Role of lipid mediators in the resistance of human melanoma to the BRAF inhibitor (Vemurafenib)

O melanoma é um câncer de pele caracterizado pela alta mortalidade devido à resistência à quimioterapia e à capacidade metastática. Aproximadamente metade dos casos de melanoma maligno apresenta mutações que ativam o gene BRAF, na maioria das vezes envolvendo a substituição V600E. Dessa forma, os inibidores de BRAF, como o Vemurafenibe, têm sido utilizados no tratamento deste tipo de câncer. Apesar da resposta inicial às opções terapêuticas ser satisfatória, a maioria dos pacientes apresenta remissão da doença em um curto período de tempo. Avaliar como mediadores lipídicos interferem nos mecanismos de resistência é uma estratégia para novos tratamentos mais efetivos ao melanoma resistente. Dessa forma, o nosso objetivo foi avaliar o papel dos mediadores lipídicos na resistência das linhagens de células de melanoma humano ao Vemurafenibe. Neste trabalho, demonstramos que as linhagens de melanoma humano (A375, SK-MEL-29, WM35 e WM278) e os melanócitos são capazes de produzir diferentes mediadores lipídicos quando estimulados in vitro e que, dentre elas, as linhagens metastáticas (A375 e SK-MEL-29), tanto as parentais (P) quanto as resistentes (R), sintetizam concentrações maiores desses compostos. Além disso, observamos através do ensaio de Azul de tripan e em modelo 3D utilizando esferoides que o tratamento das células metastáticas (A375P e R e SK-MEL-29P e R) com PGD2 diminuiu a proliferação celular e interferiu na produção das citocinas IL-6, IL-8 e IL-10 de modo independente dos receptores DP1 e DP2. Ainda, observamos que a PGJ2, metabólito da PGD2, também reduziu in vitro a proliferação das células de melanoma A375P e SK-MEL-29 P e R, por uma via independente do receptor PPAR. Dessa forma, investigamos possíveis vias metabólicas que poderiam estar perturbadas nas células A375P e R após o tratamento com PGD2. Nossas análises mostraram que vias como metabolismo de xenobióticos, metabolismo de cisteína e metionina e a transferência via carnitina foram especificamente perturbadas nesta comparação e, portanto, sugerem que as diferenças fenotípicas após tratamento com PGD2 sejam resultado de alterações nestas vias metabólicas. Portanto, nossos resultados contribuem para o desenvolvimento de novas terapêuticas que possibilitem a prevenção ou retardamento das metástases e, consequentemente, melhor prognóstico da doença.Melanoma is a skin cancer characterized by high mortality due to resistance to chemotherapy and metastatic capacity. Approximately half of the cases of malignant melanoma have mutations that activate the BRAF gene, mainly involving the V600E substitution. Thus, BRAF inhibitors, such as Vemurafenib, have been used to treat this type of cancer. Although the initial response rate is effective, disease progression and tumor resistance rapidly occurs in the majority of patients. Assessing how lipid mediators interfere with resistance mechanisms is a strategy for new, more effective treatments for resistant melanoma. Thus, our objective was to evaluate the role of lipid mediators in the resistance of human melanoma cell lines to Vemurafenib. In this work we demonstrate that human melanoma cell lines (A375, SK-MEL-29, WM35 and WM278) and melanocytes are able to produce different lipid mediators when stimulated in vitro and that, among them, metastatic cells (A375 and SK- MEL-29), both parental (P) and resistant (R), synthesize higher concentrations of these compounds. In addition, we observed through the trypan blue assay and in a 3D model using spheroids that the treatment of metastatic cells (A375P and R and SK-MEL-29P and R) with PGD2 decreased cell proliferation and interfered with the cytokines production (IL- 6, IL-8 and IL-10) independently of DP1 and DP2 receptors. Furthermore, we observed that PGJ2, a metabolite of PGD2, also reduced the proliferation of A375P and SK-MEL-29P and R melanoma cells in vitro, via a PPAR receptor-independent pathway. Thus, we investigated possible metabolic pathways that could be disturbed in A375P and R cells after treatment with PGD2. Our analyzes showed that pathways such as xenobiotic metabolism, cysteine and methionine metabolism and transfer via carnitine were specifically disturbed in this comparison and therefore suggest that phenotypic differences after treatment with PGD2 are the result of changes in these metabolic pathways. Therefore, our results contribute to the development of new therapies that enable the prevention or delay of metastasis and, consequently, a better prognosis of the disease

