B16F10 Cell Lines Research Articles

Development of 131I-ixolaris as a theranostic agent: metastatic melanoma preclinical studies.

Tissue factor (TF), a blood coagulation protein, plays an important role in tumor growth, invasion, and metastasis. Ixolaris, a tick-derived non-immunogenic molecule that binds to TF, has demonstrated in vivo inhibitory effect on murine models of melanoma, including primary growth and metastasis. This work aimed to: I) develop an efficient and stable labeling technique of ixolaris with Iodine-131(131I); II) compare the biodistribution of 131I and 131I-ixolaris in tumor-free and melanoma-bearing mice; III) evaluate whether 131I-ixolaris could serve as an antimetastatic agent. Ixolaris radioiodination was performed using iodogen, followed by liquid paper chromatography. Labeling stability and anticoagulant activity were measured. Imaging studies were performed after intravenous administration of free 131I or 131I-ixolaris in a murine melanoma model employing the B16-F10 cell line. Animals were divided in three experimental groups: the first experimental group, D0, received a single-dose of 9.25MBq of 131I-ixolaris at the same day the animals were inoculated with melanoma cells. In the second group, D15, a single-dose of 9.25MBq of 131I-ixolaris or free 131I was applied into mice on the fifteenth day after the tumor induction. The third group, D1-D15, received two therapeutic doses of 9.25MBq of 131I-ixolaris or 131I. In vitro studies demonstrated that 131I-ixolaris is stable for up to 24h and retains its inhibitory activity on blood coagulation. Biodistribution analysis and metastasis assays showed that all treatment regimens with 131I-ixolaris were effective, being the double-treatment (D1/D15) the most effective one. Remarkably, treatment with free 131I showed no anti-metastatic effect. 131I-ixolaris is a promising theranostic agent for metastatic melanoma.

Cytotoxic androstane derivatives from Sarcococca ruscifolia

Sarcorusones A–D (1–4), four new androstane (C19-steroid) derivatives were characterized from Sarcococca ruscifolia along with five known compounds. Their structures were elucidated on the basis of extensive MS and NMR spectroscopic analysis. All the new structures share common 14-hydroxyl and 17-ketone functional groups, and compounds 2–4 feature a seneciamide group connecting to C-3 position. The inhibitory activities of all the isolates against melanoma cell B16F10 and lung cancer cell H1299 were evaluated, and compounds 2, 3, 5, and 6 exhibited moderate cytotoxic activities against B16F10 and H1299 cell lines with IC50 values 2.7–8.0 μM.

Improving the function of NK cells in an anti-tumour model using fluorescent-based chemokine sorting

Background & Aim NK cells are potent antitumor leukocytes that are critical for controlling the development of tumours. NK cells directly target tumour cells for destruction and also release inflammatory mediators such as IFNy. The role of NK cells in tumour immunity makes them an encouraging target for anti-tumour immunotherapy. Recent reports suggest that increased expression of CCR2 on NK cells enhances migration into metastatic tumour lesions, resulting in increased tumour destruction. By utilising a novel GMP-compatible fluorescence-based chemokine sorting method to isolate CCR2+ NK cells, we have investigated the effects of CCR2+ NK cell infusion in both metastatic and solid tumour models using the B16F10 cell line. Methods, Results & Conclusion We have generated evidence that CCR2+ NK cell infusion 1 day post tumour induction significantly reduces the number and size of B16F10 lesions in the lung when compared to an infusion of CCR2- NK cells and that these NK cells persist in vivo for at least 2 weeks post transfer. Additionally, CCR2- NK cell infusion does not have a significant effect on tumour size or number when compared to control animals. We anticipate a similar phenotype 7 days post tumour induction and this is under investigation . By using CCR2 knock out animals as donors, we have also confirmed that CCR2 expression is paramount to the observed phenotype. We also predict that adoptive transfer of CCR2+ NK cells into mice bearing a solid tumour will result in a significant reduction in tumour growth and increase survival. We are currently investigating this hypothesis in addition to the effect of NK cell adoptive transfer on the CD8 T cells response in both metastatic and solid tumour models, as NK cells have been previously shown to have a direct impact on T cell tumour responses. In addition to murine studies we have begun to optimise a protocol for the expansion of NK cells from human peripheral blood while increasing CCR2 expression. NK cells expanded via this protocol retain their phenotype and killing capacity and increase CCR2 expression from 3% to 90% of cells and we are continuing investigate if human NK cells behave like their murine equivalents. We have optimised clinical grade cell sorting for both murine and human NK cells and we are currently testing various culture methods to further optimise NK cell expansion. Based on these data, we suggest that chemokine sorted NK cells are a potential therapeutic option that could be considered for cancer immunotherapy

