The Potential of Photodynamic Therapy Using Solid Lipid Nanoparticles with Aluminum Phthalocyanine Chloride as a Nanocarrier for Modulating Immunogenic Cell Death in Murine Melanoma In Vitro.

Photodynamic therapy (PDT) uses a photosensitizer to generate reactive oxygen species (ROS) that kill target cells. In cancer treatments, PDT can potentially induce immunogenic cell death (ICD), which is characterized by a well-controlled exposure of damage-associated molecular patterns (DAMPs) that activate dendritic cells (DCs) and consequently modulate the immune response in the tumor microenvironment. However, PDT still has limitations, such as the activity of photosensitizers in aqueous media and poor bioavailability. Therefore, a new photosensitizer system, SLN-AlPc, has been developed to improve the therapeutic efficacy of PDT. In vitro experiments showed that the light-excited nanocarrier increased ROS production in murine melanoma B16-F10 cells and modulated the profile of DCs. PDT induced cell death accompanied by the exposure of DAMPs and the formation of autophagosomes. In addition, the DCs exposed to PDT-treated B16-F10 cells exhibited morphological changes, increased expression of MHCII, CD86, CD80, and production of IL-12 and IFN-γ, suggesting immune activation towards an antitumor profile. These results indicate that the SLNs-AlPc protocol has the potential to improve PDT efficacy by inducing ICD and activating DCs.

New Evidence on the Antiproliferative Activity of Campomanesia Adamantium (Cambess.) O. Berg Extracts in Melanoma Lung Metastasis.

Given the importance of discovering plant species from the Brazilian Cerrado biome with anticancer potential, this study evaluated the antitumor activity of two extracts of Campomanesi adamantium fruits in in vitro and in vivo models of melanoma lung metastasis. Pulp and peel extracts (DEGPU and DEGPE, respectively) were extracted from fresh fruit using dichloromethane as a solvent. As cytotoxicity parameter, concentration values that inhibited 50% cell growth (GI50), total growth inhibition (TGI), and selectivity index (SI) were established. The melanoma lung metastasis model was obtained by injecting 5 × 105/50 µL B16-F10 cells via the tail vein of mice, which received treatment on the 15th day. Metastatic lungs were collected for fluorescence analysis with the IR-780 marker and also macro- and microscopic assessment. In vitro analyses showed that DEGPU was active in K562 (GI50 32.99; TGI 47.93) and U-251 (GI50 32.10; TGI 249.92), whereas DEGPE showed better cytotoxicity results for all tumor cell lines, but was more efficient in K562 (GI50 27.42; TGI 40.20) and U-251 (GI50 4.89; TGI 12.77). Both showed a cytocidal effect on B16F10 at the highest concentration tested, with approximately 25% (DEGPU) and 88% (DEGPE) of cell death. In vivo analyzes showed that both extracts showed significant activity in metastatic lung. Fluorescence images showed differences in intensity between groups owing to greater tumor involvement. Macro- and microscopic images showed that treatments with extracts limited tumor growth and prevented proliferation. The extracts tested have promising activity, thus requiring further research on their active compounds.

Molecular weight-dependent antitumor effects of prunes-derived type I arabinogalactan on human and murine triple wild-type melanomas.

The regulation of metastasis-related cellular aspects of two structurally similar AGIs from prunes tea infusion, with different molar masses, was studied in vitro against Triple Wild-Type metastatic melanoma (TWM) from murine and human origin. The higher molar mass AGI (AGI-78KDa) induced TWMs cells death and, in murine cell line, it decreased some metastasis-related cellular processes: invasiveness capacity, cell-extracellular matrix interaction, and colonies sizes. The lower molar mass AGI (AGI-12KDa) did not induce cell death but decreased TWMs proliferation rate and, in murine cell line, it decreased cell adhesion and colonies sizes. Both AGIs alter the clonogenic capacity of human cell line. In spite to understand why we saw so many differences between AGIs effects on murine and human cell lines we performed in silico analysis that demonstrated differential gene expression profiles between them. Complementary network topological predictions suggested that AGIs can modulate multiple pathways in a specie-dependent manner, which explain differential results obtained in vitro between cell lines. Our results pointed to therapeutic potential of AGIs from prunes tea against TWMs and showed that molecular weight of AGIs may influence their antitumor effects.

Paclitaxel-Loaded Lipid-Coated Magnetic Nanoparticles for Dual Chemo-Magnetic Hyperthermia Therapy of Melanoma.

