B16F10 Melanoma Cells Research Articles

Near-Infrared Bioluminescence Imaging of Macrophage Sensors for Cancer Detection In Vivo.

Melanoma is an aggressive type of skin cancer with a poor prognosis after it gets metastasized. The early detection of malignant melanoma is critical for effective therapy. Because melanoma often resembles moles, routine skin check-up may help for timely identification of suspicious areas. Recently, it has been shown that the interplay of melanoma cells with the immune system can help develop efficient therapeutic strategies. Here, we leveraged engineered macrophages (BMC2) as cell-based sensors for metastatic melanoma. To perform dual-color bioluminescence imaging (BLI) in vivo, macrophages were engineered to express a green click beetle luciferase (CBG2) and a near-infrared fluorescent dye (DiR), and B16F10 melanoma cells were instead engineered to express a near-infrared click beetle luciferase (CBR2). Using real-time in vivo dual-color BLI and near-infrared fluorescence (FL) imaging, we could demonstrate that macrophages were able to sense and substantially accumulate in subcutaneous and metastatic melanoma tissues at 72 h after systemic injections. Together, we showed the potentiality to use optical imaging technologies to track circulating macrophages for the non-invasive detection of metastatic melanoma.

Nuclear focal adhesion kinase induces APC/C activator protein CDH1-mediated cyclin-dependent kinase 4/6 degradation and inhibits melanoma proliferation

Dysregulation of cyclin-dependent kinases (CDKs) can promote unchecked cell proliferation and cancer progression. Although focal adhesion kinase (FAK) contributes to regulating cell cycle progression, the exact molecular mechanism remains unclear. Here, we found that FAK plays a key role in cell cycle progression potentially through regulation of CDK4/6 protein expression. We show that FAK inhibition increased its nuclear localization and induced G1 arrest in B16F10 melanoma cells. Mechanistically, we demonstrate nuclear FAK associated with CDK4/6 and promoted their ubiquitination and proteasomal degradation through recruitment of CDC homolog 1 (CDH1), an activator and substrate recognition subunit of the anaphase-promoting complex/cyclosome E3 ligase complex. We found the FAK N-terminal FERM domain acts as a scaffold to bring CDK4/6 and CDH1 within close proximity. However, overexpression of nonnuclear-localizing mutant FAK FERM failed to function as a scaffold for CDK4/6 and CDH1. Furthermore, shRNA knockdown of CDH1 increased CDK4/6 protein expression and blocked FAK inhibitor–induced reduction of CDK4/6 in B16F10 cells. In vivo, we show that pharmacological FAK inhibition reduced B16F10 tumor size, correlating with increased FAK nuclear localization and decreased CDK4/6 expression compared with vehicle controls. In patient-matched healthy skin and melanoma biopsies, we found FAK was mostly inactive and nuclear localized in healthy skin, whereas melanoma lesions showed increased active cytoplasmic FAK and elevated CDK4 expression. Taken together, our data demonstrate that FAK inhibition blocks tumor proliferation by inducing G1 arrest, in part through decreased CDK4/6 protein stability by nuclear FAK.

Monomeric CCTδ Interacts with Dynactin Associated Protein (dynAP)

