B16F10 Murine Melanoma Cells Research Articles

FEASIBILITY STUDY OF MAGNETIC RESONANCE IMAGINING APPLICATION IN EXPERIMENTAL RADIOLOGY FOR INTRAVITAL VERIFICATION OF LUNGS METASTASES IN MICE

Background. For preclinical studies of radiopharmaceuticals there is a high need for development new methods of in vivo metastasis diagnosis in mice after tumor cells transplantation. The study was carried out to assess feasibility of lung metastasis diagnosis with magnetic resonance imaging (MRI) visualization in mice C57Bl6.The aim of this study was to assess feasibility of MRI for verification of distant metastases of the solid B16-F10 melanoma with validation by anatomic dissection.Methods. Metastatic lesions were caused by injection of B16-F10 murine melanoma cells into cavum medullare of the tibia. Imaging studies were performed on the 21th day after transplantation using 7T magnetic resonance tomograph, coronal and axial images were acquired. Validation of metastasis was made by anatomic dissection and histological examination.Results. MRI method enables visualization of lung nodules with diameter at least 0.8 mm, because smaller nodules cannot be distinguished from heartbeat artifacts. Histological examination revealed that macroscopic anatomic dissection can precisely detect subpleural lung nodules.Conclusion. This study demonstrated feasibility of in vivo lung metastasis verification with MRI method in mice: metastases comparable in diameter to the size of large bronchi can be detected by MRI as well as by ex vivo dissection. For centrally located lung metastasis MRI method is preferable because macroscopic dissection enables to visualize only nodules which are located subpleurally.

Abstract 1577: Caloric restriction mimetics as adjuvant to immune checkpoint inhibitors for treatment of melanoma

Abstract Immune checkpoint inhibitors (ICIs) have revolutionized melanoma therapy. Still, the 5-year survival for advanced melanoma is only 50%. This leads to two questions: what makes some tumors less responsive to ICIs, and what can be done to increase responsiveness? Prior research suggests that hypoxia in the tumor microenvironment (TME) decreases the efficacy of ICIs, and metformin, an antihyperglycemic and caloric restriction mimetic (CRM), is able to reverse this condition. We hypothesize that other CRMs will also increase responsiveness to ICIs through hypoxia, and potentially other mechanisms. We chose three CRMs to study alongside metformin: hydroxycitrate, resveratrol, and sulforaphane. To identify pathways and understand more about CRMs effect on tumor hypoxia, we performed a proteomics experiment on B16F10 murine melanoma cells treated with all four CRMs. Proteomics analysis was done with a tandem mass tag labeling. To determine effect of caloric restriction mimetics on immunotherapy efficacy, B16F10 cells will be injected subcutaneously in immunocompetent male and female C57BL/6J mice, separated into four groups: 1) no treatment control, 2) ICI therapy only, 3) CRM therapy only, 4) ICI + CRM combination. Our preliminary data shows that, like metformin, additional CRMs decrease oxidative phosphorylation in cancer cells. This supports the hypothesis that the other CRMs may function similarly to metformin in the TME. We started testing the effect of four CRMs on tumor growth and hypoxia in an animal model. Our ultimate goal is to improve responsiveness to immune checkpoint inhibitors in patients with metastatic melanoma. Citation Format: Lauren C. Morehead, Brian S. Koss, Katherine F. Wallis, Alan J. Tackett, Isabelle Racine Miousse. Caloric restriction mimetics as adjuvant to immune checkpoint inhibitors for treatment of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1577.

Tumor-Penetrable Nitric Oxide-Releasing Nanoparticles Potentiate Local Antimelanoma Therapy.