Melanoma is the most aggressive and metastasis-prone form of skin cancer. Conventional therapies include chemotherapeutic agents, either as small molecules or carried by FDA-approved nanostructures. However, systemic toxicity and side effects still remain as major drawbacks. With the advancement of nanomedicine, new delivery strategies emerge at a regular pace, aiming to overcome these challenges. Stimulus-responsive drug delivery systems might considerably reduce systemic toxicity and side-effects by limiting drug release to the affected area. Herein, we report the development of paclitaxel-loaded lipid-coated manganese ferrite magnetic nanoparticles (PTX-LMNP) as magnetosomes synthetic analogs, envisaging the combined chemo-magnetic hyperthermia treatment of melanoma. PTX-LMNP physicochemical properties were verified, including their shape, size, crystallinity, FTIR spectrum, magnetization profile, and temperature profile under magnetic hyperthermia (MHT). Their diffusion in porcine ear skin (a model for human skin) was investigated after intradermal administration via fluorescence microscopy. Cumulative PTX release kinetics under different temperatures, either preceded or not by MHT, were assessed. Intrinsic cytotoxicity against B16F10 cells was determined via neutral red uptake assay after 48 h of incubation (long-term assay), as well as B16F10 cells viability after 1 h of incubation (short-term assay), followed by MHT. PTX-LMNP-mediated MHT triggers PTX release, allowing its thermal-modulated local delivery to diseased sites, within short timeframes. Moreover, half-maximal PTX inhibitory concentration (IC50) could be significantly reduced relatively to free PTX (142,500×) and Taxol® (340×). Therefore, the dual chemo-MHT therapy mediated by intratumorally injected PTX-LMNP stands out as a promising alternative to efficiently deliver PTX to melanoma cells, consequently reducing systemic side effects commonly associated with conventional chemotherapies.

Role of Annexin A1 Secreted by Neutrophils in Melanoma Metastasis.

Annexin A1 (AnxA1) is highly secreted by neutrophils and binds to formyl peptide receptors (FPRs) to trigger anti-inflammatory effects and efferocytosis. AnxA1 is also expressed in the tumor microenvironment, being mainly attributed to cancer cells. As recruited neutrophils are player cells at the tumor sites, the role of neutrophil-derived AnxA1 in lung melanoma metastasis was investigated here. Melanoma cells and neutrophils expressing AnxA1 were detected in biopsies from primary melanoma patients, which also presented higher levels of serum AnxA1 and augmented neutrophil-lymphocyte ratio (NLR) in the blood. Lung melanoma metastatic mice (C57BL/6; i.v. injected B16F10 cells) showed neutrophilia, elevated AnxA1 serum levels, and higher labeling for AnxA1 in neutrophils than in tumor cells at the lungs with metastasis. Peritoneal neutrophils collected from naïve mice were co-cultured with B16F10 cells or employed to obtain neutrophil-conditioned medium (NCM; 18 h incubation). B16F10 cells co-cultured with neutrophils or with NCM presented higher invasion, which was abolished if B16F10 cells were previously incubated with FPR antagonists or co-cultured with AnxA1 knockout (AnxA1-/-) neutrophils. The depletion of peripheral neutrophils during lung melanoma metastasis development (anti-Gr1; i.p. every 48 h for 21 days) reduced the number of metastases and AnxA1 serum levels in mice. Our findings show that AnxA1 secreted by neutrophils favors melanoma metastasis evolution via FPR pathways, addressing AnxA1 as a potential biomarker for the detection or progression of melanoma.

Esterification of p-Coumaric Acid Improves the Control over Melanoma Cell Growth.

Previous studies reported that p-coumaric acid modulates melanoma growth. Because the esterification of p-coumaric acid (p-CA) enhanced its activity as an antimelanogenic agent, we aimed to determine the antitumor potential of two derivatives, the ethyl and butyl esters, against the murine B16-F10 and the human SK-MEL-25 melanoma cells. Cell viability was determined in vitro by the lactate dehydrogenase release and violet crystal absorption assays. The cell proliferation rate and cell cycle behavior were determined by the colony formation assay and flow cytometry analysis. Although p-CA, at the concentration of 1 mM, failed to exert a significant antitumor activity, the ethyl and butyl ester derivatives caused substantial tumor cell death at doses < 1 mM. Despite a reduction in their direct cytotoxicity at minor doses, both products controlled the melanoma growth by arresting the cell cycle at the G0/G1 (B16-F10) or S/G2 (SK-MEL-25). Furthermore, the in vivo experiments showed that the butyl ester derivative suppressed the lung B16-F10 burden, compared to the p-CA-treated mice. Thus, the esterification of p-coumaric acid improved the control over the proliferation of murine and human melanoma cells and can be considered an approach for designing novel anticancer agents.

