B16F10 Murine Melanoma Cells Research Articles

감귤과 녹차의 추출액으로 제조한 콤부차의 생리 활성 및 일반 세포 독성

콤부차는 미생물의 공생체에 의해 제조되는 신맛이 강한 음료로 최근에는 “장수 식품”의 하나로 주로 러시아를 비롯한 서방 국가들에서 많이 음용되어온 기능성 음료이다. 본 연구에서는 일반적으로 제조되는 콤부차의 기능성을 제주 특산물인 감귤의 과피 추출액과 녹차의 추출액을 첨가한 후 콤부차를 발효시켜 각 성분이 가지는 기능성을 이행시킬 목적으로 감귤/녹차 콤부차를 제조하였고, 제조된 감귤/녹차 콤부차의 기능성을 항암성 중심으로 일반 콤부차와 비교 평가하였다. 또한, 콤부차의 산업화, 표준화 및 안전성 확보를 위하여 일반 세포에 미치는 독성의 정도를 RAW 264.7 마우스 대식세포를 이용하여 조사하였다. 그 결과 일반 콤부차보다 감귤/녹차 콤부차는 항산화력이 약 3배 증가하였으며, 항암성도 현저하게 증가한 것으로 나타났다. 특히 일반 세포에 대한 독성은 현저히 낮아진 것으로 나타났으며, 이는 감귤/녹차 콤부차의 안전한 항암 기능성 음료로의 개발 가능성을 시사하고 있다.

Tyrosinase inhibitory flavonoid from Juniperus communis fruits.

The fruits of Juniperus communis have been traditionally used in the treatment of skin diseases. In our preliminary experiment, the MeOH extract of J. communis effectively suppressed mushroom tyrosinase activity. Three monoflavonoids and five biflavonoids were isolated from J. communis by bioassay-guided isolation and their inhibitory effect against tyrosinase was evaluated. According to the results of all isolates, hypolaetin 7-O-β-xylopyranoside isolated from J. communis exhibited most potent effect of decreasing mushroom tyrosinase activity with an IC50 value of 45.15μM. Further study provided direct experimental evidence for hypolaetin 7-O-β-D-xylopyranoside-attenuated tyrosinase activity in α-MSH-stimulated B16F10 murine melanoma cell. Hypolaetin 7-O-β-D-xylopyranoside from the EtOAc fraction of J. communis was also effective at suppressing α-MSH-induced melanin synthesis. This is the first report of the enzyme tyrosinase inhibition by J. communis and its constituent. Therapeutic attempts with J. communis and its active component, hypolaetin 7-O-β-D-xylopyranoside, might be useful in treating melanin pigmentary disorders.

Synthesis of novel C4-benzazole naphthalimide derivatives with potent anti-tumor properties against murine melanoma

Novel C4-benzazole naphthalimide derivatives were synthesized and tested in vitro and in vivo as anti-cancer drugs. Among these synthetic molecules, compounds 9 and 10 exhibited cytotoxicity against murine B16F10 melanoma cells. In addition, the above-mentioned compounds significantly suppressed lung tumor metastasis with no visible sign of toxicity.

Skin protective effect of guava leaves against UV-induced melanogenesis via inhibition of ORAI1 channel and tyrosinase activity.

Ultraviolet (UV) irradiation is a major environmental factor affecting photoageing, which is characterized by skin wrinkle formation and hyperpigmentation. Although many factors are involved in the photoageing process, UV irradiation is thought to play a major role in melanogenesis. Tyrosinase is the key enzyme in melanin synthesis; therefore, many whitening agents target tyrosinase through various mechanisms, such as direct interference of tyrosinase catalytic activity or inhibition of tyrosinase mRNA expression. Furthermore, the highly selective calcium channel ORAI1 has been shown to be associated with UV-induced melanogenesis. Thus, ORAI1 antagonists may have applications in the prevention of melanogenesis. Here, we aimed to identify the antimelanogenesis agents from methanolic extract of guava leaves (Psidium guajava) that can inhibit tyrosinase and ORAI1 channel. The n-butanol (47.47%±7.503% inhibition at 10μg/mL) and hexane (57.88%±7.09% inhibition at 10μg/mL) fractions were found to inhibit ORAI1 channel activity. In addition, both fractions showed effective tyrosinase inhibitory activity (68.3%±0.50% and 56.9%±1.53% inhibition, respectively). We also confirmed that the hexane fraction decreased the melanin content induced by UVB irradiation and the ET-1-induced melanogenesis in murine B16F10 melanoma cells. These results suggest that the leaves of P.guajava can be used to protect against direct and indirect UV-induced melanogenesis.