Although nitric oxide (NO) has been emerging as a novel local anticancer agent because of its potent cytotoxic effects and lack of off-target side effects, its clinical applications remain a challenge because of the short effective diffusion distance of NO that limits its anticancer activity. In this study, we synthesized albumin-coated poly(lactic-co-glycolic acid) (PLGA)-conjugated linear polyethylenimine diazeniumdiolate (LP/NO) nanoparticles (Alb-PLP/NO NPs) that possess tumor-penetrating and NO-releasing properties for an effective local treatment of melanoma. Sufficient NO-loading and prolonged NO-releasing characteristics of Alb-PLP/NO NPs were acquired through PLGA-conjugated LP/NO copolymer (PLP/NO) synthesis, followed by nanoparticle fabrication. In addition, tumor penetration ability was rendered by the electrostatic adsorption of the albumin on the surface of the nanoparticles. The Alb-PLP/NO NPs showed enhanced intracellular NO delivery efficiency and cytotoxicity to B16F10 murine melanoma cells. In B16F10-tumor-bearing mice, the Alb-PLP/NO NPs showed improved extracellular matrix penetration and spatial distribution in the tumor tissue after intratumoral injection, resulting in enhanced antitumor activity. Taken together, the results suggest that Alb-PLP/NO NPs represent a promising new modality for the local treatment of melanoma.

Synthesis of cinnamoyl glucoside derivatives and their antiproliferation activities against murine melanoma B16-F10 cell line

Twelve cinnamoyl glucoside derivatives were prepared by glycosylation of glucosyl trichloroacetimidate and cinnamic acid derivatives, followed by dechloroacetylation with a pyridine/H2O mixture. Their structures were characterized by 1H and 13C NMR, as well as mass analysis. All the products were tested for their antiproliferation activities against murine melanoma B16-F10 cell line. Compounds 4e-4j were able to inhibit the proliferation of murine melanoma B16-F10 cell line with IC50 values of 17.38 ± 0.07, 9.87 ± 0.09, 9.69 ± 0.12, 29.42 ± 0.04, 32.95 ± 0.08, 25.68 ± 0.09 μM, respectively.

Poly-l-Lysine inhibits VEGF and c-Myc mediated tumor-angiogenesis and induces apoptosis in 2D and 3D tumor microenvironment of both MDA-MB-231 and B16F10 induced mice model

Cancer is a widespread disease that has shown promising mortality worldwide. Our previous study has been shown the efficacy of Poly-l-lysine (PLL) as a promising cytotoxic effect against cancer cells. However, exact-mechanism of PLL in 3D physiological relevant tumor-microenvironment and against tumor-angiogenesis has never been analysed. In this study, we have investigated apoptotic efficacy of PLL, if any in opposition to proliferative aggressive cancer cell MDA-MB-231 both 2D and-3D cell culture conditions. Furthermore, PLL was administered in B16F10 murine melanoma cells induced BALB/c mice model. The study has been designed through transcription and translation level of PLL-induced tumor-angiogenesis and apoptotic gene-expression modulation level and various relevant histological studies in comparison with untreated control. Studies have shown anti-proliferative and anti-tumor angiogenic efficacy of PLL better in in-vitro 3D tumor-microenvironment against MDA-MB-231 breast cancer cells. Furthermore, in-vivo model, PLL was found to suppress tumorigenesis process at minimum dose. PLL found to induce apoptosis through-upregulation of cytosolic-cytochrome-C, caspase-3 and PARP activations when administered in B16F10 induced in-vivo tumor. In blocking proliferation and tumor-angiogenesis, PLL was found to be effective as it significantly downregulated activity of VEGF, VEGFR2, Ki-67 and c-Myc expression. As PLL blocked tumor progression and induced DNA-break, also upregulated apoptotic process and recovered tissue architecture as revealed from histological study in comparison with untreated control. Overall PLL was found to be a promising anti-tumor angiogenic and anti-proliferative drug that was effective both in in-vitro breast cancer 3D tumor-microenvironment and in-vivo metastatic-mice-model.