Antimelanoma effect of manool in 2D cell cultures and reconstructed human skin models.

Melanoma is the most aggressive and lethal type of skin cancer, characterized by therapeutic resistance. In this context, the present study aimed to investigate the cytotoxic potential of manool, a diterpene from Salvia officinalis L., in human (A375) and murine (B16F10) melanoma cell lines. The analysis of cytotoxicity using the XTT assay showed the lowest IC50 after 48 h of treatment with the manool, being 17.6 and 18.2 µg/ml for A375 and B16F10, respectively. A selective antiproliferative effect of manool was observed on the A375 cells based on the colony formation assay, showing an IC50 equivalent to 5.6 µg/ml. The manool treatments led to 43.5% inhibition of the A375 cell migration at a concentration of 5.0 µg/ml. However, it did not affect cell migration in the B16F10 cells. Cell cycle analysis revealed that the manool interfered in the cell cycle of the A375 cells, blocking the G2/M phase. No changes in the cell cycle were observed in the B16F10 cells. Interestingly, manool did not induce apoptosis in the A375 cells, but apoptosis was observed after treatment of the B16F10 cells. Additionally, manool showed an antimelanoma effect in a reconstructed human skin model. Furthermore, in silico studies, showed that manool is stabilized in the active sites of the tubulin dimer with comparable energy concerning taxol, indicating that both structures can inhibit the proliferation of cancer cells. Altogether, it is concluded that manool, through the modulation of the cell cycle, presents a selective antiproliferative activity and a potential antimelanoma effect.

Evaluation of tumor growth remission in a murine model for subcutaneous solid tumors, benefits of associating the antitumor agent crotamine with mesoporous nanosilica particles to achieve improved dosing frequency and efficacy

Crotamine is a highly cationic polypeptide first isolated from South American rattlesnake venom, which exhibits affinity for acidic lysosomal vesicles and proliferating cells. This cationic nature is pivotal for its in vitro cytotoxicity and in vivo anticancer actions. This study aimed to enhance the antitumor efficacy of crotamine by associating it with the mesoporous SBA-15 silica, known for its controlled release of various chemical agents, including large proteins. This association aimed to mitigate the toxic effects while amplifying the pharmacological potency of several compounds. Comprehensive characterization, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis, confirmed the successful association of crotamine with the non-toxic SBA-15 nanoparticles. The TEM imaging revealed nanoparticles with a nearly spherical shape and variations in uniformity upon crotamine association. Furthermore, DLS showed a narrow unimodal size distribution, emphasizing the formation of small aggregates. Zeta potential measurements indicated a distinct shift from negative to positive values upon crotamine association, underscoring its effective adsorption onto SBA-15. Intraperitoneal or oral administration of crotamine:SBA-15 in a murine melanoma model suggested the potential to reduce the frequency of crotamine doses without compromising efficacy. Interestingly, while the oral route enhanced the antitumor efficacy of crotamine, pH-dependent release from SBA-15 was observed. Thus, associating crotamine with SBA-15 could reduce the overall required dose to inhibit solid tumor growth, bolstering the prospect of crotamine as a potent anticancer agent.

Evaluation of tumor growth remission in a murine model for subcutaneous solid tumors, benefits of associating the antitumor agent crotamine with mesoporous nanosilica particles to achieve improved dosing frequency and efficacy