The biochemical characterization, stabilization studies and the antiproliferative effect of bromelain against B16F10 murine melanoma cells

The current study aims to extract bromelain from different parts (stem, crown, peels, pulp and leaves) of Ananas comosus var. comosus AGB 772; to determine of optimum pH and temperature; to test bromelain stability in disodium EDTA and sodium benzoate, and to investigate its pharmacological activity on B16F10 murine melanoma cells in vitro. The highest enzymatic activity was found in bromelain extracted from the pulp and peel. The optimum bromelain pH among all studied pineapple parts was 6.0. The optimum temperature was above 50 °C in all bromelain extracts. The fluorescence analysis confirmed the stability of bromelain in the presence of EDTA and sodium benzoate. Bromelain was pharmacologically active against B16F10 melanoma cells and it was possible verifying approximately 100% inhibition of tumor cell proliferation in vitro. Since bromelain activity was found in different parts of pineapple plants, pineapple residues from the food industry may be used for bromelain extraction.

Tyrosinase inhibition and antioxidant properties of Asphodelus microcarpus extracts.

Background Asphodelus microcarpus belongs to the family Liliaceae that include several medicinal plants. In the traditional medicine plants of the genus Asphodelus are used to treat skin disorders such as ectodermal parasites, psoriasis, microbial infection and for lightening freckles. In order to find novel skin depigmenting agents, the present work was carry out to evaluate antioxidant activity and tyrosinase inhibitory potential of leaves, flowers and tubers extracts of A. microcarpus. The phytochemical composition of the active extract was also evaluated.MethodsThree different extracts (water, methanol and ethanol) from leaves, flowers and tubers of A. microcarpus were evaluated for their inhibitory effect on tyrosinase activity using l-3,4-dihydroxyphenylalanine (l-DOPA) as substrate. Inhibition of cellular tyrosinase activity and melanin production was also investigated in melanoma B16F10 cells. Antioxidant activity, total phenolic and flavonoids contents were determined using standard in vitro methods. HPLC-DAD-MS was used to identify phenolic profile of the active extract.ResultsThe results showed that all extracts have a direct inhibitory anti-tyrosinase activity, with ethanolic extract from flowers (FEE) exhibiting the stronger effect. Kinetic analysis revealed that FEE acts as an uncompetitive inhibitor with a Ki value of 0.19 mg/mL. The same effect was observed in murine melanoma B16F10 cells. Cellular tyrosinase activity as well as melanin content were reduced in FEE-treated cells. The results were comparable to that of the standard tyrosinase inhibitor (kojic acid). Furthermore, the same extract showed the highest antioxidant activity and an elevated levels of total phenolics and flavonoid content. Eleven phenolic components were identified as chlorogenic acid, luteolin derivates, naringenin and apigenin.ConclusionsOur findings showed that FEE from A. microcarpus inhibits tyrosinase and exerted antimelanogenesis effect in B16F10 cells. This extract also showed the highest scavenging activity, which could be mainly attributed to its high levels of total polyphenols and flavonoids. These results suggest that A. microcarpus has a great potential as sources of bioactive compounds which could be used as depigmenting agents in skin disorders.

Growth hormone is increased in the lungs and enhances experimental lung metastasis of melanoma in DJ-1 KO mice.