Endophytic actinobacteria of Hymenachne amplexicaulis from the Brazilian Pantanal wetland produce compounds with antibacterial and antitumor activities

The increase in the number of deaths from infections caused by multidrug-resistant bacteria and cancer diseases highlights the need for new molecules with biological activity. Actinobacteria represent a potential source of new compounds, as these microorganisms have already produced a great diversity of clinically employed antibiotics. Endophytes from unexplored biomes, such as the Pantanal (the largest wetland in the world), can be a source of new molecules. Hymenachne amplexicaulis is among the unexplored native plants of the Pantanal in terms of its endophytic community. This plant is considered a weed in other countries due to its ability to adapt and compete with native plants, and there is evidence to suggest that the endophytic community of H. amplexicaulis plays an important role in this competitiveness. To explore its therapeutic potential, the present study isolated, identified (using partial sequence of the 16S rDNA) and bioprospected H. amplexicaulis endophytic actinobacteria. Ten isolates belonging to the genera Streptomyces, Microbispora, Leifsonia, and Verrucosispora were obtained from root fragments. The susceptibility profile of the isolates to the different classes of antibiotics was evaluated, with 80 % of the isolates showing resistance to the antibiotics Nalidixic Acid, Ampicillin, Chloramphenicol, Oxacillin, and Rifampicin. To assess antibacterial and antitumor activities, methanolic extracts were obtained by fermentation in SG culture medium at 36 °C at 180 rpm for 10 days. The extract produced from the S. albidoflavus CMRP4854 isolate was the only one to show activity against the Gram-negative bacterium Acinetobacter baumanii. Due to the great clinical importance of this pathogen and the difficulty in obtaining active compounds against it, the CMRP4854 isolate should be further investigated for the identification of active compounds and mode of action. We also emphasize the results obtained by the extract of the isolates Streptomyces albidoflavus CMRP4852 and Verrucosispora sp. CMRP4860 that presented antibacterial effect against Methicilin-resistant Staphylococcus aureus (MRSA) (MIC: 1.5 μg/mL and 13 μg/mL, respectively) and Vancomycin-resistant Enterococcus (VRE) (MIC: 40 μg/mL for both extracts). Extracts (200 μg/mL) of these two endophytes also showed selective cytotoxicity action against murine B16-F10 melanoma cells. However, the CMRP4852 extract also affected the density of normal cells. Due to these results, the crude extract of isolate CMRP4860 Verrucosispora sp., which was the only one that presented cytotoxicity and reduced cell density only in tumor cells, was selected for subsequent analysis involving scale-up fermentation of the CMRP4860 resulting in 9 fractions that were tested against both bacteria and tumor cells, with particular fractions showing promise and meriting further investigation. Taken together, the results of this study not only show for the first time that the endophytic community of H. amplexicaulis actinobacteria can produce secondary metabolites that potentially possess important antibacterial and cytotoxic properties, but also reinforce the pressing need to conserve biomes such as the Brazilian Pantanal.

Antioxidant Compounds, Kirenol and Methyl ent-16α, 17-dihydroxy-kauran-19-oate Bioactivity-Guided Isolated from Siegesbeckia glabrescens Attenuates MITF-Mediated Melanogenesis via Inhibition of Intracellular ROS Production.

Siegesbeckia glabrescens (Compositae), an annual herb indigenous to Korean mountainous regions and has been eaten as a food in Korea. This study investigated ABTS, DPPH and nitric oxide (NO) radical-scavenging activities, and melanin production and TYR inhibitory effects-guided fractionation to identify therapeutic phytochemicals from S. glabrescens that can attenuate oxidation and melanogenesis in murine melanoma B16F10 cells. Nine compounds with inhibitory effects on melanin production, and TYR activity, and ABTS, DPPH, and NO radical scavenging activity were isolated from the 100% ethanol fraction from S. glabrescens. Among the nine compounds, kirenol (K), methyl ent-16α, 17-dihydroxy-kauran-19-oate (MDK) had strong inhibitory effects on melanin production and TYR activity with antioxidant effects. Western blot analysis revealed that K and MDK suppressed tyrosinase-related protein (TYRP)-1, TYRP-2 and microphthalmia-associated transcription factor (MITF) expression. Moreover, these two compounds inhibited intracellular reactive oxygen species (ROS) level in tert-butyl hydroperoxide (t-BHP)-treated B16F10 cells. Our results suggest that S. glabrescens containing active compounds such as K and MDK, which has antioxidant and antimelanogenesis effects, is the potent therapeutic and functional material for the prevention of oxidation-induced hyperpigmentation.