Introduction‐The chaperonin containing tailless complex polypeptide 1 (CCT) is a molecular chaperone consisting of eight distinct protein subunits. In the monomeric form, several CCT subunits possess additional functions distinct from their oligomeric function [1]. Over‐expression of CCTδ, but not the other seven CCT subunits, results in the appearance of numerous protrusions at the cell surface termed a “protrusion phenotype” which is abrogated by mutations in CCTδ apical domain (G357D) and the ATP binding pocket (D104E) [2]. In addition, it is dependent on CCTδ binding the p150Glued of the dynactin complex. Depletion of either p150Glued or the dynactin complex‐associated transmembrane protein, dynAP prevents GFP‐CCTδ localization to the plasma membrane and blocks protrusion phenotype formation [3]. The work of others [4] shows dynAP binds p150Glued. However, biochemical evidence of CCTδ interacting with dynAP has not been shown. We hypothesized either a direct transient interaction between CCTδ and dynAP or a trimer with p150Glued occurring.Materials & Method‐Mouse melanoma B16F1 cells were cultured as described in [2], transiently transfected with CCTδ constructs, and mutants thereof fused to GFP as described in [2], and full‐length human; p150Glued with C‐terminal Myc and dynAP with an N‐terminal FLAG epitope tags as described in [3]. To stabilize transient interactions in‐vivo, confluent cells were crosslinked with 0.5 mM 3, 3′‐Dithiodipropionic acid di (N‐hydroxysuccinimide ester). Post‐nuclear supernatants from lysed cells were applied to GFP‐trap beads (Chromotek), followed by Western Blotting to detect precipitating proteins.Results‐Following the double transfection of B16 cells with Flag‐dynAP and wild type (WT) GFP‐CCTδ, there was a significant interaction between dynAP and WT CCTδ versus control, D104E. In addition, following the triple transfection of GFP‐CCTδ/p150Glued‐Myc/Flag‐dynAP, there was a significant interaction between dynAP and WT CCTδ versus control, D104E. Furthermore, following the double transfection of WT CCTδ apical domain of (AD) and Flag‐dynAP, there was a significant interaction between dynAP and WT AD versus control, G357D.Conclusion‐Here, we show for the first time an interaction between CCTδ and dynAP via immunoprecipitation representing either a direct interaction or as part of a trimeric complex with p150Glued. This provides biochemical evidence for the mechanism underlying the formation of the protrusion phenotype. This work lends more information on monomeric CCTδ function in cells and would contribute to more understanding of CCTδ’ s role in basic cell and cancer biology. More research is needed to understand the molecular effects of these interactions in cells and in‐vivo.References Svanstrom & Grantham (2015) Cell Stress and Chaperones; 21:55–62. Spiess, M. et al (2015) Journal of Molecular Biology; 427:2757‐2764. Echbarthi M. et al (2018) Experimental Cell Research; 370:137‐149. Kunoh T. et al (2010) Molecular Cancer Therapeutics; 9:2934‐2942.

Exercise Alters Ceramide Metabolism in Melanoma

IntroductionWe previously demonstrated that exercise enhanced the efficacy of chemotherapy against B16F10 melanoma in mice. Exercise likely impacts both the tumor microenvironment and the tumor cells. Exercise has been demonstrated to modulate ceramide metabolism in healthy tissue, but the relationship between exercise and ceramide in tumors has not been evaluated. Ceramides are essential sphingolipid metabolites involved in cell death and survival pathways. Low content of ceramides in melanoma is a potential mechanism for drug resistance. Thus, increasing ceramides content in tumor cells is likely to increase sensitivity to chemotherapy. We hypothesized that exercise would increase accumulation of ceramide in tumor by regulating ceramide metabolism.MethodsC57BL/6 male mice were subcutaneously injected with B16F10 or BP melanoma cells and randomly assigned into sedentary control (No Ex) and exercise (Ex) groups. After tumor establishment, Ex mice performed treadmill running at 12 meter/min for 45 min for 5 consecutive exercise days with two days off then 5 additional exercise days. Ceramides were measured in tumor homogenates by liquid chromatography‐tandem mass spectrometry (LC/MS). Ceramide metabolizing enzymes were analyzed in tumors by immunofluorescence staining and western blotting. Cultured B16F10 or BP cells were treated with serum collected from corresponding melanoma tumor bearing mouse to examine effect of circulating factors on ceramide regulating enzymes.ResultsSeveral ceramide species were increased in Ex compared to No Ex B16F10 tumors, with the largest exercise‐induced increases in C16‐, dihydro‐C16‐, and C20‐ ceramide. Significantly increased expression of ceramide synthase 6 (CerS6), which generates C16‐ceramide using sphinganine or sphingosine, was observed in Ex B16F10 tumors by immunofluorescence staining and western blotting. Surprisingly, there was a trend toward an increase in the ceramide degrading enzyme acid ceramidase (aCDase) in Ex tumors. This may suggest a feedback loop. In contrast, Ex significantly suppressed expression of sphingosine kinase 1 (Sphk1), which converts sphingosine to the key survival sphingolipid S1P, in B16F10 tumors. Likewise, Sphk1 protein expression decreased in B16F10 cells treated in vitro with serum from Ex. Unlike the findings in B16F10 tumors and cultured B16F10 cells, no change in C16‐Ceramide, CerS6, or Sphk1 was observed in Ex BP tumors and in in vitro treatment of BP cells with Ex mice serum.DiscussionOur data suggests that exercise may contribute to B16F10 tumor cell death in response to chemotherapy by modulating ceramide metabolism toward a pro‐apoptotic ceramide/sphingosine‐1‐phosphate balance. However, these results were not consistent in BP tumors. This raises the important finding that exercise can have different effects between tumors of different patient or mouse origin, even with the same diagnosis (i.e. B16F10 vs BP melanoma models). Future work to determine the extent to which exercise regulates ceramide‐sphingolipid signaling in other melanoma tumor models, and in patients, is necessary. Future work will also determine whether exercise‐induced changes in ceramide levels are responsible for the previously reported exercise‐induced increased chemotherapy efficacy against melanoma tumors.