BackgroundGrowth hormone (GH) mainly serves an endocrine function to regulate somatic growth, but also serves an autocrine function in lung growth and pulmonary function. Several recent studies have demonstrated the role of autocrine GH in tumor progression in some organs. However, it is not clear whether excessive secretion of GH in the lungs is related to pulmonary nodule formation.MethodsFirstly, the lung tissues dissected from mice were used for Western blotting and PCR measurement. Secondly, the cultured cells were used for examining effects of GH on B16F10 murine melanoma cells. Thirdly, male C57BL/6 mice were intravenously injected with B16F10 cells and then subcutaneously injected with recombinant GH twice per week for three weeks. Finally, stably transfected pool of B16F10 cells with knockdown of growth hormone receptor (GHR) was used to be injected into mice.ResultsWe found that expression of GH was elevated in the lungs of DJ-1 knockout (KO) mice. We also examined the effects of GH on the growth of cultured melanoma cells. The results showed that GH increased proliferation, colony formation, and invasive capacity of B16F10 cells. In addition, GH also increased the expression of matrix metalloproteinases (MMPs) in B16F10 cells. Administration of GH in vivo enhanced lung nodule formation in C57/B6 mice. Increased lung nodule formation in DJ-1 KO mice following intravenous injection of melanoma cells was inhibited by GHR knockdown in B16F10 cells.ConclusionsThese results indicate that up-regulation of GH in the lungs of DJ-1 KO mice may enhance the malignancy of B16F10 cells and nodule formation in pulmonary metastasis of melanoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2898-5) contains supplementary material, which is available to authorized users.

159 - Identification of Antimelanogenic Peptides through Positional Scanning of a Synthetic Peptide Combinatorial Library

Antimelanogenic peptides are potentially useful as cosmeceutical ingredients in skin whitening agents. The purpose of this study was to identify peptide inhibitors of cellular melanogenesis through positional scanning of a synthetic peptide combinatorial library (PS-SPCL). A synthetic hexapeptide combinatorial library consisted of 6 positional sub-libraries, namely OXXXXX, XOXXXX, XXOXXX, XXXOXX, XXXXOX, and XXXXXO, where O positions are any one of the 19 amino acids excluding cysteine, and other X positions are equimolar mixtures of the 19 amino acids. The antimelanogenic effects of peptide mixtures were evaluated in murine melanoma B16-F10 cells stimulated with α-melanocyte-stimulating hormone (α-MSH) to identify the sequence of antimelanogenic hexapeptides. PS-SPCL identified the active hexapeptide sequences as (F/I)(N/S)(D/H/N)(H/N)L(G/F)-NH 2 . Of the hexapeptide mixtures and individual hexapeptides evaluated, the strongest antimelanogenic activity was seen with hexapeptide B6 (FSHHLG-NH 2 ). Hexapeptide B6 decreased melanin content and levels of melanogenic enzymes in B16-F10 cells stimulated with α-MSH. Additionally, it decreased melanin content in human epidermal melanocytes stimulated with L-tyrosine. The present study identified novel antimelanogenic hexapeptides that might be useful for controlling skin pigmentation.

Jolkinolide B induces apoptosis and inhibits tumor growth in mouse melanoma B16F10 cells by altering glycolysis.

Most cancer cells preferentially rely on glycolysis to produce the energy (adenosine triphosphate, ATP) for growth and proliferation. Emerging evidence demonstrates that the apoptosis in cancer cells could be closely associated with the inhibition of glycolysis. In this study, we have found that jolkinolide B (JB), a bioactive diterpenoid extracted from the root of Euphorbia fischeriana Steud, induced tumor cells apoptosis and decreased the production of ATP and lactic acid in mouse melanoma B16F10 cells. Furthermore, we found that JB downregulated the mRNA expression of glucose transporter genes (Glut1, Glut3 and Glut4) and glycolysis-related kinase genes (Hk2 and Ldha) in B16F10 cells. Moreover, treatment with JB upregulated the mRNA expression of pro-apoptosis genes (Bax), downregulated the mRNA expression of anti-apoptosis genes (Bcl-2, Caspase-3 and Caspase-9), decreased the potential of mitochondrial membrane and increased reactive oxygen species (ROS) levels in B16F10 cells. Finally, intragastric administration of JB suppressed tumor growth and induced tumor apoptosis in mouse xenograft model of murine melanoma B16F10 cells. Taken together, these results suggest that JB could induce apoptosis through the mitochondrial pathway and inhibit tumor growth. The inhibition of glycolysis could play a crucial role in the induction of apoptosis in JB-treated B16F10 cells.

