoma cells / 眼科新进展

Objective To construct the eukaryotic expression vector of human BRE1B labeled with pCMV-Tag-2B and detect its biological activity in melanoma cells preliminarily.Methods Ocular B16 melanoma cells were randomly divided into the experimental group,in which the cells were transfected with pCMV-Tag-2B-BRE1B and the control group,which was transfected with pCMV plasmid.The CDS coding region of human BRE1B gene was amplified by PCR using human mammary gland cDNA as a template for construction of the recombinant plasmid pCMV-Tag-2B-BRE1B.After transfected with pCMV-Tag-2B-BRE1B and pCMV plasmid in the experimental and control group,respectively,Western blot was applied to detect the expression of BRE1B protein,while cell counting kit-8 (CCK8) and colony assays were used to analyze the effects of recombinant plasmid pCMV-Tag-2B-BRE1B on the growth of B16 melanoma cells.Results The CDS coding sequence of human BRE1B gene was amplified by PCR successfully,which was equal to the expected size.Compared with the control group,the sequence from bacteria PCR was identified as positive,with the length of 4000 bp and 3050 bp by double enzyme digestion respectively.Moreover,the coding sequence of the human BRE1B gene was exactly the same as the inserted DNA sequence.Western blot results showed that the expression of recombinant plasmid pCMV-Tag-2B-BRE1B was successfully expressed in the experimental group,but there was no specific fragments in the control group.And cell counting kit-8 (CCK8) and colony assays showed that pCMV-Tag-2B-BRE1B recombinant plasmid could inhibit the growth of B16 melanoma cells.Conclusion The eukaryotic expression vector of pCMV-Tag-2B-BRE1B labeled with pCMV-Tag-2B is constructed successfully,and it has inhibitory effects on the growth of ocular B16 melanoma cells.

Piceatannol induced apoptosis through up-regulation of microRNA-181a in melanoma cells

BACKGROUND: Melanoma took top position among the lethal cancers and, despite there have been some great attempts made to increase the natural life of patients with metastatic disease, long-lasting and complete remissions are few. Piceatannol, owns the similar function as resveratrol, has been defined as an anti-cancer agent playing important role in inhibition of proliferation, migration and metastasis in various cancer. Thus, we aim to investigate the anti-cancer effect and mechanisms of piceatannol in melanoma cells. METHODS: Melanoma cell lines WM266-4 and A2058 were treated either with or without piceatannol. Cell viability and cell apoptosis were assessed by using MTT and Annexin V/PI assay, respectively. Cells were transfected with specific miRNA using Lipfectamine 2000. miRNA bingding ability to 3'-UTR region within specific gene was assed by firefly luciferase analysis. Gene and protein expression was eveluated by qRT-PCR and western blot analysis, respectively. RESULTS: Our study showed that piceatannol inhibited WM266-4 and A2058 cells growth and induced apoptosis. Totally, 16 differentially expressed miRNAs were screened out including 8 up-regulated and 8 down-regulated miRNAs. Expression level of miR-181a is significantly higher in piceatannol-treated cells than normal control and is lower in melanoma cancer tissues than its adjacent normal tissues. Bcl-2 is a target gene of miR-181a. Moreover, silencing of miR-181a reverses the decrease of cell viability induced by piceatannol in WM266-4 and A2058 cells. Taken together, present study uncovered the ability of piceatannol to repress melanoma cell growth and clarified the contribution of miR-181a in the anticancer role of piceatannol. CONCLUSION: The present study proposes that piceatannol can be taken into account to be a hopeful anticancer agent for melanoma.

A preliminary study on the MATP gene knockout in a mouse melanoma cell line using CRISPR-Cas9 system / 中国比较医学杂志

Objective To knockout the MATP gene of mouse melanoma cell line B16F10 using CRISPR/Cas9 system,and to lay foundation for the functional study of MATP gene.Methods Specific primers of MATP were designed according to the report in http://crispr.mit.edu/ website.The primers were linked to pCAS9/gRNA1 vector.Then the positive vector was transfected into mouse melanoma B16F10 cells,and monoclonal cell lines were obtained by the infinite dilution method.After the genomes of different monoclonal cell lines were extracted and sequenced,the cell lines with MATP gene cleavage were screened,and the expression of MATP in these cell lines was verified by Western-blot analysis.Results Three MATP gene knockout cell lines were successfully obtained.The western-blot results showed that the cell lines did not express MATP protein.Conclusions The knockout of MATP gene in B16F10 cell line can be successfully achieved using the pCAS9/gRNA1 vector.