Targeting CISH enhances natural cytotoxicity receptor signaling and reduces NK cell exhaustion to improve solid tumor immunity

BackgroundThe success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK)...

Crosstalk between β2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation.

Adrenergic receptors (AR) belong to the G protein-coupled receptor superfamily and regulate migration and proliferation in various cell types. The objective of this study was to evaluate whether β-AR stimulation affects the antiproliferative action of α2-AR agonists on B16F10 cells and, if so, to determine the relative contribution of β-AR subtypes. Using pharmacological approaches, evaluation of Ki-67 expression by flow cytometry and luciferase-based cAMP assay, we found that treatment with isoproterenol, a β-AR agonist, increased cAMP levels in B16F10 melanoma cells without affecting cell proliferation. Propranolol inhibited the cAMP response to isoproterenol. In addition, stimulation of α2-ARs with agonists such as clonidine, a well-known antihypertensive drug, decreased cancer cell proliferation. This effect on cell proliferation was suppressed by treatment with isoproterenol. In turn, the suppressive effects of isoproterenol were abolished by the treatment with either ICI 118,551, a β2-AR antagonist, or propranolol, suggesting that isoproterenol effects are mainly mediated by the β2-AR stimulation. We conclude that the crosstalk between the β2-AR and α2-AR signaling pathways regulates the proliferative activity of B16F10 cells and may therefore represent a therapeutic target for melanoma therapy.

Sodium alginate nanofibers loaded Terminalia catappa scaffold regulates intrinsic apoptosis signaling in skin melanoma cancer

Drug delivery by nanofibrous scaffold is a promising approach for treating cancer disease. In this study, bioactive compounds were extracted from the medicinal plant Terminalia catappa and loaded into sodium alginate (SA) polymer to prepare a bioactive electrospun nanofibers. Initially, SA polymeric fibers alone was prepared by electrospinning approach and characterized by FT-IR spectroscopy, XRD and SEM techniques. Further, the antioxidant properties of SA, TC, and TC loaded SA (SA@TC) were evaluated using DPPH assay and the results indicated that the SA@TC has good antioxidant activity than unloaded carrier. Further, the cytotoxic activity showed the SA and SA@TC has excellent inhibiting activity of human skin melanoma B16F10 cells. As prepared SA@TC scaffold increased the ROS generation in the mitochondria and damaged the nuclear of B16F10 cells. Further, the SA@TC scaffold increases the expression of apoptosis marker genes and proteins such as Bax, Cyt C, p21, p53, caspase 9 & 3. The LC-MS analysis indicated the presence of ten anticancer compounds in T. cattapa extract that could influence the anticancer activity. Hence, the prepared SA@TC scaffolds having the ability to inhibit the cancer cells through intrinsic apoptosis signaling and can be potential candidates for skin cancer treatment.