Interleukin-33 pretreatment promotes metastatic growth of murine melanoma by reducing the cytotoxic capacity of CD8+ T cells and enhancing regulatory T cells.

Interleukin-33 (IL-33) regulates innate and acquired immune response to pathogens, self-antigens and tumors. IL-33 effects on tumors depend on the dose and mode of administration along with the type of malignancy. We studied the effects of IL-33 on the development of primary and metastatic melanoma induced by B16-F1 cell line in C57BL/6 mice. Intraperitoneally applied IL-33 restricts primary tumor growth. When administered intranasally 3days prior to the intravenous injection of the tumor cells, IL-33 promoted growth of B16-F1 melanoma metastases, while B16-F10 gave massive metastases independently of IL-33. To mimic natural dissemination, we next used a limited number (5 × 104) of B16-F1 cells intravenously followed by application of IL-33 intraperitoneally. IL-33 increased the size of metastases (10.96 ± 3.96mm2) when compared to the control group (0.86 ± 0.39mm2), without changing incidence and number of metastases. IL-33 increased expression of ST2 on both tumor and immune cells in metastases. Also, IL-33 enhanced eosinophils and anti-tumor NK cells in the lung. The striking finding was reduced cytotoxicity of CD8+ T cells derived from metastatic lung of IL-33 injected mice. IL-33 reduced the percentage of TNF-α+ and IFN-γ+ CD8+ T cells while increasing the frequency of CD8+ T cells that express inhibitory molecules (PD-1, KLRG-1 and CTLA-4). There was a significant accumulation of CD11b+Gr-1+ myeloid suppressor cells and FoxP3+, IL-10+ and CTLA-4+ regulatory T cells in the metastatic lung of IL-33 injected mice. The relevance of IL-33 for melanoma metastases was also documented in a significantly increased level of serum IL-33 in stage III melanoma patients.

Identification of very small cancer stem cells expressing hallmarks of pluripotency in B16F10 melanoma cells and their reoccurrence in B16F10-derived clones

Melanoma is characterized by high heterogeneity and plasticity, most likely due to the presence of mutated melanocyte stem cells or immature progenitor cells in the skin that serves as precursors to melanoma. In the present study, for the first time, we identified rare cells in the murine melanoma B16F10, and human A2058 and SK-MEL-28 cell lines that express pluripotency markers, including Oct4, Nanog, Sox2 and a marker of melanoma cancer cells (ALDH1/2). These cells are very small with round morphology and they grow onto melanoma cells, thereby demonstrating feeder layer dependence similar to that of other pluripotent cells. These cells underwent self-renewal, symmetric and asymmetric division. We called these cells murine very small cancer stem cells (VSCSC). VSCSC were also found in B16F10-derived clones after 3–5 consecutive passages, where they occur as single cells or as small colonies, nevertheless, always using melanoma cells as feeders. These cells formed melanospheres enriched with Oct4-and ALDH1/2-positive cells. We also evaluated the possible effect of VSCSC that presented in the parental cell line (B16F10) and in clones based on their functional characteristics. We found that VCSCS present in the B16F10 cell line reappearing in their clones were required for continuous tumor growth and were responsible for melanoma cell heterogeneity and plasticity rather than directly affecting functional characteristics of melanoma cells. Our data, together with those of previous reports suggested the existence of melanoma-competent melanocyte stem cells, which corroborate the hypothesis of the existence of tumor-initiating cells and cancer stem cell hierarchies, at least in melanoma.