Crotamine is a highly cationic polypeptide first isolated from South American rattlesnake venom, which exhibits affinity for acidic lysosomal vesicles and proliferating cells. This cationic nature is pivotal for its in vitro cytotoxicity and in vivo anticancer actions. This study aimed to enhance the antitumor efficacy of crotamine by associating it with the mesoporous SBA-15 silica, known for its controlled release of various chemical agents, including large proteins. This association aimed to mitigate the toxic effects while amplifying the pharmacological potency of several compounds. Comprehensive characterization, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis, confirmed the successful association of crotamine with the non-toxic SBA-15 nanoparticles. The TEM imaging revealed nanoparticles with a nearly spherical shape and variations in uniformity upon crotamine association. Furthermore, DLS showed a narrow unimodal size distribution, emphasizing the formation of small aggregates. Zeta potential measurements indicated a distinct shift from negative to positive values upon crotamine association, underscoring its effective adsorption onto SBA-15. Intraperitoneal or oral administration of crotamine:SBA-15 in a murine melanoma model suggested the potential to reduce the frequency of crotamine doses without compromising efficacy. Interestingly, while the oral route enhanced the antitumor efficacy of crotamine, pH-dependent release from SBA-15 was observed. Thus, associating crotamine with SBA-15 could reduce the overall required dose to inhibit solid tumor growth, bolstering the prospect of crotamine as a potent anticancer agent.

Istradefylline modulates purinergic enzymes and reduces malignancy-associated factors in B16F10 melanoma cells.

ATP and adenosine exert pivotal roles in the development, maintenance, and metastatic spreading of melanoma. The action of such key melanoma tumor microenvironment (TME) constituents might be complementary or opposed, and their effects are not exclusive to immune cells but also to other host cells and tumor cells. The effects of ATP are controlled by the axis CD39/73, resulting in adenosine, the main actor in the TME, and A2A is the crucial mediator of its effects. We evaluated ATP and adenosine signaling through A2A on B16F10 melanoma cells using istradefylline (IST) (antiparkinsonian A2A antagonist) and caffeine (CAF) treatments after exposure to ATP and adenosine. Adenosine increased melanoma cell viability and proliferation in a concentration-dependent manner. ATP increases viability only as a substrate by CD39 to produce adenosine. Both IST and CAF are toxic to B16F10 cells, but only IST potentialized paclitaxel-induced cytotoxic effects, even decreasing its IC50 value. IST positively modulated CD39 and CD73 expression. CD39 activity was increased, and E-ADA was reduced, indicating that the melanoma cells promoted compensatory feedback in the production and maintenance of adenosine levels. A2A antagonism by IST reduced the factors associated with malignancy, like migration, adhesion, colony formation, and the capacity to produce melanin. Moreover, IST significantly increases nitric oxide (NO) production, which correlates to a decline in melanoma cell viability by apoptotic events. Altogether, our results suggest that adenosine signaling through A2A is essential for B16F10 cells, and its inhibition by IST causes compensatory purinergic enzymatic modulations. Furthermore, IST is a promising therapy that provides new ways to improve current melanoma treatments.

Impaired expression of serine/arginine protein kinase 2 (SRPK2) affects melanoma progression.

Melanoma is one of the most aggressive tumors, and its lethality is associated with the ability of malignant cells to migrate and invade surrounding tissues to colonize distant organs and to generate widespread metastasis. The serine/arginine protein kinases 1 and 2 (SRPK1 and SRPK2) are classically related to the control of pre-mRNA splicing through SR protein phosphorylation and have been found overexpressed in many types of cancer, including melanoma. Previously, we have demonstrated that the pharmacological inhibition of SRPKs impairs pulmonary colonization of metastatic melanoma in mice. As the used compounds could target at least both SRPK1 and SRPK2, here we sought to obtain additional clues regarding the involvement of these paralogs in melanoma progression. We analyzed single-cell RNA sequencing data of melanoma patient cohorts and found that SRPK2 expression in melanoma cells is associated with poor prognosis. Consistently, CRISPR-Cas9 genome targeting of SRPK2, but not SRPK1, impaired actin polymerization dynamics as well as the proliferative and invasive capacity of B16F10 cells in vitro. In further in vivo experiments, genetic targeting of SRPK2, but not SRPK1, reduced tumor progression in both subcutaneous and caudal vein melanoma induction models. Taken together, these findings suggest different functional roles for SRPK1/2 in metastatic melanoma and highlight the relevance of pursuing selective pharmacological inhibitors of SRPK2.

Folate-Targeted PEGylated Magnetoliposomes for Hyperthermia-Mediated Controlled Release of Doxorubicin.