Antityrosinase and antioxidant activity of asparagus and its inhibition on B16F10 melanoma cells before and after hydrothermal treatment

In this study, we compared the effects of asparagus polyphenol extract (ASE) and asparagus polyphenol extract after hydrothermal treatments (ASEH) on antityrosinase and antioxidant activity, in addition to murine melanoma B16F10 cells. The intracellular experiments of four fractions, including cell morphology, cell viability, melanin content and intracellular tyrosinase activity were evaluated after 24, 48, and 72 h of treatment. The results indicated that the enhancement of gallic acid, coumaric acid, and hydroxybenzoic acid contents could be major reasons for the increased antityrosinase ability. Hydrothermal treatments could significantly increase the antityrosinase and antioxidant activity of asparagus polyphenol extracts. Meanwhile, ASEH (25–200 μg/mL) decreased the melanin content in B16F10 cells from 56.18% to 21.99% in a concentration-dependent manner. Furthermore, the intracellular tyrosinase activity of B16F10 cells was 39.78% at an ASEH concentration of 200 μg/mL. These results demonstrated that hydrothermal treatment was an efficient method to enhance the antityrosinase and antioxidant activity of asparagus polyphenols.

Microscopy of Eugenia involucrata, Chemical Composition and Biological Activities of the Volatile Oil

This paper studied the chemical profile, cytotoxicity, and insecticidal activity of the volatile oil, as well as provided a detailed anatomy of the leaves and stems from Eugenia involucrata DC., Myrtaceae. Its volatile oil was identified by gas chromatography coupled with mass spectrometry. The insecticidal activity was performed using the volatile oil against two strains of Cimex lectularius L., using topical, residual, and fumigant methods. Cytotoxicity was verified against B16F10, MCF7, and McCoy cell lines. The cytotoxic mechanism was studied by the morphology feature changes. The microscopic analysis was performed by usual microscopy techniques. The main volatile compounds of E. involucrata were germacrene B (22.17%), bicyclogermacrene (19.76%), and β-elemene (10.86%). Volatile oil demonstrated low toxicity against two strains of the bed bug in the topical test and did not present toxicity in fumigation and residual methods. Cytotoxicity for the B16F10 murine melanoma cells was demonstrated. Moreover, the volatile oil induces morphological characteristic changes of the cell death process of apoptosis. The microscopic data of the leaf and stem provided information to support the identification of E. involucrata.

Novel (Phenothiazinyl)Vinyl-Pyridinium Dyes and Their Potential Applications as Cellular Staining Agents.

We report here the synthesis and structural characterization of novel cationic (phenothiazinyl)vinyl-pyridinium (PVP) dyes, together with optical (absorption/emission) properties and their potential applicability as fluorescent labels. Convective heating, ultrasound irradiation and mechanochemical synthesis were considered as alternative synthetic methodologies proficient for overcoming drawbacks such as long reaction time, nonsatisfactory yields or solvent requirements in the synthesis of novel dye (E)-1-(3-chloropropyl)-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium bromide 3d and its N-alkyl-2-methylpyridinium precursor 1c. The trans geometry of the newly synthesized (E)-4-(2-(7-bromo-10-ethyl-10H-phenothiazin-3-yl)vinyl)-1-methylpyridin-1-ium iodide 3b and (E)-1-methyl-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium tetrafluoroborate 3a′ was confirmed by single crystal X-ray diffraction. A negative solvatochromism of the dyes in polar solvents was highlighted by UV-Vis spectroscopy and explanatory insights were supported by molecular modeling which suggested a better stabilization of the lowest unoccupied molecular orbitals (LUMO). The photostability of the dye 3b was investigated by irradiation at 365 nm in different solvents, while the steady-state and time-resolved fluorescence properties of dye 3b and 3a′ in solid state were evaluated under one-photon excitation at 485 nm. The in vitro cytotoxicity of the new PVP dyes on B16-F10 melanoma cells was evaluated by WST-1 assay, while their intracellular localization was assessed by epi-fluorescence conventional microscopy imaging as well as one- and two-photon excited confocal fluorescence lifetime imaging microscopy (FLIM). PVP dyes displayed low cytotoxicity, good internalization inside melanoma cells and intense fluorescence emission inside the B16-F10 murine melanoma cells, making them suitable staining agents for imaging applications.