Sesquiterpenes from the whole plants of Parasenecio roborowskii

Six eremophilane-type (parasenolide A–F) and an eudesmane-type (parasenin) sesquiterpenoids, along with eight known sesquiterpenes, were isolated from the whole plants of Parasenecio roborowskii. The structures and absolute configurations of new compounds were elucidated using extensive spectroscopic analysis, including HRESIMS, 1D and 2D NMR experiments, the CD exciton chirality methods, and single-crystal X-ray crystallography. All isolated compounds were evaluated for cytotoxicity against five human cancer (HeLa, HepG2, K562, MDA231, and NCI-H460) cell lines and a murine melanoma B16 F10 cell line by MTT assay. Compounds 1–15 showed cytotoxic activities, especially compounds 3, 4, 8, 10, and 12. These five compounds showed broad spectrum activities against all the tested cancer cell lines with IC50 ranging from 9.2 to 35.5μM. The study supports that eremophilenolides and eudesmane-type sesquiterpenes occur mainly in the genus Parasenecio and can be used as a chemosystematic marker of the genus.

Enhancement of radiosensitivity of melanoma cells by pegylated gold nanoparticles under irradiation of megavoltage electrons

Purpose: Gold nanoparticles (GNP) have significant potential as radiosensitizer agents due to their distinctive properties. Several studies have shown that the surface modification of nanoparticles with methyl polyethylene glycol (mPEG) can increase their biocompatibility. However, the present study investigated the radiosensitization effects of mPEG-coated GNP (mPEG-GNP) in B16F10 murine melanoma cells under irradiation of 6 MeV Electron beam.Materials and methods: The synthesized GNP were characterized by UV-Visible spectroscopy, dynamic light scattering, transmission electron microscopy, and zeta potential. Enhancement of radiosensitization was evaluated by the clonogenic assay at different radiation doses of megavoltage electron beams.Results: It was observed that mPEG-GNP with a hydrodynamic size of approximately 50 nm are almost spherical and cellular uptake occurred at all concentrations. Both proliferation efficiency and survival fraction decreased with increasing mPEG-GNP concentration. Furthermore, significant GNP sensitization occurred with a maximum dose enhancement factor of 1.22 at a concentration of 30 μM.Conclusions: Pegylated-GNP are taken up by B16F10 cancer cells and cause radiosensitization in the presence of 6 MeV electrons. The radiosensitization effects of GNP may probably be due to biological processes. Therefore, the underlying biological mechanisms beyond the physical dose enhancement need to be further clarified.

Foeniculum vulgare extract and its constituent, trans-anethole, inhibit UV-induced melanogenesis via ORAI1 channel inhibition