High performance liquid chromatography coupled with tandem mass spectrometry to investigate eicosanoid profile in peripheral blood after stimulation: comparison between sickle cell anemia patients with healthy individuals

Os eicosanoides, produtos do metabolismo do ácido araquidônico, apresentam papel importante na homeostasia e na patogênese de diversas doenças humanas. A biossíntese desses compostos pode ser estimulada por agentes farmacológicos como ionóforos e inibidores da Ca2+-ATPase, e também por agonistas naturais como o formil-metionil-leucil-fenialanina (fMLP). Considerando os interesses em avaliar e comparar o perfil de mediadores lipídicos, como os leucotrienos (LTs), as prostaglandinas (PGs), os ácidos epoxieicosatrienoicos (EETs), os ácidos dihidroxitetraenoicos (DiHETEs) e os ácidos hidroxieicosatetraenoicos (HETEs), na saúde e na doença, o objetivo deste trabalho foi padronizar um método analítico para determinar do perfil de eicosanoides em plasma humano após estimulação do sangue total, e assim observar diferenças entre indivíduos saudáveis e doentes. Dessa forma, um método por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (HPLC-MS/MS) foi validado para quantificação de 22 eicosanoides em plasma de indivíduos saudáveis. A análise por HPLCMS/ MS foi realizada em modo negativo pelo modo de varredura por monitoramento de reações múltiplas (MRM). A linearidade do método apresentou coeficiente de correlação (r) maior que 0,98 para todos os eicosanoides analisados. A precisão e exatidão intra e inter-ensaios tiveram desvio padrão e erro relativo menores que 15%, exceto para o limite inferior de quantificação cujos valores foram menores que 20%. Para estimulação das células do sangue total, quatro estímulos (fMLP, ionomicina, A23187 e tapsigargina) foram utilizados. A análise estatística mostrou que o A23187 e a tapsigargina foram os estímulos mais potentes na indução da produção de eicosanoides. Em seguida, comparamos o perfil de eicosanoides em amostras de plasma de indivíduos saudáveis com pacientes com anemia falciforme (AF), em tratamento com hidroxiureia (HU) ou transfusão sanguínea crônica. Os resultados demonstraram que o método é preciso para determinação de diferenças entre os pacientes e indivíduos saudáveis quanto à produção dos mediadores lipídicos 5-HETE, 12-HETE, LTB4, LTE4, TXB2 e PGE2. Portanto, nosso método analítico é sensível, específico e reprodutível para identificar e quantificar diferenças no perfil de eicosanoides em amostras de sangue estimuladas in vitro, e poderá contribuir para o estabelecimento do perfil de mediadores lipídicos em diferentes doenças inflamatórias e infecciosas.Eicosanoids, products from arachidonic acid metabolism, play an important role in the homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca2+ ionophores and Ca2+-ATPase inhibitors, as well as natural agonists such as fMet-leu-Phe (fMLP) can stimulate eicosanoid biosynthesis. Considering the interests in evaluate and compare the profile of lipid mediators, as leukotriens (LTs), prostaglandins (PGs), epoxyeicosatrienoic acids (EETs), dihydroxytetraenoic acids (DiHETEs) and hydroxyeicosatetraenoic acids (HETEs), in healthy and disease, the aim of this work was to standardize a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation, and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatographytandem mass spectrometry (LC-MS/MS) method was validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient > 0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of < 15%, except for the lower limit of quantification, where these values were < 20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were used. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli to induce the production of eicosanoids. We next compared the eicosanoid profiles of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients compared to healthy subjects for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method is sensitive, specific and reproducible for indentify and quantify changes in eicosanoid profiles in whole blood stimulated in vitro, which can contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases

Lipoxin A4 encapsulated in PLGA microparticles accelerates wound healing of skin ulcers.