Doxorubicin (DOX) is a chemotherapeutic agent commonly used for the treatment of solid tumors. However, the cardiotoxicity associated with its prolonged use prevents further adherence and therapeutic efficacy. By encapsulating DOX within a PEGylated liposome, Doxil® considerably decreased DOX cardiotoxicity. By using thermally sensitive lysolipids in its bilayer composition, ThermoDox® implemented a heat-induced controlled release of DOX. However, both ThermoDox® and Doxil® rely on their passive retention in tumors, depending on their half-lives in blood. Moreover, ThermoDox® ordinarily depend on invasive radiofrequency-generating metallic probes for local heating. In this study, we prepare, characterize, and evaluate the antitumoral capabilities of DOX-loaded folate-targeted PEGylated magnetoliposomes (DFPML). Unlike ThermoDox®, DOX delivery via DFPML is mediated by the heat released through dynamic hysteresis losses from magnetothermal converting systems composed by MnFe2O4 nanoparticles (NPs) under AC magnetic field excitation-a non-invasive technique designated magnetic hyperthermia (MHT). Moreover, DFPML dismisses the use of thermally sensitive lysolipids, allowing the use of simpler and cheaper alternative lipids. MnFe2O4 NPs and DFPML are fully characterized in terms of their size, morphology, polydispersion, magnetic, and magnetothermal properties. About 50% of the DOX load is released from DFPML after 30 min under MHT conditions. Being folate-targeted, in vitro DFPML antitumoral activity is higher (IC50 ≈ 1 µg/ml) for folate receptor-overexpressing B16F10 murine melanoma cells, compared to MCF7 human breast adenocarcinoma cells (IC50 ≈ 4 µg/ml). Taken together, our results indicate that DFPML are strong candidates for folate-targeted anticancer therapies based on DOX controlled release.

C-phycocyanin decreases proliferation and migration of melanoma cells: In silico and in vitro evidences.

The incidence and number of deaths caused by melanoma have been increasing in recent years, and the pigment C-phycocyanin (C-PC) appears as a possible alternative to treat this disease. So, the objective of this study was to combine in silico and in vitro analysis to understand the main anti-melanoma pathways exerted by C-PC. We evaluated the ability of C-PC to bind to the main cellular targets related in the progression of melanoma through molecular docking, and the reflection of this bind in the biological effects in the B16F10 cell line through in vitro analysis. Our results showed that C-PC was able to bind BRAF and MEK, which are related to the signal transduction pathway for proliferation and survival. There was also an interaction between C-PC and cyclin-dependent kinase 4 and 6. In vitro analysis demonstrated that C-PC decreased B16F10 cell proliferation, as observed by cell viability and mitotic index assays. C-PC also interacted with matrix metalloproteinase 2 and 9 and N-cadherin, which may have caused the decrease in cell migration observed in vitro. Besides that, C-PC interacts with VEGF, a factor responsible for regulating the proliferation and cellular invasion pathways. Finally, C-PC did not alter the cell viability of the non-tumoral melanocytes. Therefore, C-PC is a strong anti-tumor candidate for the treatment of melanoma, since it acts in different cellular pathways of melanoma, without causing damage to non-tumoral cells.

Synthesis of cinnamic acid ester derivatives with antiproliferative and antimetastatic activities on murine melanoma cells.

Melanoma is the most aggressive skin cancer, and its incidence has continued to rise during the past decades. Conventional treatments present severe side effects in cancer patients, and melanoma can be refractory to commonly used anticancer drugs, which justify the efforts to find new potential anti-melanoma drugs. An alternative to promote the discovery of new pharmacological substances would be modifying chemical groups from a bioactive compound. Here we describe the synthesis of seventeen compounds derived from cinnamic acid and their bioactivity evaluation against melanoma cells. The compound phenyl 2,3-dibromo-3-phenylpropanoate (3q) was the most effective against murine B16-F10 cells, as observed in cytotoxicity and cell migration assays. Simultaneously, this compound showed low cytotoxic activity on non-tumor cells. At the highest concentration, the compound 3q was able to trigger apoptosis, whereas, at lower concentrations, it affected the cell cycle and melanoma cell proliferation. Furthermore, cinnamate 3q impaired cell invasion, adhesion, colonization, and actin polymerization. In conclusion, these results highlight the antiproliferative and antimetastatic potential of cinnamic acid derivatives on melanoma.

Polybrominated diphenyl ethers BDE-47 and BDE-99 modulate murine melanoma cell phenotype in vitro.