Luteolin binds Src, promotes STAT3 protein ubiquitination and exerts anti-melanoma effects in cell and mouse models

Signal transducer and activator of transcription 3 (STAT3) has been proposed as a target for melanoma prevention. Luteolin, a bioactive flavonoid abundant inmedicinal herbs, has been reported to have anti-melanoma activity in vitro. However, its in vivo anti-melanoma effects and underlying mechanisms have not been fully elucidated. In this study, ten cell lines and two mouse models (B16F10 allograft and A375 xenograft models) were used for assessing the in vitro and in vivo anti-melanoma effects of luteolin. A STAT3 over-activated stable A375 cell line was used to determine the contribution of STAT3 signaling in luteolin’s anti-melanoma effects. Results showed that luteolin dose-dependently reduced viability of melanoma cells. Luteolin also induced apoptosis in, and suppressed migration and invasion of, A375 and B16F10 melanoma cells. Mechanistically, luteolin inhibited phosphorylation of STAT3 and Src (an upstream kinase of STAT3), accelerated ubiquitin–proteasome pathway-mediated STAT3 degradation, and downregulated the expression of STAT3-targeted genes involved in cell survival and invasion in melanoma cells. Molecular modelling and surface plasmon resonance imaging showed that luteolin stably bound to the protein kinase domain of Src. Animal studies demonstrated that prophylactic administration of luteolin restrained melanoma growth and Src/STAT3 signaling in both A375 and B16F10 melanoma-bearing mice. Moreover, luteolin’s anti-melanoma effects were diminished by STAT3 over-activation in A375 cells. Our findings indicate that luteolin inhibits STAT3 signaling by suppressing STAT3 activation and promoting STAT3 protein degradation in melanoma cells, thereby exhibiting anti-melanoma effects. This study provides further pharmacological groundwork for developing luteolin as a chemopreventive agent against melanoma.

Mechanistic Insights into the Ameliorating Effect of Melanogenesis of Psoralen Derivatives in B16F10 Melanoma Cells.

The objectives of this study were to investigate the melanogenetic potential of the psoralen derivatives 5-hydroxypsoralen, 5-methoxypsoralen, 8-hydroxypsoralen, 8-methoxypsoralen, and 5,8-dimethoxypsoralen in B16F10 melanoma cells. The results indicated that melanin production is significantly stimulated in B16F10 melanoma cells with 5-methoxypsoralen, 8-methoxypsoralen, and 5,8-dimethoxypsoralen, especially for 5-methoxypsoralen (bergapten), as reported previously. In addition, Western blot results showed that the protein levels of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2) increase after bergapten treatment for the first time. The results also showed that bergapten promotes the phosphorylation of Akt at Ser 473 and glycogen synthase kinase-3β at Ser 9. Moreover, bergapten increased the content of β-catenin in the cell cytoplasm and nucleus by reducing the phosphorylated β-catenin (p-β-catenin) content. The results also indicated that bergapten regulates melanogenesis by upregulating the phosphorylation of p38 and JNK-mitogen-activated protein kinase. Taken together, these findings suggest that the regulation of melanogenesis by bergapten may be mediated by the β-catenin and MAPK signaling pathways and that bergapten might provide new insights into the pathogenesis of pigmented diseases.

Characterization of Tyrosinase Inhibitors in Dryopteris crassirhizoma Rhizome Using a Combination of High-Speed Counter-Current Chromatography, Affinity-Based Ultrafiltration, and Liquid Chromatography-Tandem Mass Spectrometry.