Baicalein Inhibits the Migration and Invasion of B16F10 Mouse Melanoma Cells through Inactivation of the PI3K/Akt Signaling Pathway

Baicalein, a natural flavonoid obtained from the rhizome of Scutellaria baicalensis Georgi, has been reported to have anticancer activities in several human cancer cell lines. However, its antimetastatic effects and associated mechanisms in melanoma cells have not been extensively studied. The current study examined the effects of baicalein on cell motility and anti-invasive activity using mouse melanoma B16F10 cells. Within the noncytotoxic concentration range, baicalein significantly inhibited the cell motility and invasiveness of B16F10 cells in a concentration-dependent manner. Baicalein also reduced the activity and expression of matrix metalloproteinase (MMP)-2 and -9; however, the levels of tissue inhibitor of metalloproteinase-1 and -2 were concomitantly increased. The inhibitory effects of baicalein on cell motility and invasiveness were found to be associated with its tightening of tight junction (TJ), which was demonstrated by an increase in transepithelial electrical resistance and downregulation of the claudin family of proteins. Additionally, treatment with baicalein markedly reduced the expression levels of lipopolysaccharide-induced phosphorylated Akt and the invasive activity in B16F10 cells. Taken together, these results suggest that baicalein inhibits B16F10 melanoma cell migration and invasion by reducing the expression of MMPs and tightening TJ through the suppression of claudin expression, possibly in association with a suppression of the phosphoinositide 3-kinase/Akt signaling pathway.

Effects of isolated compound from Sonneratia caseolaris leaf: a validation of traditional utilization by melanin biosynthesis and antioxidant assays.

In continuing to search new materials for skin whitening agent from natural resources, we have focused on plants used by Dayak tribe (native people in East Kalimantan, Indonesia) for skin care treatment. The ethanol extract of the leaves of Sonneratia caseolaris showed anti-melanogenesis activity in melanin biosynthesis assays using B16 melanoma cells. By activity-guided fractionation, luteolin-7-O--glucoside (compound 1) was isolated as an active compound. In melanin formation inhibition assay, luteolin-7-O--glucoside showed the inhibitory activity in a dose-dependent manner and reach 44% of inhibition at 223.2 μM with 92% of cells viability. The compound also showed potent anti-oxidative stress ability with a significant intracellular H2O2-scavenging activity in HaCaT cells. These results showed a validation of traditional use for skin care treatment by Dayak tribe in East Kalimantan.

Antiproliferative evaluation of tall-oil docosanol and tetracosanol over CHO-K1 and human melanoma cells

Background Polycosanols derived from plant species have traditionally been used in medicine as antiproliferative agents for treating various viruses (primarily the herpes simplex virus). However, few studies have studied their effects on hyperproliferative cell lines. In this work, the antiproliferative capacity of polycosanols from tall-oil pitch, obtained from black liquor soaps in the kraft pulping process of cellulose (specifically from Pinus radiata, Pinus taede, and Eucalyptus globulus), was evaluated on CHO-K1 and CRL-1974 human melanoma cell lines. Results The proliferative capacities and cell viabilities were measured for 72 and 140 h, respectively. Treatment with docosanol produced differential effects on the CHO-K1 and human melanoma cells and significantly affected their proliferation rates, but not their cell viabilities. Tetracosanol produced a significant negative effect on the proliferation of human melanoma cells, and this effect was less than that caused by docosanol. However, it had no effect on the proliferation of CHO-K1 cells and did not induce any significant effect on the viability of the studied cell lines. Conclusion Docosanol and tetracosanol induced antiproliferative effects on the studied cell lines and exhibited significantly greater effects on the oncogenic cell lines. Prior to this study, the capacity of these polycosanols has never been investigated. Future studies will be necessary to determine their mechanisms of action on these cell systems.

Isoliquiritigenin induced apoptosis in human melanoma A375 cells / 中国药理学通报

Aim To evaluate the mechanism of apopto-sis induced by the isoliquiritigenin in A375 human ma-lignant melanoma cells. Methods Sulforhodamine B ( SRB) method was used to determine the A375 cell viability;acridine orange/ethidium bromide ( AO/EB) and Hoechst 33258 staining were used to observe the morphological changes of apoptotic cells; flow cytome-try was used to detect A375 cell apoptotic rate;DCFH-DA was applied to determine the changes of total intra-cellular ROS in A375 cells;JC-1 method was used to measure the changes of mitochondrial membrane poten-tial;the kits methods were used to determine the con-tent of ATP, lactic acid and glucose in A375 cell which was treated with different concentrations of isoliquiritigenin. Results Isoliquiritigenin could in-hibit A375 cell proliferation in a concentration-depend-ent manner; A375 cells showed obvious apoptosis charateristics after treatment by isoliquiritigenin, and the apoptosis rate increased with increasing concentra-tion of isoliquiritigenin. The level of total intracellular ROS in A375 cells increased obviously after dealing with different concentrations of isoliquiritigenin;in ad-dition, the mitochondrial membrane potential, the lev-els of intracellular ATP,lactic acid and the level of glu-cose uptake all declined. Conclusions These find-ings demonstrate that isoliquiritigenin can induce apop-tosis of A375 cells. The mechanism may be related to elevation of ROS level and reduction of aerobic glycoly-sis level.