BackgroundUltraviolet radiation exposure is the most important cause of extrinsic skin aging (photoaging), which causes skin wrinkling and hyperpigmentation. Although many factors are involved in the photoaging process, calcium release-activated calcium channel protein 1 (ORAI1) has been reported to be involved in UV-induced melanogenesis. ObjectiveThe aim of the present study was to find inhibitory effects of the extract of Foeniculum vulgare (fennel) fruits on ORAI1 ion channels and UV-induced melanogenesis in melanoma cells and to identify its active constituents. MethodsActive compounds were isolated and quantitatively analyzed. An electrophysiological assay was performed by using the whole-cell patch-clamp technique. Intracellular free calcium concentration was measured by Fura-2. Tyrosinase activity was evaluated by levodopa colorimetry. Effects of the most active compound on cell viability of murine B16F10 melanoma cells and inhibition of melanin content after UVB irradiation were determined. ResultsF. vulgare fruits extract and its hexane fraction strongly blocked ORAI1 currents and tyrosinase activity and significantly inhibited UV-induced melanogenesis. Of the 13 compounds isolated from the hexane fraction, trans-anethole (TA) exhibited inhibitory effects on ORAI1 (IC50=8.954±1.36μM) and increased cytoplasmic Ca2+ concentrations in response. TA inhibited UV-induced melanogenesis without affecting tyrosinase activity. ConclusionOur findings suggest that the fruits extract of F. vulgare and its active constituent, TA, provide a possible novel approach for treating and preventing UV-induced melanogenesis.

A scaffold-free surface culture of B16F10 murine melanoma cells based on magnetic levitation.

Multicellular spheroids are obtained in a variety of three-dimensional (3D) culture systems without the use of supporting scaffold. We present here a 3D culture method that resulted in a multicellular sheet under scaffold-free conditions. A floating disk-shaped 3D culture was prepared by magnetic levitation of B16F10 cells that has ingested Fe3O4-containing fibroin microspheres. The melanoma disk grew up to 19mm in diameter and the thickness was ranged between 80 and 100μm. The 3D culture was filled with closely packed cells that were proliferating exponentially at a specific growth rate of µ=0.015h-1. Approximately half of the cells were Ki-67 positive with no detectable levels of apoptotic or autophagic cells. However, the percentage of propidium iodide-permeable cells was 8.5±1.2%, which was probably due to physical damage in the cell membrane caused by Fe3O4-containing microspheres under a strong magnetic field. Melanin production increased by a factor of 3.0-3.7 in the 3D culture, due to an increased population of pigmented cells. This study presented a surface 3D culture of B16F10 cells without the use of a scaffold based on magnetic levitation.

English

Pachycereus marginatus (DC.) Britton & Rose is a species belonging to the family Cactaceae. In traditional medicine, it is recommended to treat diabetes and gastrointestinal infections; however, there are no studies related to its use in cancer treatment. The in vitro antitumor effect of P. marginatus hexane, chloroform, methanol, and methanol-aqueous partition stem extracts, against murine lymphoma L5178Y-R and skin melanoma B16F10 cells, was evaluated in liquid medium by the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The extracts resulted in up to 84, 85, 84, and 82% cytotoxicity (p < 0.05) to L5178Y-R cells, respectively, and up to 39, 51, 48, and 42% cytotoxicity (p < 0.05) to B16F10 cells, respectively. Vehicle controls were not cytotoxic for tumor cells, and along with the extracts they did not affect viability of resident murine thymus and spleen lymphocytes. Taken together, the present results showed that P. marginatus extracts possess antitumor potential against L5178Y-R lymphoma and B16F10 skin melanoma cells. Key words: Cancer, cacti, Pachycereus marginatus, lymphoma, melanoma, medicinal plants.

Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors.

We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown in syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy.

PRDM5 promotes the proliferation and invasion of murine melanoma cells through up-regulating JNK expression.

PRDM (PRDI‐BF1 and RIZ domain‐containing) proteins constitute a family of zinc finger proteins and play important roles in multiple cellular processes by acting as epigenetic modifiers. PRDM5 is a recently identified member of the PRDM family and may function as a tumor suppressor in several types of cancer. However, the role of PRDM5 in murine melanoma remains largely unknown. In our study, effect of PRDM5 on murine melanoma cells was determined and results showed that PRDM5 overexpression significantly promoted proliferation, migration, and invasion of murine melanoma B16F10 cells. Consistently, silencing of PRDM5 expression significantly inhibited proliferation, invasion, and migration of B16F10 cells. In vivo study also showed that PRDM5 silencing significantly inhibited the growth and metastasis of melanoma in mice. PRDM5 was then found to increase the expression and activation of JNK in B16F10 cells. JNK silencing significantly reduced PRDM5‐mediated up‐regulation of JNK expression and blocked the PRDM5‐induced proliferation and invasion of B16F10 cells. To further verify the involvement of JNK signaling in PRDM5‐induced progression of B16F10 cells, a specific JNK inhibitor was employed to inhibit the JNK signaling pathway, and results showed that PRDM5‐induced proliferation and invasion of B16F10 cells were abolished. We conclude that PRDM5 promotes the proliferation and invasion of murine melanoma cells through up‐regulating JNK expression and strategies targeting PRDM5 may be promising for the therapy of melanoma.