Dryopteris crassirhizoma rhizome (DCR) inhibits melanin production in B16F10 melanoma cells and tyrosinase activity. The melanin content and tyrosinase activity of DCR-treated zebrafish embryos were determined to evaluate the in vivo inhibitory effect of DCR on melanogenesis. Moreover, an off-line hyphenated method combining the high-speed counter-current chromatography, affinity-based ultrafiltration, and liquid chromatography–tandem mass spectrometry was used to identify and characterize the DCR compounds with tyrosinase inhibitory activity. Our results indicated that DCR significantly decreased the melanin content and tyrosinase activity in zebrafish embryos in a dose-dependent manner; moreover, 22 compounds in DCR presented tyrosinase inhibitory activity. In silico molecular docking prediction data indicated that the 22 compounds in DCR can form stable conformations in the active site pocket of tyrosinase.

Silibinin promotes melanogenesis through the PKA and p38 MAPK signaling pathways in melanoma cells.

Silibinin is a flavonolignan isolated from milk thistle (Silybum marianum). Silibinin has been reported to possess multiple biological activities; however, its effect on melanogenesis remains unclear. This study investigated the effect of silibinin on melanogenesis in melanoma cells and the associated molecular mechanism. Our findings demonstrated that silibinin markedly increased melanin content in murine B16-F1 and human HMV-II melanoma cells. Silibinin activated intracellular tyrosinase activity and expression of tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, and microphthalmia-associated transcription factor (MITF). Furthermore, silibinin enhanced the phosphorylation of cyclic AMP-responsive element-binding protein (CREB), protein kinase A (PKA), and p38 mitogen-activated protein kinase (MAPK) but not of Akt and extracellular signal-regulated kinase (ERK). The specific PKA (H-89) and p38 (SB203580) inhibitors significantly attenuated silibinin-mediated melanin synthesis. These results suggest that silibinin is an effective stimulator of melanogenesis through upregulation of the protein expression of melanogenic enzymes activated by the PKA and p38 pathways, leading to CREB phosphorylation and MITF expression. Therefore, silibinin may have potential for use in the treatment of hypopigmentation disorders.

RIG-I acts as a tumor suppressor in melanoma via regulating the activation of the MKK/p38MAPK signaling pathway

Studies have indicated that RIG-I may act as a tumor suppressor and participate in the tumorigenesis of some malignant diseases. However, RIG-I induces distinct cellular responses via different downstream signaling pathways depending on the cell type. To investigate the biological function and underlying molecular mechanism of RIG-I in the tumorigenesis of melanoma, we constructed RIG-I knockout, RIG-I-overexpressing B16-F10 and RIG-I knockdown A375 melanoma cell lines, and analyzed the RIG-I-mediated change in the biological behavior of tumor cells in spontaneous and poly (I:C)-induced RIG-I activation. Cell proliferation, cell cycling, apoptosis and migration were detected by CCK-8 assay, BrdU incorporation assay, Annexin V–PI staining assay and Transwell assay, respectively. In vivo tumorigenicity was evaluated by tumor xenograft growth in nude mice and subsequently by Ki67 staining and TUNEL assays. Furthermore, Western blotting was utilized to explore the underlying mechanism of RIG-I in melanoma cells. Our data showed that RIG-I promotes apoptosis and inhibits proliferation by G1 phase cell cycle arrest in the melanoma cell lines. Mechanistically, RIG-I induced the phosphorylation of p38 MAPK and MAPK kinases MKK3 and MKK4. In conclusion, the current study demonstrated that RIG-I suppressed the development of melanoma by regulating the activity of the MKK/p38 MAPK signaling pathway, which is relevant to research on novel therapeutic targets for this malignant disease.

Combined Action of Hyper-Harmonized Hydroxylated Fullerene Water Complex and Hyperpolarized Light Leads to Melanoma Cell Reprogramming In Vitro.