Effect of Different Purity Galangin on Melanocyte Proliferation and Melanin Synthesis of A375-HaCaT Co-culture System / 中国中医药信息杂志

Objective To investigate the effect of Galangin of different purity on melanocyte proliferation and melanin synthesis of A375-HaCaT co-culture system. Methods The A375-HaCaT co-culture system was established with Transwell technology, and 0.1, 0.5, 1.0, 5.0, 10 μg/mL Galangin of the purity of 70%and 90%was used to act on the co-culture system. Cell proliferation, melanin content and tyrosinase of A375 were measured by MTT assay, NaOH lysis assay and L-DOPA oxidation assay respectively. The cytokines such as ET-1 and SCF in HaCaT were detected by ELISA. Results A375 cells in co-culture system grew well, and 0.1, 0.5, 1.0, 5.0, 10 μg/mL Galangin of the purity of 70% and 90% had up-regulation effect on cell proliferation, melanin content and tyrosinase of A375. Moreover, Galangin could increase the expression level of ET-1 and SCF of HaCaT at more than 0.5 μg/mL, and the effect of Galangin of 70% purity was better than 90% purity. Conclusion Galangin could promote the cell proliferation, melanin content and tyrosinase of A375, and mechanism of the pathways is probably related to the up-regulation on the expression of ET-1 and SCF of HaCaT.

Inhibition of tyrosinase activity and melanine pigmentation by 2-hydroxytyrosol

2-Hydroxytyrosol (2-HT), originally reported as a synthetic compound, was isolated for the first time as a fungal metabolite. 2-HT was found to inhibit mushroom tyrosinase with an IC50 value of 13.0 µmol/L. Furthermore, 2-HT dose-dependently inhibited tyrosinase activity (IC50, 32.5 µmol/L) in the cell-free extract of B16 melanoma cells and α-melanocyte stimulating hormone (α-MSH)-stimulated melanin formation in intact B16 melanoma cells.

Involvement of Transglutaminase-2 in alpha-MSH-Induced Melanogenesis in SK-MEL-2 Human Melanoma Cells

Skin hyperpigmentation is one of the most common skin disorders caused by abnormal melanogenesis. The mechanism and key factors at play are not fully understood. Previous reports have indicated that cystamine (CTM) inhibits melanin synthesis, though its molecular mechanism in melanogenesis remains unclear. In the present study, we investigated the effect of CTM on melanin production using ELISA reader and the expression of proteins involved in melanogenesis by Western blotting, and examined the involvement of transglutaminase-2 (Tgase-2) in SK-MEL-2 human melanoma cells by gene silencing. In the results, CTM dose-dependently suppressed melanin production and dendrite extension in alpha-MSH-induced melanogenesis of SK-MEL-2 human melanoma cells. CTM also suppressed alpha-MSH-induced chemotactic migration as well as the expressions of melanogenesis factors TRP-1, TRP-2 and MITF in alpha-MSH-treated SK-MEL-2 cells. Meanwhile, gene silencing of Tgase-2 suppressed dendrite extension and the expressions of TRP-1 and TRP-2 in alpha-MSH-treated SK-MEL-2 cells. Overall, these findings suggested that CTM suppresses alpha-MSH-induced melanogenesis via Tgase-2 inhibition and that therefore, Tgase-2 might be a new target in hyperpigmentation disorder therapy.

Purification of butin and butein from the Vernonia anthelmintica Willd and their effects on proliferation and melanogenesis of A375 human melanoma cells / 中国药学杂志

OBJECTIVE: To purify butin and butein from the Vernonia anthelmintica Willd, and preliminary study their effects on proliferation and melanogenesis of A375 human melanoma cells. METHODS: Selica gel and Sephadex LH-20 column chromatography methods were used to separate butin and butein; their effects on proliferation and melanogenesis of A375 human melanoma cells were studied by MTT method, enzyme method and NaOH method. RESULTS: Butin and butein were isolated and identified. In the concentration range of 0.50-10.00 μg · mL-1, butin and butein enhanced the proliferation of A375 human melanoma cells, and the effect of butein was stronger than butin(P < 0.05) ; in the concentration range of 0.10-1.00 μg · mL-1, both butin and butein increased the activity of tyrosinase; in the concentration range of 0.50-5.00 μg · mL-1, butein stimulated melanogenesis, but butin inhibited melanogenesis. CONCLUSION: Butin and butein may be the active components of Vernonia anthelmintica Willd; butein promotes the proliferation and stimulates the melanogenesis of A375 human melanoma cells, however, butin mildly inhibites the melanogenesis.