In vitro and in vivo studies of the antineoplastic activity of copper (II) compounds against human leukemia THP-1 and murine melanoma B16-F10 cell lines

We investigated the antineoplastic activities of a previously reported copper (II) coordination compound, [Cu(BMPA)Cl2]CH3OH (1), and a new compound, [Cu(HBPA)Cl2]H2O (2), where BMPA is bis(pyridin-2-ylmethyl)amine and HBPA is (2-hydroxybenzyl)(2-pyridylmethyl)amine, using various cellular models of human leukemia (THP-1, U937, HL60, Molt-4, JURKAT) and human colon cancer (COLO 205), as well as a murine highly metastatic melanoma (B16-F10) cell line. Compound (2) was characterized using several physical and chemical techniques, including X-ray diffraction studies. The IC50 values of the copper coordination complexes in the human leukemia cell lines ranged from 87.63 ± 1.02 to ≥400 μM at high cell concentrations and from 19.17 ± 1.06 to 97.67 ± 1.23 μM at low cell concentrations. Both compounds induced cell death, which was determined by cell cycle analyses and phosphatidylserine exposure studies. THP-1 cells released cytochrome c to the cytoplasm 12 h after treatment with 400 μM of compound (2). To evaluate the apoptosis pathway induced by compound (2), we measured the activities of initiator caspases 8 and 9 and executioner caspases 3 and 6. The results were suggestive of the activation of both intrinsic and extrinsic apoptosis pathways. To investigate the activities of the compounds in vivo, we selected two sensitive cell lines from leukemia (THP-1) and solid tumor (B16-F10) lineages. BALB/c nude bearing THP-1 tumors treated with 12 mg·kg−1 of compound (2) showed a 92.4% inhibition of tumor growth compared with the control group.

Matricellular TSP-1 as a target of interest for impeding melanoma spreading: towards a therapeutic use for TAX2 peptide.

Thrombospondin-1 (TSP-1) is a matricellular glycoprotein known for being highly expressed within a tumor microenvironment, where it promotes an aggressive phenotype particularly by interacting with the CD47 cell-surface receptor. While it originates from the stromal compartment in many malignancies, melanoma is an exception as invasive and metastatic melanoma cells overexpress TSP-1. We recently demonstrated that a new molecular agent that selectively prevents TSP-1 binding to CD47, called TAX2, exhibits anti-cancer properties when administered systemically by decreasing viable tumor tissue within subcutaneous B16 melanoma allografts. At the same time, emerging evidence was published suggesting a contribution of TSP-1 in melanoma metastatic dissemination and resistance to treatment. Through a comprehensive systems biology approach based on multiple genomics and proteomics databases analyses, we first identified a TSP-1-centered interaction network that is overexpressed in metastatic melanoma. Then, we investigated the effects of disrupting TSP-1:CD47 interaction in A375 human malignant melanoma xenografts. In this model, TAX2 systemic administrations induce tumor necrosis by decreasing intra-tumoral blood flow, while concomitantly making tumors less infiltrative. Besides, TAX2 treatment also drastically inhibits B16F10 murine melanoma cells metastatic dissemination and growth in a syngeneic experimental model of lung metastasis, as demonstrated by histopathological analyses as well as longitudinal and quantitative µCT follow-up of metastatic progression. Altogether, the results obtained by combining bioinformatics and preclinical studies strongly suggest that targeting TSP-1/CD47 axis may represent a valuable therapeutic alternative for hampering melanoma spreading.