Lipoxin A4 (LXA4) is involved in the resolution of inflammation and wound healing; however, it is extremely unstable. Thus, to preserve its biological activities and confer stability, we encapsulated LXA4 in poly-lactic-co-glycolic acid (PLGA) microparticles (LXA4-MS) and assessed its application in treating dorsal rat skin lesions. Ulcers were sealed with fibrin adhesive and treated with either LXA4-MS, unloaded microparticles (Un-MS), soluble LXA4, or PBS/glue (vehicle). All groups were compared at 0, 2, 7, and 14 days post-lesions. Our results revealed that LXA4-MS accelerated wound healing from day 7 and reduced initial ulcer diameters by 80%. Soluble LXA4, Un-MS, or PBS closed wounds by 60%, 45%, and 39%, respectively. LXA4-MS reduced IL-1β and TNF-α, but increased TGF-β, collagen deposition, and the number of blood vessels. Compared to other treatments, LXA4-MS reduced inflammatory cell numbers, myeloperoxidase (MPO) concentration, and metalloproteinase-8 (MMP8) mRNA in scar tissue, indicating decreased neutrophil chemotaxis. In addition, LXA4-MS treatment increased macrophages and IL-4, suggesting a positive impact on wound healing. Finally, we demonstrated that WRW4, a selective LXA4 receptor (ALX) antagonist, reversed healing by 50%, indicating that LXA4 must interact with ALX to induce wound healing. Our results show that LXA4-MS could be used as a pharmaceutical formulation for the treatment of skin ulcers

Scanning electron microscopy (SEM) of microparticles and <i>in vitro</i> release of LXA₄ from MS.

<p>Representative images (2,000×) of (A) Unloaded and (B) LXA₄-MS morphologies. (C) <i>In vitro</i> cumulative release of LXA₄ from LXA₄-MS. LXA₄ concentration was determined by mass spectrometry over 48 h. Data are representative of two batches.</p

WRW4, a selective LXA₄ receptor antagonist, reversed wound healing properties of LXA₄-MS.

<p>(A) qRT-PCR analysis was performed to assess the LXA₄ receptor <i>ALX</i> mRNA’s abundance in skin ulcers collected on days 2, 7, and 14 in the control (vehicle—PBS/glue), Un-MS, soluble LXA₄, and LXA₄-MS groups. Data represent means ± SEM (n = 5 ulcers/group). One-way ANOVA was done to determine statistical significance (<i>p</i> < 0.05), which is indicated as follows: *, LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^#, LXA₄-MS <i>vs</i>. Un-MS; and ^$, LXA₄-MS <i>vs</i>. soluble LXA₄. (B) Representative images of 1.5 cm dorsal wounds at days 2 and 7, with day 0 images serving as pre-injury images, are presented for the following groups: WRW4 (25 μl per animal—from a main peptide solution of 1 mg/ml), WRW4 + LXA₄-MS (WRW4 applied 10 minutes before MS application– 10 mg of LXA₄-MS), and LXA₄-MS (10 mg). (C) Wound healing index values for the groups outlined in (B). Index values range from 0 to 1, where a value of 0 indicates the original wound, and a value of 1 represents a completely closed wound. Data represent means ± SEM (n = 9 ulcers/group); One-way ANOVA was done to determine statistical significance (*<i>p</i> < 0.05).</p

LXA₄-MS modulated cytokines generation in the skin.

<p>Skin ulcer tissues collected on days 0, 2, 7, and 14 from the control (vehicle—PBS/glue), Un-MS, soluble LXA₄, and LXA₄-MS groups were homogenized to assess (A) IL-1β, (B) TNF-α, (C) IL-6, and (D) TGF-β production by ELISAs. Data (represented as bars) represent means ± SEM (n = 5 ulcers/group). One-way ANOVA was done to determine statistical significance (<i>p</i> < 0.05) and indicated as follows: *, demonstrated significant differences compared to normal tissues (dashed line); ^#, soluble LXA₄ or LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^$, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^&, LXA₄-MS <i>vs</i>. soluble LXA₄.</p

Topical application of LXA₄-MS to skin ulcers accelerated wound closure and attenuated neutrophil chemotaxis.