Distinct rhodamine B derivatives exhibiting dual effect of anticancer activity and fluorescence property

Design and application of novel Rhodamine B amide derivatives achieved especially with simple secondary amines, and furthermore exhibiting fluorescence property and anticancer activity is not reported elsewhere. Further functionalization of rhodamine B at the carboxylic acid group with selected secondary amines generated amides, avoiding cyclization and simultaneously sustaining fluorescence. Currently, we have designed and developed three well defined novel rhodamine B amide derivatives by reacting rhodamine B (RHB) with thiomorpholine, bis-(2-chloroethyl) amine and morpholine (the side chains of several anticancer drugs) via rhodamine B acid chloride which resulted in fluorophores namely rhodamine B thiomorpholine amide (1), rhodamine B bis(2-chloroethyl)amine amide (2) and rhodamine B morpholine amide (3). All the three RHB amides were thoroughly characterized using analytical techniques like FT-IR, Mass, 1H NMR, 13C NMR spectroscopy and HPLC. Obtained compounds displayed molecular material attributes as well as anticancer activity. The wavelength of maximum absorption and emission of solutions occurred at ~560 nm and ~583 nm. Quantum yields (Φf = 0.40) and average fluorescence decay (~1.60 ns) of amides was comparable to RHB (1.72 ns). Optical bandgap of solids revealed semiconducting property (~ 2.9 eV). Acquired rhodamine B amides were found to be insensitive over a wide range of pH i.e. from 2 to 10 suggesting their plausible utility in biological labeling, moreover, showed potential anticancer activity against B16F10 (murine melanoma cells), MDA-MB231 (human breast cancer cells), A549 (human lung cancer cells) while they displayed less toxicity to normal HEK 293 (human embryonic kidney) cells. Interestingly, the cellular uptake study manifested that more quantity of the compound were taken up by the cancer cells compared to normal cells which indicate that these amides might become potential candidates to be developed as theranostic agents.

Expression of MITF Gene in Melanogenesis with Psoralea corylifolia, Zingiber officinale, Terminalia chebula, Punica granatum and Eclipta alba Based on Poly-Herbal Formulation

Pigmentation is an important physical characteristic of human beings due to the presence of a biopolymer molecule called melanin. Melanin pigment in the human skin and several other tissues were due to the activation and inhibition of different genes at the downstream signaling pathway like Tyrosinase-related protein 1 (TYRP1) and Tyrosinase-related protein 2 (TYRP2) and Microphthalmia-associated transcription factor (MITF) genes. In this study, we tried to deliver the molecular approach of TYRP1, TYRP2, and MITF by activating kinases in the process of Melanogenesis by treating with Unani formulations on the B16F10 murine melanoma cell line. RNA isolation of B16F10 cells was performed followed by cDNA conversion and expression studies at mRNA levels by RT-PCR method. To explore further western blot was performed at the protein level for the significant results which we obtained with UNIM regimes in the expression studies. The bioactive ingredient of UNIM-004 and UNIM-005 of Methanolic extracts up-regulates the expression and protein levels of the MITF gene which leads to the synthesis of melanin pigment in human beings. Therefore from this study, it indicates that the application and administration of UNIM-004 and UNIM-005 of methanolic extract will result in activating melanin in B16F10 cells by increasing mRNA expression levels in the MITF gene when compared with TYRP1and TYRP2 genes followed by Western blot experiment and found to be producing melanocytes in the progression of Melanogenesis in a dose-dependent manner at 10 μg and 100 μg concerning for to GAPDH control gene at mRNA expression level in Vitiligo patients.