Role of β-catenin signaling in the anti-invasive effect of the omega-3 fatty acid DHA in human melanoma cells

BackgroundWe previously found that docosahexaenoic acid (DHA), a dietary polyunsaturated fatty acid present at high level in fatty fish, inhibited cell growth and induced differentiation of melanoma cells in vitro by increasing nuclear β-catenin content. An anti-neoplastic role of nuclear β-catenin was suggested in melanoma, and related to the presence in the melanocyte lineage of the microphtalmia transcription factor (MITF), which interferes with the transcription of β-catenin/TCF/LEF pro-invasive target genes. ObjectiveIn the present work we investigated if DHA could inhibit the invasive potential of melanoma cells, and if this effect could be related to DHA-induced alterations of the Wnt/β-catenin signaling, including changes in MITF expression. MethodsWM115 and WM266-4 human melanoma, and B16-F10 murine melanoma cell lines were used. Cell invasion was evaluated by Wound Healing and Matrigel transwell assays. Protein expression was analyzed by Western Blotting and β-catenin phosphorylation by immunoprecipitation. The role of MITF in the anti-invasive effect of DHA was analyzed by siRNA gene silencing. ResultsWe found that DHA inhibited anchorage-independent cell growth, reduced their migration/invasion in vitro and down-regulated several Matrix Metalloproteinases (MMP: MMP-2, MT1-MMP and MMP-13), known to be involved in melanoma invasion. We related these effects to the β-catenin increased nuclear expression and PKA-dependent phosphorylation, as well as to the increased expression of MITF. ConclusionThe data obtained further support the potential role of dietary DHA as suppressor of melanoma progression to invasive malignancy through its ability to enhance MITF expression and PKA-dependent nuclear β-catenin phosphorylation.

Transcutaneous iontophoretic delivery of STAT3 siRNA using layer-by-layer chitosan coated gold nanoparticles to treat melanoma

Overexpression of signal transducer and activator of transcription 3 (STAT3) protein prevents apoptosis and enhances proliferation of melanocytes. The aim of this study was to investigate the feasibility of using layer-by-layer assembled gold nanoparticles (LbL-AuNP) as a carrier for iontophoretic delivery of STAT3 siRNA to treat melanoma. Chitosan coated AuNP (AuNP-CS) were prepared by direct reduction of HAuCl4 in the presence of chitosan. The AuNP-CS were then sequentially layered with siRNA and chitosan to form AuNP-CS/siRNA/CS. STAT3 siRNA replaced with scrambled siRNA or sodium alginate were used as controls. The average particle size and zeta-potential of the prepared LbL-AuNP were 150±10nm (PDI: 0.41±0.06) and 35±6mV, respectively. In vitro studies in B16F10 murine melanoma cells showed that AuNP-CS/siRNA/CS inhibited the cell growth by 49.0±0.6% and 66.0±0.2% at 0.25nM and 0.5nM STAT3 siRNA concentration, respectively. Fluorescence microscopy and flow cytometry studies showed a time dependent cell uptake of the LbL-AuNP up to 120min. Clathrin mediated endocytosis was found to be the predominant cell uptake mechanism for LbL-AuNP. STAT3 siRNA loaded LbL-AuNP reduced the STAT3 protein expression by 47.3% in B16F10 cells. Similarly, apoptosis assay showed 29% and 44% of early and late apoptotic events, respectively after treatment with STAT3 siRNA loaded LbL-AuNP. Confocal microscope and skin cryosections showed that application of 0.47mA/cm2 of anodal iontophoresis enhanced the skin penetration of LbL-AuNP to reach viable epidermis. In conclusion, layer-by-layer chitosan coated AuNP can be developed as a carrier for iontophoretic delivery of STAT3 siRNA to treat melanoma.