(1) Background: Their unique structure and electron deficiency have brought fullerenes into the focus of research in many fields, including medicine. The hyper-harmonized hydroxylated fullerene water complex (3HFWC) formulation has solved the limitations of the poor solubility and bioavailability of fullerenes. To achieve better antitumor activity, 3HFWC was combined with short-term irradiation of cells with hyperpolarized light (HPL) generated by the application of a nanophotonic fullerene filter in a Bioptron® device. The benefits of HPL were confirmed in the microcirculation, wound healing and immunological function. (2) Methods: B16, B16-F10 and A375 melanoma cells were exposed to a wide spectrum of 3HFWC doses and to a single short-term HPL irradiation. (3) Results: Apart from the differences in the redox status and level of invasiveness, the effects of the treatments were quite similar. Decreased viability, morphological alteration, signs of melanocytic differentiation and cellular senescence were observed upon the successful internalization of the nanoquantum substance. (4) Conclusions: Overall, 3HFWC/HPL promoted melanoma cell reprogramming toward a normal phenotype.

Gut Microbial Shifts Indicate Melanoma Presence and Bacterial Interactions in a Murine Model.

Through a multitude of studies, the gut microbiota has been recognized as a significant influencer of both homeostasis and pathophysiology. Certain microbial taxa can even affect treatments such as cancer immunotherapies, including the immune checkpoint blockade. These taxa can impact such processes both individually as well as collectively through mechanisms from quorum sensing to metabolite production. Due to this overarching presence of the gut microbiota in many physiological processes distal to the GI tract, we hypothesized that mice bearing tumors at extraintestinal sites would display a distinct intestinal microbial signature from non-tumor-bearing mice, and that such a signature would involve taxa that collectively shift with tumor presence. Microbial OTUs were determined from 16S rRNA genes isolated from the fecal samples of C57BL/6 mice challenged with either B16-F10 melanoma cells or PBS control and analyzed using QIIME. Relative proportions of bacteria were determined for each mouse and, using machine-learning approaches, significantly altered taxa and co-occurrence patterns between tumor- and non-tumor-bearing mice were found. Mice with a tumor had elevated proportions of Ruminococcaceae, Peptococcaceae.g_rc4.4, and Christensenellaceae, as well as significant information gains and ReliefF weights for Bacteroidales.f__S24.7, Ruminococcaceae, Clostridiales, and Erysipelotrichaceae. Bacteroidales.f__S24.7, Ruminococcaceae, and Clostridiales were also implicated through shifting co-occurrences and PCA values. Using these seven taxa as a melanoma signature, a neural network reached an 80% tumor detection accuracy in a 10-fold stratified random sampling validation. These results indicated gut microbial proportions as a biosensor for tumor detection, and that shifting co-occurrences could be used to reveal relevant taxa.

CD47-SIRPα Checkpoint Disruption in Metastases Requires Tumor-Targeting Antibody for Molecular and Engineered Macrophage Therapies.

Simple SummaryImmunotherapies offer broad potential to treat many diseases, including cancer. Macrophages are highly abundant immune cells, but are generally inhibited from eliminating tumor cells by ‘self’ signaling. Blocking this macrophage signaling checkpoint with antibodies has gone through clinical trials, which show that efficacy remains elusive especially for solid tumors. Here we show that CRISPR-based deletion of this inhibitory checkpoint is also insufficient to suppress solid metastatic tumors unless combined with a pro-phagocytic tumor-opsonizing antibody. Our tumor model is immunocompetent and resistant to T cell checkpoint blockade, which are typical characteristics of human cancer. We further demonstrate an equally effective cell therapy with antibody-engineered macrophages, and the approach also suppresses model metastatic human tumors.The macrophage checkpoint interaction CD47-SIRPα is an emerging target for cancer therapy, but clinical trials of monoclonal anti-CD47 show efficacy only in liquid tumors when combined with tumor-opsonizing IgG. Here, in challenging metastatic solid tumors, CD47 deletion shows no effect on tumor growth unless combined with otherwise ineffective tumor-opsonization, and we likewise show wild-type metastases are suppressed by SIRPα-blocked macrophages plus tumor-opsonization. Lung tumor nodules of syngeneic B16F10 melanoma cells with CD47 deletion show opsonization drives macrophage phagocytosis of B16F10s, consistent with growth versus phagocytosis calculus for exponential suppression of cancer. Wild-type CD47 levels on metastases in lungs of immunocompetent mice and on human metastases in livers of immunodeficient mice show that systemic injection of antibody-engineered macrophages also suppresses growth. Such in vivo functionality can be modulated by particle pre-loading of the macrophages. Thus, even though CD47-SIRPα disruption and tumor-opsonizing IgG are separately ineffective against established metastatic solid tumors, their combination in molecular and cellular therapies prolongs survival.