Apoptotic Effects of Co-Treatment with a Chios Gum Mastic and Eugenol on G361 Human Melanoma Cells

We investigated the synergistic apoptotic effects of co-treatments with Chios gum mastic (CGM) and eugenol on G361 human melanoma cells. An MTT assay was conducted to investigate whether this co-treatment efficiently reduces the viability of G361 cells compared with each single treatment. The induction and augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining, and analyses of DNA hypoploidy. Western blot analysis and immunofluorescent staining were also performed to evaluate expression and translocation of apoptosis-related proteins following CGM and eugenol co-treatment. Proteasome activity and mitochondrial membrane potential (MMP) changes were also assayed.The results indicated that the co-treatment of CGM and eugenol induces multiple pathways and processes associated with an apoptotic response in G361 cells. These include nuclear condensation, DNA fragmentation, a reduction in MMP and proteasome activity, an increase of Bax and decrease of Bcl-2, a decreased DNA content, cytochrome c release into the cytosol, the translocation of AIF and DFF40 (CAD) into the nucleus, and the activation of caspase-9, caspase-7, caspase-3, PARP and DFF45 (ICAD). In contrast, separate treatments of 40 microg/ml CGM or 300 microM eugenol for 24 hours did not induce apoptosis. Our present data thus suggest that a combination therapy of CGM and eugenol is a potential treatment strategy for human melanoma.

ETM study of electroporation influence on cell morphology in human malignant melanoma and human primary gingival fibroblast cells

Objective:To estimate electroporation (EP) influence on malignant and normal cells.Methods:Two cell lines including human malignant melanoma (Me-45) and normal human gingival fibroblast (HGFs) were used. EP parameters were the following:250,1000,1750,2500 V/cm;50 μs by5 impulses for every case. The viability of cells after EP was estimated byMTT assay. The ultrastructural analysis was observed by transmission electron microscope (ZeissEM900). Results:In the current study we observed the intracellular effect followingEP on Me-45 and HGF cells. At the conditions applied, we did not observe any significant damage of mitochondrial activity in both cell lines treated byEP. Conversely, we showed thatEP in some conditions can stimulate cells to proliferation. Some changes induced byEP were only visible in electron microscopy. In fibroblast cells we observed significant changes in lower parameters ofEP (250 and1000 V/cm). After applying higher electric field intensities (2500 V/cm) we detected many vacuoles, myelin-like bodies and swallowed endoplasmic reticulum. In melanoma cells such strong pathological modifications afterEP were not observed, in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters ofEP.Conclusions:We can claim thatEP conditions are cell line dependent. In terms of the intracellular morphology, human fibroblasts are more sensitive to electric field as compared with melanoma cells. Optimal conditions should be determined for each cell line. Summarizing our study, we can conclude thatEP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells.

ETM study of electroporation influence on cell morphology in human malignant melanoma and human primary gingival fibroblast cells

<p><b>OBJECTIVE</b>To estimate electroporation (EP) influence on malignant and normal cells.</p><p><b>METHODS</b>Two cell lines including human malignant melanoma (Me-45) and normal human gingival fibroblast (HGFs) were used. EP parameters were the following: 250, 1 000, 1 750, 2 500 V/cm; 50 µs by 5 impulses for every case. The viability of cells after EP was estimated by MTT assay. The ultrastructural analysis was observed by transmission electron microscope (Zeiss EM 900).</p><p><b>RESULTS</b>In the current study we observed the intracellular effect following EP on Me-45 and HGF cells. At the conditions applied, we did not observe any significant damage of mitochondrial activity in both cell lines treated by EP. Conversely, we showed that EP in some conditions can stimulate cells to proliferation. Some changes induced by EP were only visible in electron microscopy. In fibroblast cells we observed significant changes in lower parameters of EP (250 and 1 000 V/cm). After applying higher electric field intensities (2 500 V/cm) we detected many vacuoles, myelin-like bodies and swallowed endoplasmic reticulum. In melanoma cells such strong pathological modifications after EP were not observed, in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters of EP.</p><p><b>CONCLUSIONS</b>We can claim that EP conditions are cell line dependent. In terms of the intracellular morphology, human fibroblasts are more sensitive to electric field as compared with melanoma cells. Optimal conditions should be determined for each cell line. Summarizing our study, we can conclude that EP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells.</p>