In Vitro Inhibition of Melanoma (B16f10) Viability and Colonization through Combining Metformin and Dacarbazine

Background: Dacarbazine is considered as a standard treatment for melanoma, but resistance to anticancer therapy is a major cause of cancer stem cells invasion. In vitro assays have shown that metformin interferes with cell viability, proliferation, and apoptosis. Method: Melanoma cell line B16f10 was treated with dacarbazine IC50, metformin in different doses (0.5, 2 and 8 mM) and combination therapy. The influence of treating and cell viability was determined with MTT assay, and the effect of treat on colonization was quantified. Changes in cleaved PARP were investigated using immunoblotting. The cytotoxicity effect of Dacarbazine was further analyzed. Result: Metformin induced cytotoxicity on B16-F10 cells; cell viability, determined at various time intervals (24 and 48 h) and in the presence of different drug concentrations (≅0.7μM), was reduced by ~50% following 24 h. The proliferation rate was evaluated over 24-48 hours and 12 days using varying subcytotoxic and cytotoxic concentrations of metformin (2-8μM), which was reduced in a dose-dependent manner. Resistance cells resulted in slender spindles and better colonization. Finally, metformin decreased the cytotoxicity of dacarbazine and increased apoptosis. Conclusion: A study with B16-F10 cells showed that the drug combination induces significantly more apoptosis compared with when each drug is individually used. B16F10 was the most sensitive and resistant at a normal dose of metformin and dacarbazine, which is a very encouraging result with regards to the possibility of metformin becoming a new tool for melanoma research and treatment.

Anti-proliferative profile of Anacardium occidentale polysaccharide and characterization by AFM

This paper explores the application of cashew gum (CG) as an in vitro antiproliferative, firstly by isolating and characterizing the gum using elemental analysis, gel-permeation chromatography, nuclear magnetic resonance (NMR) and atomic force microscopy (AFM). The molar mass of isolated CG was in the order of 103–104 g/mol, with small protein traces present. Polymer characterization by NMR identified key signals correlating to galactose, glucose, rhamnose and acid-related groups. Three distinct conformational stages were observed by AFM. The impact of CG on cell morphology and viability with both tumor and non-tumor cell lines was studied by AFM and 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay respectively. Antiproliferative activity was confirmed for HCT116 (colorectal carcinoma), B16F10 (melanoma) and HL60 (promyelocytic leukemia) cancer cell lines. A change in cell morphology was demonstrated as an increased surface roughness for HL60. Considering that a CG does not exhibit cytotoxicity to non-tumor lines, it can be seen that the CG shows selectivity for tumor cells and can be a promising biomaterial for future studies.

Abstract A79: Inhibition of p53-MDM2 protein interaction reduces tumor growth in a mouse melanoma model

Abstract Tumor suppressor protein p53 is mutated in close to 50% of human tumors. Under steady state conditions, the two E3 ligases MDM2 and MDM4 interact with and inhibit the transcriptional activity of p53. Inhibition of p53-MDM2/4 interaction to reactivate p53 has therefore been considered a therapeutic strategy. In this study, we tested p53-MDM2 protein-protein interaction inhibition using the small molecule AMG 232, which is currently being tested in phase 1b/2a clinical trials. In vitro, AMG 232 induced a significant, p53-dependent growth arrest in the mouse melanoma cell line B16-F10. Using mass spectrometry-based proteomics, we identified differential protein expression patterns following AMG 232 treatment of B16-F10 melanoma cells. In vivo, the growth of B16-F10 melanoma cells implanted in WT C57BL/6 mice was significantly reduced by AMG 232 treatment. Our data demonstrate that AMG 232 induces a p53-dependent tumor growth arrest in an immunocompetent mouse model, and we are currently testing whether AMG232 can synergize with checkpoint immunotherapy. Citation Format: Katrine Ingelshed, Danai Lianoudaki, Dilraj Lama, Silke Sohn, Nicolas Fritz, Long Jiang, Fredrik Wermeling, Mikael Karlsson, David P. Lane, Saikiran K. Sedimbi. Inhibition of p53-MDM2 protein interaction reduces tumor growth in a mouse melanoma model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A79.