Low-Temperature Argon Plasma Regulates Skin Moisturizing and Melanogenesis-Regulating Markers through Yes-Associated Protein.

Extensive water loss and melanin hyperproduction can cause various skin disorders. Low-temperature argon plasma (LTAP) has shown the possibility of being used for the treatment of various skin diseases, such as atopic dermatitis and skin cancer. However, the role of LTAP in regulating skin moisturizing and melanogenesis has not been investigated. In this study, we aimed to determine the effect of LTAP on yes-associated protein (YAP), a major transcriptional coactivator in the Hippo signaling pathway that is involved in skin moisturizing and melanogenesis-regulating markers. In normal human epidermal keratinocytes (NHEKs), the human epidermal keratinocyte line HaCaT, and human dermal fibroblasts (HDFs), we found that LTAP exhibited increased expression levels of YAP protein. In addition, the expression levels of filaggrin (FLG), which is involved in natural moisturizing factors (NMFs), and hyaluronic acid synthase (HAS), transglutaminase (TGM), and involucrin (IVL), which regulate skin barrier and moisturizing, were also increased after exposure to LTAP. Furthermore, collagen type I alpha 1 and type III alpha 1 (COL1A1, COL3A1) were increased after LTAP exposure, but the expression level of matrix metalloproteinase-3 (MMP-3) was reduced. Moreover, LTAP was found to suppress alpha-melanocyte stimulating hormone (α-MSH)-induced melanogenesis in murine melanoma B16F10 cells and normal human melanocytes (NHEMs). LTAP regulates melanogenesis of the melanocytes through decreased YAP pathway activation in a melanocortin 1 receptor (MC1R)-dependent manner. Taken together, our data show that LTAP regulates skin moisturizing and melanogenesis through modulation of the YAP pathway, and the effect of LTAP on the expression level of YAP varies from cell to cell. Thus, LTAP might be developed as a treatment method to improve the skin barrier, moisture content, and wrinkle formation, and to reduce melanin generation.

Identification of Mushroom and Murine Tyrosinase Inhibitors from Achillea biebersteinii Afan. Extract.

Growing scientific evidence indicates that Achillea biebersteinii is a valuable source of active ingredients with potential cosmetic applications. However, the data on its composition and pharmacological properties are still insufficient. This study aims to optimize the extraction procedure of the plant material, evaluate its phytochemical composition, and compare anti-tyrosinase potential of A. biebersteinii extracts obtained by various methods. In order to identify compounds responsible for the tyrosinase inhibitory activity of A. biebersteinii, the most active anti-tyrosinase extract was fractionated by column chromatography. The fractions were examined for their skin lightening potential by mushroom and murine tyrosinase inhibitory assays and melanin release assay. HPLC-ESI-Q-TOF-MS/MS analysis of the total extract revealed the presence of several phenolic acids, flavonoids, flavonoid glucosides, and carboxylic acid. Among them, fraxetin-8-O-glucoside, quercetin-O-glucopyranose, schaftoside/isoschaftoside, gmelinin B, 1,3-dicaffeoylquinic acid (1,3-DCQA), and ferulic acid were found in the fractions with the highest skin lightening potential. Based on obtained qualitative and quantitative analysis of the fractions, it was assumed that the caffeoylquinic acid derivatives and dicaffeoylquinic acid derivatives are more likely responsible for mushroom tyrosinase inhibitory activity of A. biebersteinii extracts and fractions. Ferulic acid was proposed as the most active murine tyrosinase inhibitor, responsible also for the reduced melanin release from B16F10 murine melanoma cells.

Monosubstituted Acetophenone Thiosemicarbazones as Potent Inhibitors of Tyrosinase: Synthesis, Inhibitory Studies, and Molecular Docking.