Prooxidative effect of cardols is involved in their cytotoxic activity against murine B16–F10 melanoma cells

Cardols are resorcinolic lipids, available in many natural sources including cashew nut, pistachio, macadamia, and mango. Despite of several beneficial biological activities of cardols, cytotoxic activities of cardols are not fully understood. In preliminary studies, 5[8(Z),11(Z),14-pentadecatrienyl]resorcinol, known as cardol (C15:3) was found to inhibit tyrosinase-catalyzed melanin formation in cell-free system. In the case of cultured cell analysis, cardol (C15:3) showed intense cytotoxicity but not anti-melanogenic activity against B16–F10 melanoma cells. Subsequently, cardol (C10:0) and cardol (C5:0), containing shorter alkyl side chain, exhibited inferior cytotoxicity compared to cardol (C15:3). The cytotoxicity via cardol (C15:3) was reversed by the addition of antioxidants, indicating that intracellular prooxidative activity was involved. Furthermore, cardol (C15:3) produced significant levels of reactive oxygen species (ROS) while cardol (C5:0) generated lesser ROS levels. Our findings suggest the cytotoxic activity of cardols is their prooxidative effect depending on the length of alkyl side chain.

Elastase inhibitory activity of quinoline Analogues: Synthesis, kinetic mechanism, cytotoxicity, chemoinformatics and molecular docking studies

Herein, we have synthesized quinoline united various Schiff base derivatives (Q1-Q13) and systematically characterized them using diverse analytical practices such as 1H NMR, 13C NMR, FT-IR and LC-MS respectively. All of the target compounds that have been synthesized were tested for elastase inhibition, and the findings were compared to the standard drug oleanolic acid. Among the entire series, compound Q11 (IC50 = 0.897 ± 0.015 µM) exhibit most promising elastase inhibitory activity than oleanolic acid (Standard) having an IC50 value of 13.426 ± 0.015 µM. Also, the utmost effectivecompound Q11 was used for kinetic mechanism investigation based on in-vitro data, from which it has been concluded that compound Q11 inhibits elastase competitively. Furthermore, utilizing the MTT test approach, the most effective compounds were assessed for cytotoxicity on B16F10 melanoma cells. From the cytotoxicity experiment, the most potent compound did not display any hazardous response against B16F10 melanoma cells despite being treated at high concentrations. Additionally, the molecular docking study was settled to govern the binding interaction pattern among an enzyme and inhibitors.

Caryocar brasiliense Camb. fruit peel butanolic fraction induces antiproliferative effects against murine melanoma cell line