Paclitaxel-Loaded Macrophage Membrane Camouflaged Albumin Nanoparticles for Targeted Cancer Therapy.

BackgroundMelanoma is the most common symptom of aggressive skin cancer, and it has become a serious health concern worldwide in recent years. The metastasis rate of malignant melanoma remains high, and it is highly difficult to cure with the currently available treatment options. Effective yet safe therapeutic options are still lacking. Alternative treatment options are in great demand to improve the therapeutic outcome against advanced melanoma. This study aimed to develop albumin nanoparticles (ANPs) coated with macrophage plasma membranes (RANPs) loaded with paclitaxel (PTX) to achieve targeted therapy against malignant melanoma.MethodsMembrane derivations were achieved by using a combination of hypotonic lysis, mechanical membrane fragmentation, and differential centrifugation to empty the harvested cells of their intracellular contents. The collected membrane was then physically extruded through a 400 nm porous polycarbonate membrane to form macrophage cell membrane vesicles. Albumin nanoparticles were prepared through a well-studied nanoprecipitation process. At last, the two components were then coextruded through a 200 nm porous polycarbonate membrane.ResultsUsing paclitaxel as the model drug, PTX-loaded RANPs displayed significantly enhanced cytotoxicity and apoptosis rates compared to albumin nanoparticles without membrane coating in the murine melanoma cell line B16F10. RANPs also exhibited significantly higher internalization efficiency in B16F10 cells than albumin nanoparticles without a membrane coating. Next, a B16F10 tumor xenograft mouse model was established to explore the biodistribution profiles of RANPs, which showed prolonged blood circulation and selective accumulation at the tumor site. PTX-loaded RANPs also demonstrated greatly improved antitumor efficacy in B16F10 tumor-bearing mouse xenografts.ConclusionAlbumin-based nanoscale delivery systems coated with macrophage plasma membranes offer a highly promising approach to achieve tumor-targeted therapy following systemic administration.

Preventive effect of Lactobacillus reuteri on melanoma

Lactobacillus is one of the bacteria strains which has been widely used in human life, has valuable probiotic properties. In the present study, L. reuteri FLRE5K1 was isolated from healthy mouse kidney and tested for its anti-melanoma activity in cell assays and animal models. The results showed that L. reuteri FLRE5K1 could significantly reduce the incidence of melanoma from 70 % to 30 % and significantly prolonged the survival of tumor-bearing mice. Despite of its significant effect on melanoma prevention, the tumor mass remained uneliminated. The alanine aminotransferase (ALT) level remained normal in tumor-bearing mice with intragastric administration of L. reuteri FLRE5K1, indicating that L. reuteri FLRE5K1 could prevent melanoma cells from damaging the liver. Cell assays showed that melanoma cells were inhibited by serum from mice with intragastric administration of L. reuteri FLRE5K1, but not by the bacteria itself. Further examination revealed that the levels of TNF-α and INF-γ in this serum were significantly increased. In summary, it was speculated that L. reuteri FLRE5K1 could stimulate the body to produce anti-cancer cytokines, inhibit the migration and colonization of melanoma cell line B16-F10, so as to prevent the onset of melanoma and prolong the survival of mice. The experiment was a useful complement to functional studies of endogenous probiotics and provided theoretical data support for the development of new microbioecologics.

Characterization and anti-tumor activity of saponin-rich fractions of South Korean sea cucumbers (Apostichopus japonicus).