A set of 12 monosubstituted acetophenone thiosemicarbazone derivatives (TSCs) were synthesized and their inhibitory properties toward tyrosinase activity were tested. Moreover, their ability to inhibit melanogenesis in the B16F10 murine melanoma cell line was studied. In order to investigate the nature of interactions between the enzyme and the inhibitors, molecular docking to the active site was performed. TSCs 5, 6, 8, and 9 revealed a half maximal inhibitory concentration (IC50) below 1 µM. Compound 6 turned out to be the most potent tyrosinase inhibitor. All investigated compounds showed reversible inhibition of competitive or mixed type. The para-substituted TSCs had higher affinity for the enzyme as compared to their ortho- and meta-analogues. All investigated compounds inhibited melanin production in B16F10 cells at the micromolar level. Molecular docking showed that the sulfur atom of the thiourea moiety penetrates the active site and interacts with copper ions. The above outcomes might be helpful in the design of new tyrosinase inhibitors in the food and cosmetic industries.

The Influence of Some Axial Ligands on Ruthenium-Phthalocyanine Complexes: Chemical, Photochemical, and Photobiological Properties.

This work presents a new procedure to synthesize ruthenium–phthalocyanine complexes and uses diverse spectroscopic techniques to characterize trans-[RuCl(Pc)DMSO] (I) (Pc = phthalocyanine) and trans-[Ru(Pc)(4-ampy)2] (II) (4-ampy = 4-aminopyridine). The triplet excited-state lifetimes of (I) measured by nanosecond transient absorption showed that two processes occurred, one around 15 ns and the other around 3.8 μs. Axial ligands seemed to affect the singlet oxygen quantum yield. Yields of 0.62 and 0.14 were achieved for (I) and (II), respectively. The lower value obtained for (II) probably resulted from secondary reactions of singlet oxygen in the presence of the ruthenium complex. We also investigate how axial ligands in the ruthenium–phthalocyanine complexes affect their photo-bioactivity in B16F10 murine melanoma cells. In the case of (I) at 1 μmol/L, photosensitization with 5.95 J/cm2 provided B16F10 cell viability of 6%, showing that (I) was more active than (II) at the same concentration. Furthermore, (II) was detected intracellularly in B16F10 cell extracts. The behavior of the evaluated ruthenium–phthalocyanine complexes point to the potential use of (I) as a metal-based drug in clinical therapy. Changes in axial ligands can modulate the photosensitizer activity of the ruthenium phthalocyanine complexes.

Synthesis of Novel Cinnamides and a Bis Cinnamate Bearing 1,2,3-Triazole Functionalities with Antiproliferative and Antimetastatic Activities on Melanoma Cells

The present investigation describes the synthesis of novel cinnamides and a bis cinnamate bearing 1,2,3-triazole functionalities and investigation of their antiproliferative and antimetastatic effects on melanoma cells. The necessity for the development of new chemotherapeutic agents for melanoma treatment motivated this work. Sixteen derivatives were obtained with yields ranging from 23-81% and fully characterized by spectroscopic (1H and 13C nuclear magnetic resonance, infrared) and spectrometric high resolution mass spectrometry (HRMS) techniques. The derivatives were in vitro evaluated against B16-F10 murine melanoma cell line. The most effective compound (a bis cinnamate) (6b) reduced the melanoma cell viability, generated cell cycle arrest, and influenced the metastatic behavior of melanoma cells by decreasing migration, invasion, and colony formation. Based on these findings, it is believed that compound 6b may represent an interesting scaffold to be explored toward the development of new antimelanoma agents.