Cancer is one of the leading causes of mortality worldwide. Even with the advances of pharmaceutical industry and treatments, the mortality rate for various types of cancer remains high. In particular, phenotypic alterations of tumor cells concerning drug efflux, migratory and invasive capabilities may represent a hurdle for cancer treatment and contribute to poor prognosis. In the present study, we investigated the effects of polybrominated diphenyl ethers (PBDEs) used as flame retardants on phenotypic features of melanoma cells that are important for cancer. Murine melanoma B16-F1 (less metastatic) and B16-F10 (more metastatic) cells were exposed to 0.01-1.0 nM of BDE-47 (2,2',4,4'-tetrabromodiphenyl ether), BDE-99 (2,2',4,4',5-pentabromodiphenyl ether), and the mixture of both (at 0.01 nM) for 24 h (acute exposure) and 15 days (chronic exposure). The polybrominated diphenyl ethers (PBDEs) did not affect cell viability but led to increased drug efflux transporter activity, cell migration, and colony formation, as well as overexpression of Abcc2 (ATP-binding cassette subfamily C member 2), Mmp-2 (matrix metalloproteinase-2), Mmp-9 (matrix metalloproteinase-9), and Tp53 (tumor protein p53) genes and downregulation of Timp-3 (tissue inhibitor of metalloproteinase 3) gene in B16-F10 cells. These effects are consistent with increased aggressiveness and malignancy of tumors due to exposure to the flame retardants and raise some concerns on the effects such chemicals may have on melanoma treatment and cancer prognosis.

Therapeutic potential of Smilax fluminensis ethanolic extract: antitumoral activity in murine melanoma cells.

The aim of the study was to investigate the in vitro and in vivo antitumor activity of leaves ethanol extract from Smilax fluminensis on murine melanoma. The extract was performed by ethylic alcohol and submitted to classical chemical analysis. Cytotoxicity test were performed on neoplastic cells, where antitumor activity was expressed in GI50 (concentration that inhibits 50% of cell growth) and the determination of selectivity index using a normal cell line. In addition, BALB/c mice models were used to evaluate the in vivo antitumor activity of extract in two different concentrations against B16-F10 melanoma cells. The tumor inhibition ratio was determined and the histopathological analyses of nodules and liver were compared. The chemical analysis indicated a major presence of phenolic compounds and flavonoids. Cytotoxicity test results that S. fluminensis extract was active in B16-F10 line (GI50: 4.37 µg/mL), being the extract considered a promising antineoplastic agent. In the experimental model, the inhibition percentage of tumoral growth was between 78.77 and 83.49%. Histopathology analysis of nodules showed necrotic cells reduction, adipocytes presence, melanin deposition, vascularization, and inflammatory process in a concentration-dependent manner. On the liver, the animals treated with the extract on both concentrations showed normal hepatic organization, normal hepatocytes, and absence of inflammatory focus. The results indicate that S. fluminensis extract demonstrated both in vitro and in vivo antitumor activity, reducing the tumoral growth in B16-F10 and could therefore be a promising antineoplastic agent.

Antiproliferative activity of aqueous and polyphenol-rich extracts of Larrea divaricata Cav. on a melanoma cell line.

Most of the deaths from skin cancer are caused by melanoma, a malignancy in which STAT3 plays a crucial role. The inhibition of STAT3 is considered a potential target to induce cell death, tumor regression and metastasis inhibition. The objective of this work was to evaluate the activity of the aqueous extract of Larrea divaricata (Aq), a fraction rich in polyphenols (EA),and the isolated compound quercetin-3-methyl ether (Q3ME) on B16F10 melanoma cells. The effects of Aq, EA and Q3ME were assessed on B16F10 cells by determining the proliferation, viability, apoptosis induction and the expression and phosphorylation of STAT3. The phytochemical composition of the extracts was determined by High Performance Liquid Chromatography. Aq, EA and Q3ME presented antiproliferative activity on B6F10 cells through p-STAT3 inhibition and early and late apoptosis induction (EC50 EA= ≤0.1 µg/ml; Aq= 316 ± 30 µg/ml; Q3ME= <0.1 µg/ml). L. divaricata could be considered for the development of adjuvant phytotherapies in melanoma treatment.

Methylene blue and photodynamic therapy for melanomas: Inducing different rates of cell death (necrosis and apoptosis) in B16-F10 melanoma cells according to methylene blue concentration and energy dose.