In this study, the saponin-rich fractions of five individual (two Red and three Black) sea cucumbers (Apostichopus japonicus) in South Korea were investigated for their antiproliferative effect against HL-60, B16F10, MCF-7, and Hep3B tumor cell lines. The red sea cucumber saponin-rich fraction (SSC) from Jeju Island (JRe) decreased the growth of HL-60 with an IC50 value of 23.55 ± 3.40μg/mL, which represented the strongest anticancer activity among the extracts. Further, SSC downregulated B-cell lymphoma extra-large (Bcl-xL), while upregulating, to different degrees, Bcl-2-associated X protein (Bax), caspase-9, caspase-3, PARP cleavage, and apoptotic bodies in cancer cells. Evidence for SSC inducing apoptosis via the mitochondria-mediated pathway was found. The contents of SSCs were determined using ultra high-performance liquid chromatography coupled with a quadrupole orbitrap mass spectrometry to comparatively evaluate the regional influence. In West Sea, the total SSC content of A. japonicus was 15.5mg/g, representing the highest content, while A. japonicus in the South Sea yielded the lowest content at 8mg/g. The major saponin constituent in SSC was identified as Holotoxin A1, which may the anti-tumor compound in A. japonicus.

Structure, DFT studies, magnetism and biological activity of bis[(µ-azido)-chloro-(1,10-phenanthroline)-copper(II)] complex

The dinuclear complex Bis[(µ-azido)-chloro-(1,10-phenanthroline)-copper(II)] (1) was synthesized, and characterized by X-ray. Complex (1) crystallizes in the monoclinic system, it consists of centrosymmetric [CuCl(phen)µ-N3]2 dimers bridged by azide groups. The phenanthroline ligand, chloride ion, and ƞ-N of equatorial bridging azide ligand are coplanar and the square pyramidal copper ion is displaced slightly out of this basal plane, toward the axial bridging azide. The presence of H-bonds, CH-π and π-π interactions form layers of type ABAB within the supramolecular structure of (1). The magnetic susceptibility of (1) versus temperature data showed a weak antiferromagnetic coupling between Cu(II) ions. The best fitting parameters were for g = 2.01 ± 0.01 and J = −0.55 ± 0.01 cm−1. Thermal analysis of (1) exhibited an exothermic peak due to decomposition of azide ligands. The calculated HOMO–LUMO gap from DFT is 0.03098 a.u. (0.08430 eV).Besides, complex (1) exhibited potent chemotherapeutic potential with strong activity after 48 h against MDA-MB-32 (breast adenocarcinoma), HT-29 (colon adenocarcinoma), A549 (lung adenocarcinoma), SF (astrocytoma) and B16F10 (melanoma) cell lines, with IC50 value 0.78 μg/ml (1.21 µM) which is several folds higher than cisplatin 4.88 μg/ml (16.3 µM). (1) also has a better therapeutic index and toxicological profile compared to cisplatin, as evidenced by the median lethal dose (LD50).

Exploring the structure of a ruthenium acetate cluster for biological purposes

This work evaluates the cytotoxicity of [Ru3(μ3-O)(μ-OAc)5{μ-η1(C),η2(N,N)-phen}(py)2](PF6) (1), a triruthenium acetate cluster combined with an ortho-metallated 1,10-phenanthroline (phen) ligand, along with its interactions with biomolecules. In vitro cytotoxicity tests against the B16F10 (murine melanoma) and L929 (fibroblast) cell lines showed that 1 decreased cancer cell viability by 50% at 25 μM, while it requires 50 μM of free phen ligand to achieve the same effect. Importantly, 1 is not active against non-tumor cell model L929 up to 100 μM. Spectrophotometric titrations suggest that 1 and DNA interacted weakly through electrostatic attraction and semi-intercalation. Despite the presence of a planar and aromatic ligand, relative viscosity measurements are not consistent with the DNA intercalation of 1, presumably due to the small size of phenanthroline. The protein-binding ability of compound 1 was evaluated using human serum albumin (HSA) as a model and monitored by spectroscopic techniques, including steady-state emission and absorption, time-resolved luminescence, and circular dichroism, CD. Compound 1 quenches the HSA emission efficiently (Kap = 4.72 × 108 M−1, 298 K) mostly by static quenching, consistent with its relatively strong binding to HSA (Kb = 3.70 × 106 M−1, 298 K). Analysis of ΔH and ΔS values (604 kJ/mol and 2162 J/mol K, respectively) suggest the contribution of hydrophobic interactions. CD measurements demonstrated that the protein α-helical structure decreased in the presence of l, indicating a partial conformation change of HSA. Although the interaction of 1 with DNA is weak, its binding to the model protein HSA is strong, such that the cytotoxicity of the complex may be attributed to protein-complex interactions. This work provides a framework to design new triruthenium acetates to achieve stronger and more selective binding to biomolecules. Importantly, the selective activity of 1 towards a cancer cell line, while remaining inactive against healthy cells, represents a critical finding for the design of new cancer therapeutics based on triruthenium acetate clusters.