Identification of metastasis-related genes by genomic and transcriptomic studies in murine melanoma

AimsWe systematically characterized metastatic murine B16-F10 melanoma, a sub-line derived from murine melanoma B16-F1 cells. Materials and methodsRNA-sequencing and network analyses (Ingenuity Pathway Analysis) were performed to identify novel potential metastasis mechanisms. Chromosomal aberrations were identified by multicolor fluorescence in situ hybridization (mFISH) using all 21 murine whole chromosome painting probes. Key findingsNumerous genes were overexpressed in B16-F10 cells, some of which have been already described as being metastasis-linked. Nr5a1/sf1, a known prognostic marker for adrenal tumors, was 177-fold upregulated in B16-F10 cells compared to B16-F1 cells. Hoxb8 was 75-fold upregulated, which was previously associated with gastric cancer progression and metastasis. Ptk7, which is linked with tumorigenesis and metastasis of esophageal squamous carcinoma, was 67-fold upregulated. B16-F10 cells acquired additional chromosomal aberrations compared to B16-F1 cells, including dic(4)(pter->qter:qter->pter), +dic(6;15), +der(10)t(10;?1;16). SignificanceIn addition to well-known metastatic genes, numerous novel genes and genomic aberrations were identified, which may serve as targets for treatment in the future. Transcriptomic and genetic analyses in B16-F10 cells unraveled a range of novel metastasis mechanisms, which may also have important implications for future treatment strategies.

Sodium dichloroacetate attenuates the growth of B16-F10 melanoma in vitro and in vivo: an opportunity for drug repurposing.

Sodium dichloroacetate (DCA) is a metabolic regulator used to treat diabetes. Since DCA inhibits pyruvate dehydrogenase kinase, decreasing lactic acid formation, it can reverse the Warburg effect in cancer cells, promoting apoptosis. Therefore, this study aimed to investigate the potential of DCA as a drug repurposing candidate for the treatment of melanoma. For the in-vitro assay, murine B16-F10 melanoma cells were treated with 0.5, 1, 5, 10, 20 or 50 mM DCA for 3 days, analyzed with the crystal violet method. The in-vivo effect of DCA was evaluated in B16-F10 tumor-bearing C57BL/6 mice treated with different doses of DCA (0, 25, 75 or 150 mg/kg) by gavage for 10 days, followed by measurement of tumor volume. Upon necropsy, representative slices of lung, liver, kidney, spleen and intestine were collected, processed and submitted for histopathological examination. The DCA concentrations of 10, 20 and 50 mM reduced B16-F10 cell viability after 48 and 72 h of treatment, whereas 20 and 50 mM were effective after 24 h of treatment. A significant reduction in tumor growth was observed in B16-F10 melanoma bearing mice at all doses, with no change in body weight or histology. DCA attenuates the growth of B16-F10 melanoma in vitro and in vivo, without systemic toxic effects. Therefore, DCA is a candidate for drug repurposing against melanomas.

Multiple Forms of Angiostatin Induce Apoptosis in Endothelial Cells

AbstractAngiostatin is a circulating inhibitor of angiogenesis generated by proteolytic cleavage of plasminogen. In this study we have used recombinant human and murine angiostatins (kringles 1-4) as well as native human angiostatin (prepared by elastase digestion of plasminogen [kringles 1-3] or by plasmin autocatalysis in the presence of a free sulfhydryl donor [kringles 1-4]). We report that angiostatin reduces endothelial cell number in a 4-day proliferation assay without affecting cell cycle progression into S-phase (as determined by bromodeoxyuridine labeling). This suggested that the reduction in cell number in the proliferation assay might in part be due to cytotoxicity. This was confirmed by the observation that ethidium homodimer incorporation (a measure of plasma membrane integrity) into endothelial cells was increased by angiostatin in a manner similar to that seen with tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1), both of which induce apoptosis in endothelial cells. In contrast to TNF-α and TGF-β1, angiostatin did not induce cytotoxicity in human MRC-5 fibroblast, rat smooth muscle, canine MDCK epithelial, or murine B16-F10 melanoma cell lines. Angiostatin-induced apoptosis was confirmed by endothelial cell nuclear acridine orange incorporation as well as by annexin V and TUNEL staining. These in vitro findings point to endothelial cell apoptosis as a mechanism for the antiangiogenic effect of angiostatin in vivo.