BACKGROUND: Photodynamic therapy (PDT) is a cancer treatment based on the interaction between the photosensitizing agent methylene blue (MB), light, and molecular oxygen. MB has antibacterial properties and can to bind to melanin. Here, we investigated whether MB based PDT (MB-PDT) could decrease viability and induce death of murine melanoma B16-F10 cells. METHODS: B16-F10 cells were incubated with different concentrations of MB (0, 1, or 2 µg/mL) and exposed to a diode red laser with a wavelength of 660 nm and power output of 100 mW/cm2. The energy dose and density varied from 0 J and 0 J/cm2 to 100.8 J and 720 J/cm². Cell viability was measured using the trypan blue exclusion assay of cell viability and confirmed by performing an MTT assay. The morphological type and cell death rates were determined using fluorescence microscopy with acridine orange and ethidium bromide. The presence and rate of apoptosis were evaluated via Annexin V-Alexa Fluor/propidium iodide staining and flow cytometry analysis. RESULTS: MB-PDT decreased cell viability and increased cell death (necrosis and apoptosis) in a drug- and light-dose dependent manner. Morphological characteristics of necrosis were observed immediately after treatment, and apoptotic characteristics were observed after 3 h. The apoptosis and necrosis rates varied with the drug and light doses, with 2 µg/mL MB and a 100.8 J energy dose inducing the highest rates. CONCLUSIONS: We demonstrated that MB-PDT reduced murine melanoma B16-F10 cell viability and induced cell death in a drug- and light-dose dependent manner.

Metformin pretreatment reduces effect to dacarbazine and suppresses melanoma cell resistance.

Oxidative stress role on metformin process of dacarbazine (DTIC) inducing resistance of B16F10 melanoma murine cells are investigated. To induce resistance to DTIC, murine melanoma cells were exposed to increasing concentrations of dacarabazine (DTIC-res group). Metformin was administered before and during the induction of resistance to DTIC (MET-DTIC). The oxidative stress parameters of the DTIC-res group showed increased levels of malondialdehyde (MDA), thiol, and reduced nuclear p53, 8-hydroxy-2'-deoxyguanosine (8-OH-DG), nuclear factor kappa B (NF-ĸB), and Nrf2. In presence of metformin in the resistant induction process to DTIC, (MET-DTIC) cells had increased antioxidant thiols, MDA, nuclear p53, 8-OH-DG, Nrf2, and reducing NF-ĸB, weakening the DTIC-resistant phenotype. The exclusive administration of metformin (MET group) also induced the cellular resistance to DTIC. The MET group presented high levels of total thiols, MDA, and reduced percentage of nuclear p53. It also presented reduced nuclear 8-OH-DG, NF-ĸB, and Nrf2 when compared with the control. Oxidative stress and the studied biomarkers seem to be part of the alterations evidenced in DTIC-resistant B16F10 cells. In addition, metformin administration is able to play a dual role according to the experimental protocol, preventing or inducing a DTIC-resistant phenotype. These findings should help future research with the aim of investigating DTIC resistance in melanoma.

Anti-genotoxic and anti-mutagenic effects of melatonin supplementation in a mouse model of melanoma.

Melanoma, an aggressive skin cancer originating from melanocytes, can metastasize to the lungs, liver, cortex, femur, and spinal cord, ultimately resulting in DNA mutagenic effects. Melatonin is an endogenous hormone and free radical scavenger that possesses the ability to protect the DNA and to exert anti-proliferative effects in melanoma cells. The aim of this study was to evaluate the effects of B16F10 melanoma cells and the effects of melatonin supplementation on genotoxic parameters in murine melanoma models. Thirty-two male C57Bl/6 mice were divided in the following four groups: PBS + vehicle (n = 6), melanoma + vehicle (n = 10), PBS + melatonin (n = 6), and melanoma + melatonin (n = 10). The melanoma groups received a B16F10 cell injection, and melatonin was administered during 60 days. After treatment, tumor sizes were evaluated. DNA damage within the peripheral blood, lungs, liver, cortex, and spinal cord was determined using comet assay, and the mutagenicity within the bone marrow was determined using the micronucleus test. B16F10 cells effectively induced DNA damage in all tissues, and melatonin supplementation decreased DNA damage in the blood, liver, cortex, and spinal cord. This hormone exerts anti-tumor activity via its anti-proliferative, antioxidative, and pro-apoptotic effects. As this result was not observed within the lungs, we hypothesized that melatonin can induce apoptosis in cancer cells, and this was not evaluated by comet assay. This study provides evidence that melatonin can reduce the genotoxicity and mutagenicity caused by B16F10 cells.