Anti-tumor immune response varies among individuals: A gene expression profiling of mouse melanoma

Melanoma is amongst the most aggressive malignant tumors. The purpose of this study is to detect the tumor microenvironment systematically using multi-omics analyses and to propose strategies for precision medicine. Multiple factors of tumor microenvironment contribute to the drug resistance and immune surveillance failure. Here we analyzed genome mutations and characterized the immune state of tumor microenvironments in mouse melanoma by whole exome sequencing (WES) and RNA sequencing (RNA-Seq) approaches. Somatic mutation analysis revealed 35.1% novel mutations in mouse tumors when compared with B16F10 cell line, provided a basis for multi-site sequencing for accurate neoantigen selection. Mutation cluster, gene expression comparison, and gene ontology (GO) analyses by R packages proved DNA repair damage, inflammation, slower cell division, and metabolic change in tumor microenvironment. Further analyses of T-cell receptor (TCR) sequences, immune signaling pathway activation, tumor infiltrated immune cells and chemokine expression revealed extensive difference of antitumor immune response among individuals. Our study revealed the characteristics of tumor microenvironment with mouse melanoma model, suggested the need of comprehensive genome mutations and personal immune state analyses for cancer precision medicine.

STAT3 inhibitory stattic enhances immunogenic cell death induced by chemotherapy in cancer cells.

Induction of immunogenic cell death (ICD) is considered a promising strategy for cancer immunotherapy. Stattic is an inhibitor of STAT3, which is found constitutively active in many cancers and plays a major role in cancer progression. In the present study, we proposed to evaluate whether stattic can enhance the effects of chemotherapy in the induction of ICD in cancer cells harboring hyperactive STAT3. The growth inhibitory effects of stattic and chemo agents including doxorubicin (DOX) and oxaliplatin (OXP) were evaluated using MTT assay in B16F10 and CT26 cell lines. Flow cytometry was applied to study cell apoptosis and calreticulin (CRT) surface exposure. The levels of high mobility group box1 (HGMB1), heat shock protein70 (HSP70) and interleukin-12 (IL-12) were measured using ELISA. Treatment of B16F10 and CT26 cells with stattic in combination with DOX resulted in synergistic antitumor effects with combination index being 0.82 and 0.87, respectively. Interestingly, we found a higher level of ICD markers including CRT expression as well as HMGB1 and HSP70 secretion in the cells received combination therapy of stattic and DOX as compared with monotherapies. Moreover, exposure of dendritic cells (DCs) to conditioned media (CM) from cancer cells treated with stattic and/or DOX resulted in secretion of IL-12, which is an indicator of DCs maturation and induction of Th1 response. OXP and stattic monotherapy induced ICD in CT26 cells and stimulated IL-12 secretion by DCs; however, we did not observe a significant increase in the level of ICD in CT26 cells and IL-12 secretion by DCs when CT26 cells were treated with stattic and OXP combination as compared with monotherapy groups. These findings indicate that STAT3 inhibitory stattic can increase ICD induced by DOX. Graphical abstract.

Melatonin-Induced Cytoskeleton Reorganization Leads to Inhibition of Melanoma Cancer Cell Proliferation.