BackgroundCutaneous malignant melanoma is a skin cancer type highly resistant to standard cancer therapies. Natural compounds have been reported as important sources for the creation of new drugs for cancer treatment. Caryocar brasiliense Camb., popularly known as Pequi, is often used in Brazilian folk medicine, with anticancer effects reported. Objectives: The present study evaluated the antiproliferative activity of the crude extract butanolic fraction of the C. brasiliense Camb. peel on the B16F10 cell linage MethodC. brasiliense peel fraction was analyzed by Gas Chromatography coupled to massa spectra, and its biological effects were evaluated on the melanoma cell line B16F10. ResultsThe chromatography analysis of the butanolic fraction of the C. brasiliense peel fraction identified a majority presence of gallic acid and sarothrin compounds. These compounds might have been responsible for an antiproliferative effect on B16F10, with the inhibitory concentration (IC50) equal to 390.9 µg/mL (24 h) and 226.4 µg/mL (48 h) after treatments. Our results revealed that cell death assay via bromide and acridine orange tests indicated an increase in cell death observed after 24 h treatment with the pequi fraction at 250 μg/mL (p < 0.05) and 500 μg/mL (p < 0.01). In addition, a significant increase in cell death at 250 μg/mL (p < 0.01) and 500 μg/mL (p < 0.0001) occurred after 48 h. Furthermore, a significant reduction in migratory activity in cells treated at 250 μg/mL (p < 0.05) and 500 μg/mL (p < 0.01) occurred and was enhanced by the 48 h treatment (p < 0.001). ConclusionsThe present study is the first to demonstrate the use of "Pequi" residual by product as a potential reservoir of bioactive compounds with antiproliferative activity on B16F10 melanoma cells.

New Poly(N-isopropylacrylamide-butylacrylate) Copolymer Biointerfaces and Their Characteristic Influence on Cell Behavior In Vitro.

Designing and obtaining new synthetic smart biointerfaces with specific and controlled characteristics relevant for applications in biomedical and bioengineering domains represents one of the main challenges in these fields. In this work, Matrix-Assisted Pulsed Laser Evaporation (MAPLE) is used to obtain synthetic biointerfaces of poly(N-isopropyl acrylamide-butyl acrylate) p(NIPAM-BA) copolymer with different characteristics (i.e., roughness, porosity, wettability), and their effect on normal HEK 293 T and murine melanoma B16-F1 cells is studied. For this, the influence of various solvents (chloroform, dimethylsulfoxide, water) and fluence variation (250–450 mJ/cm2) on the morphological, roughness, wettability, and physico–chemical characteristics of the coatings are evaluated by atomic force microscopy, scanning electron microscopy, contact angle measurements, Fourier-transform-IR spectroscopy, and X-ray photoelectron spectroscopy. Coatings obtained by the spin coating method are used for reference. No significant alteration in the chemistry of the surfaces is observed for the coatings obtained by both methods. All p(NIPAM-BA) coatings show hydrophilic character, with the exception of those obtained with chloroform at 250 mJ/cm2. The surface morphology is shown to depend on both solvent type and laser fluence and it ranges from smooth surfaces to rough and porous ones. Physico–chemical and biological analysis reveal that the MAPLE deposition method with fluences of 350–450 mJ/cm2 when using DMSO solvent is more appropriate for bioengineering applications due to the surface characteristics (i.e., pore presence) and to the good compatibility with normal cells and cytotoxicity against melanoma cells.

Carbon Ion Induces Cell Death and G2/M Arrest Through pRb/E2F1Chk2/Cdc2 Signaling Pathway in X-ray Resistant B16F10 Melanoma Cells.

To explore the effect of high-LET carbon ion (C-ion) radiation on malignant melanoma, we systematically compared the radiobiological effects of C-ion with that of X-rays in B16F10 melanoma cells. Results showed that C-ion radiation statistically inhibited clonogenic survival capacity of B16F10 melanoma cells. The RBE was 3.7 at D10 levels, meaning 1.0 Gy C-ion should cause the same biological effect as ≥ 3.0 Gy X-rays. In addition, we also observed a stronger proliferation-inhibiting and higher ratio of cell apoptosis and necrosis in B16F10 cells treated with C-ion than X-rays. Moreover, C-ion radiation exhibited stronger and long-lasting G2/M arrest than X-rays. As an underlying mechanism, we speculated that C-ion radiation-induced G2/M block through activating pRb/E2F1/Chk2 pathway. With these results, we highlighted the potential of C-ion in treatment of cutaneous melanoma. Further, in vitro experiments as well as clinical trials are needed to further evaluate the effect of carbon ion radiotherapy in melanoma.