<p>(A) Representative images of 1.5 cm dorsal wounds were collected on days 0, 2, 7, and 14 for the following groups: control (vehicle—PBS/glue), Un-MS, soluble LXA₄, and LXA₄-MS. (B) Wound healing index values for the groups outlined in (A). Index values range from 0 to 1, where a value of 0 indicates the original wound, and a value of 1 represents a completely closed wound. Values are means ± SEM (n = 10 ulcers/group). One-way ANOVA was done to determine statistical significance (<i>p</i> < 0.05), which is as follows: *, soluble LXA₄ or LXA₄-MS <i>vs</i>. vehicle (PBS/glue); ^#, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^{, LXA₄-MS vs. soluble LXA₄. (C) ImageJ software was used to count inflammatory cells on day 2 in at least 12 random optical 400× fields per group. (D) Neutrophil accumulation (represented as bars) as measured by MPO. Values are means ± SEM (n = 5 wounds/group). One-way ANOVA was done to determine statistical significance (p < 0.05), which is indicated as follows: *, demonstrated significant increase compared to normal tissue (dashed line); ^#, soluble LXA₄ or LXA₄-MS vs. Vehicle (PBS/glue);}, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and &, LXA₄-MS <i>vs</i>. soluble LXA₄. (E) qRT-PCR was performed to assess <i>MMP8</i> mRNA transcript abundance in skin ulcers collected on days 2, 7, and 14 from the vehicle (PBS/glue), Un-MS, soluble LXA₄, and LXA₄-MS groups. Data represent means ± SEM (n = 5 ulcers/group). One-way ANOVA was done to determine statistical significance (<i>p</i> < 0.05) and indicated as follows: *, soluble LXA₄ or LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^#, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^$, LXA₄-MS <i>vs</i>. soluble LXA₄.</p

LXA₄-MS increased collagen deposition and angiogenesis and affected VEGF production.

<p>(A) Collagen deposition was measured using the ImageJ software with the Color Deconvolution plug-in, which measured densitometry in at least 12 random 400× fields in all groups, at days 2, 7 and 14. Percentages of collagen deposition are represented as means ± SEM (n = 6 ulcers/group). One-way ANOVA was used to determine statistical significance (<i>p</i> < 0.05) and is indicated as follows: *, soluble LXA₄ or LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^#, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^{, LXA₄-MS vs. soluble LXA₄. (B) Photomicrographs of wounds stained with Picro Sirius Red (200×) show collagen deposition at days 2, 7, and 14. (C) Histogram showing quantitative analysis of vascular density using the ImageJ software with the Cell Counter plug-in on 200× images. One-way ANOVA was performed to determine statistical significance (p < 0.05), which is indicated as follows: *, significant VEGF increase as compared to normal tissue (dashed line); ^#, soluble LXA₄ or LXA₄-MS vs. Vehicle (PBS/glue);}, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^&, LXA₄-MS <i>vs</i>. soluble LXA₄. (D) VEGF was quantified in all groups at days 2, 7 and 14 (represented as bars) in wounds via ELISAs as proxy to blood vessel density. Values are means ± SEM (n = 6 wounds/group). One-way ANOVA was performed to determine statistical significance (<i>p</i>< 0.05), which is indicated as follows: *, significant VEGF increase as compared to normal tissue (dashed line); ^#, soluble LXA₄ or LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^$, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^&, LXA₄-MS <i>vs</i>. soluble LXA₄.</p

Lipoxin A₄ encapsulated in PLGA microparticles accelerates wound healing of skin ulcers

<div><p>Lipoxin A₄ (LXA₄) is involved in the resolution of inflammation and wound healing; however, it is extremely unstable. Thus, to preserve its biological activities and confer stability, we encapsulated LXA₄ in poly-lactic-co-glycolic acid (PLGA) microparticles (LXA₄-MS) and assessed its application in treating dorsal rat skin lesions. Ulcers were sealed with fibrin adhesive and treated with either LXA₄-MS, unloaded microparticles (Un-MS), soluble LXA₄, or PBS/glue (vehicle). All groups were compared at 0, 2, 7, and 14 days post-lesions. Our results revealed that LXA₄-MS accelerated wound healing from day 7 and reduced initial ulcer diameters by 80%. Soluble LXA₄, Un-MS, or PBS closed wounds by 60%, 45%, and 39%, respectively. LXA₄-MS reduced IL-1β and TNF-α, but increased TGF-β, collagen deposition, and the number of blood vessels. Compared to other treatments, LXA₄-MS reduced inflammatory cell numbers, myeloperoxidase (MPO) concentration, and metalloproteinase-8 (<i>MMP8</i>) mRNA in scar tissue, indicating decreased neutrophil chemotaxis. In addition, LXA₄-MS treatment increased macrophages and IL-4, suggesting a positive impact on wound healing. Finally, we demonstrated that WRW4, a selective LXA₄ receptor (ALX) antagonist, reversed healing by 50%, indicating that LXA₄ must interact with ALX to induce wound healing. Our results show that LXA₄-MS could be used as a pharmaceutical formulation for the treatment of skin ulcers.</p></div