Neuroindole melatonin, a hormone synthesized during the night mainly—but not exclusively—by the pineal gland of all vertebrates, functions as an adapting signal to the light-dark cycle. Its antioxidant, neuroprotective, anti-inflammatory, and antitumor properties are all well-known and widely reported. Melanoma is one of the most common carcinomas among developed countries and a type of tumor particularly difficult to fight back in medium/advanced stages. In contrast to other types of cancer, influence of melatonin on melanoma has been scarcely investigated. Thus, we have chosen the murine melanoma model B16-F10 cell line to study antiproliferative and antitumoral actions of melatonin. For this purpose, we combined both, cell culture and in vivo models. Melatonin reduced either, growth rate or migration of B16-F10 cells. Furthermore, melanin synthesis was altered by melatonin, promoting its synthesis. Melatonin also induced a G2/M cell cycle arrest and altered the cytoskeletal organization. To corroborate these results, we tested the effect of melatonin in the in vivo model of B16-F10 cell injection in the tail vein, which causes numerous lung metastases. Two different strategies of melatonin administration were used, namely, in drinking water, or daily intraperitoneal injection. However, contrary to what occurred in cell culture, no differences were observed between control and melatonin treated groups. Results obtained led us to conclude that melatonin exerts an antiproliferative and anti-migrating effect on this melanoma model by interfering with the cytoskeleton organization, but this pharmacological effect cannot be translated in vivo as the indole did not prevent metastasis in the murine model, suggesting that further insights into the effects of the indole in melanoma cells should be approached to understand this apparent paradox.

The activation of cancer cells by a nanosecond-pulsed magnetic field generator

Cancer treatment is a challenge in modern medicine. Several novel modalities have been invented to selectively treat cancer cells. The reactive species such as reactive oxygen species and reactive nitrogen species play important role in the anti-cancer capacity of several modalities, such as cold atmospheric plasma. In this study, we first demonstrated that a nanosecond-pulsed magnetic field (NMF) generator could sensitize the melanoma cell line B16-F10 to the cytotoxicity of an important ROS, H2O2. The magnetic field strength and the frequency were two key factors to control the activation level of NMF treatment. This study provided a novel strategy to use electromagnetic modality in cancer treatment particularly for the treatment on deep tumors such as brain, pancreatic, etc.

Effective copper(II) and nickel(II) complexes with N3O and ON3 thiosemicarbazidato ligands. Synthesis, structural analysis and in vitro cytotoxity on melanoma B16F10 cells

Novel copper(II) and nickel(II) complexes were newly synthesized by template condensation using 2-amino-3,5-dibromo- or 2-hydroxy-5-bromo-benzaldehyde S-propyl thiosemicarbazones as starting materials. The complexes with asymmetric donor atom set were identified through use of analytical and spectroscopic methods (IR, 1H NMR, ESI-MS and X-ray crystallography). Cytotoxicity potents of the N3O and ON3-type complexes were determined on melanoma B16F10 and fibroblast BJ ATCC CRL-2522 cell lines. The cell lines were exposed to the complexes in the concentration range of 6–500 µM for 24 h. The results indicated selective effect of the nickel and copper complexes in favor of healthy cells. Namely, relative low concentrations of the test molecules resulted in cytotoxic effects on malignant cells while higher concentrations were required for normal cell death. The nickel complexes were more effective than the copper complexes and a nickel complex (1a) showed selectively cytotoxcity by IC50 value of 1978 μg/ml on B16F10 cells compared to the 5464 μg/ml value of CRL-2522 cells.

Antitumour Effect of a Mixture of N-Propyl Polysulfides In Vitro

Abstract Copper serves as a limiting factor for multiple steps of tumour progression, including angiogenesis, growth and metastasis. High levels of copper have been found in a wide spectrum of human cancers. Antitumour activities of copper-chelating drugs have been reported in animal models. Organosulfur compounds (diallyl sulfide, DAS; diallyl disulfide, DADS; S-ethylcysteine, SEC; N-acetylcysteine, NAC) derived from garlic exhibit marked copper-chelating activity. We analysed a mixture of fifteen n-propyl polysulfides (DPPS) for potential antitumour activity against several murine tumour cell lines, including colon carcinoma (CT26), mammary carcinoma (4T1) and melanoma cell lines (B16F10), and compared the effects with the antiproliferative effect in highly proliferative murine mesenchymal stem cells (mMSCs). The effects of the mixture of n-propyl polysulfides (100%) on cell viability were determined using MTT assays. Cell apoptosis was analysed using Annexin V-FITC/PI assays. The results of the MTT assays indicate that this standardized mixture of n-propyl polysulfides has a strong, dose-dependent cytotoxic effect against all three of the tested tumour cell lines (CT26, 4T1, B16F10). The cytotoxic effect of the n-propyl polysulfide mixture against the CT26 and B16F10 cell lines was much stronger than that of cisplatin and was significantly weaker in mMSCs, which are non-cancerous and highly proliferative cells, than in cancer cells. Flow cytometric analysis of CT26 and 4T1 cells revealed that apoptosis was not the dominant mechanism of cell death induced by the n-propyl polysulfide mixture. The n-propyl polysulfide mixture exerted highly cytotoxic activity against murine colon carcinoma and melanoma cell lines, but its antiproliferative activity against mMSCs was significantly lower than that of cisplatin.

Establishment of cisplatin-resistant melanoma cell line with stable expression of T-cadherin and its biological characteristics

To establish cisplatin (CDDP)-resistant melanoma B16F10 (CDDP-R B16F10) cell line with stable expression of T-cadherin, and to study its biological characteristics. Methods: CDDP-R B16F10 cell line was exposure to high and gradually increased dose of CDDP. 3-(4.5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was used to test the proliferation of CDDP-R B16F10 cell line, and the sensitivity of CDDP-R B16F10 cell line to CDDP and paclitaxel was examined. The pEGFP-N1-T-cadherin, a plasmid vector encoding human T-cadherin, was generated by inserting T-cadherin cDNA into a pEGFP-N1 vector. The pEGFP-N1-T-cadherin was transfected into CDDP-R B16F10 cell line. The expression of T-cadherin mRNA and protein were measured by reverse transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry SP method, respectively. The effect of T-cadherin combined with CDDP on proliferation of CDDP-R B16F10 cell line was determined by MTT assay. The sensitivity of CDDP-R B16F10 cell line with stably transfected T-cadherin to paclitaxel was examined by MTT assay. Results: The CDDP-R B16F10 cell line was established successfully. There was no difference in proliferation between the CDDP-R B16F10 cell line and B16F10 cell line (P>0.05). The IC50 of CDDP-R B16F10 cell line and B16F10 cell line to CDDP were 268.706 and 19.748 mg/L, respectively, and the resistance index was 13.61. The IC50 of CDDP-R B16F10 cell line and B16F10 cells to paclitaxel were 11.415 and 7.799 mg/L, respectively, and the resistance index was 1.46. The expression vector pEGFP-N1-T-cadherin was constructed successfully. RT-PCR, Western blotting and immunohistochemistry SP method showed that T-cadherin could be transcribed and expressed. MTT assay showed that T-cadherin combined with CDDP could inhibit the proliferation of CDDP-R B16F10 cell line (P<0.05). Factorial analysis showed that there was interaction between T-cadherin and CDDP in inhibiting the proliferation of CDDP-R B16F10 cell line (P<0.05). T-cadherin combined with paclitaxel could inhibit the proliferation of CDDP-R B16F10 cell line (P<0.05). Factorial analysis showed that there was no interaction between T-cadherin and paclitaxel in inhibiting the proliferation of CDDP-R B16F10 cell line (P>0.05). Conclusion: The CDDP-R B16F10 cell line with stable expression of T-cadherin is established successfully. T-cadherin can reverse the CDDP resistance to CDDP-R B16F10 cell line, and promote its sensitivity to paclitaxel.