Mechanism of growth inhibition of melanoma cells by 4-S-cysteaminylphenol and its analogues

Our previous studies have shown that 4-S-cysteaminylphenol (4-S-CAP) causes a significant inhibition of in vivo melanoma growth and a marked depigmentation of black skin and hair follicles. These studies have suggested a role of tyrosinase in the manifestation of these in vivo effects. In this study 4-S-CAP and its analogues were examined for their effects on the growth of human melanoma cells in vitro. 4-S-CAP and 4-S-HomoCAP exhibited strong cytotoxicity with effects much greater than those of alpha-methyl-4-S-CAP and N,N-dimethyl-4-S-CAP. The cytotoxicity of the former two amines was completely prevented by semicarbazide, an inhibitor of plasma monoamine oxidase, while that of the latter two was not prevented by semicarbazide, catalase, and phenylthiourea, a tyrosinase inhibitor. In culture medium 4-S-CAP was rapidly converted by the action of monoamine oxidase present in fetal bovine serum to the aldehyde which was then metabolized to the alcohol and the carboxylic acid when cells were present. alpha-Methyl-4-S-CAP was found to exert higher cytotoxicity to cells with higher tyrosinase activity and melanin content. These results suggest that the in vitro cytotoxicity of 4-S-CAP and 4-S-HomoCAP is mediated through conversion to the aldehydes while that of alpha-methyl-4-S-CAP appears to be dependent on tyrosinase activity to some extent.     

Mechanism of putative neo-antigen formation from N-propionyl-4-S-cysteaminylphenol, a tyrosinase substrate, in melanoma models

Metastatic melanoma is resistant to conventional therapies. N-propionyl-4-S-cysteaminylphenol (NPrCAP), an N-protected sulfur-amine analog of tyrosine, is a good substrate for tyrosinase and is selectively incorporated into melanoma cells, causing cytotoxicity in vitro and in vivo. We have recently shown that intratumoral injections of NPrCAP suppress not only the growth of primary B16F1 melanoma tumors but also of secondary, re-challenged tumors. The participation of CD8(+) T cells has been suggested for the NPrCAP-mediated anti-B16 melanoma immunity. In this study, the molecular mechanism of the NPrCAP cytotoxicity and immunogenicity was examined. The phenol NPrCAP was shown to be activated by mushroom tyrosinase to the ortho-quinone N-propionyl-4-S-cysteaminyl-1,2-benzoquinone (NPrCAQ), and the structure was confirmed by reducing it to the corresponding catechol. NPrCAQ reacted rapidly with biologically relevant sulfhydryl compounds such as cysteine, glutathione and bovine serum albumin. The NPrCAQ-thiol adduct formation was proven with a model thiol N-acetylcysteine by spectroscopic methods. The production and release of NPrCAQ-protein adducts was verified in B16F1 melanoma cells in vitro and in B16F1 melanoma-bearing mice in vivo through the detection of 5-S-cysteaminyl-3-S-cysteinylcatechol after acid hydrolysis of the protein fraction. These results suggest that the phenol NPrCAP, acting as a prohapten, can be activated in melanoma cells by tyrosinase to the quinone-hapten NPrCAQ, which binds to melanosomal proteins through their cysteine residues to form possible neo-antigens, thus triggering the immunological response. NPrCAP thus represents a potential new approach to immunotherapy against metastatic melanoma.     

4-硫-5-(2-噻吩基)尿苷体外对黑色素肿瘤细胞增殖作用的影响

目的探究新化合物4-硫-5-(2-噻吩基)尿嘧啶核苷酸体外对黑色素瘤细胞的增殖作用影响。方法利用MTT法和磺酰罗丹明B染色法对比考察该化合物对人黑色素瘤细胞(A375)、小鼠黑色素肿瘤细胞(B16)和人正常皮肤细胞体外增殖的影响,并利用流式细胞术检测不同药物浓度下该化合物对人黑色素瘤细胞(A375)周期的影响。结果 4-硫-5-(2-噻吩基)尿嘧啶核苷酸苷体外对人和小鼠的黑色素瘤细胞无种属特异性,均表现出剂量依赖性的抑制作用,但对正常皮肤细胞却无抑制作用;流式细胞术显示:4-硫-5-(2-噻吩基)尿苷导致剂量依赖性的G2细胞累积,抑制细胞有丝分裂;同时具有诱导细胞凋亡的作用。结论 4-硫-5-(2-噻吩基)尿嘧啶核糖核苷酸对黑色素瘤有明显抑制作用,对正常皮肤细胞无抑制作用表明该化合物对细胞作用时具有选择性,有可能成为新型靶向抗癌药物的候选者。     

Inhibitory effects of alpha-arbutin on melanin synthesis in cultured human melanoma cells and a three-dimensional human skin model

We studied the inhibitory effects of 4-hydroxyphenyl alpha-glucopyranoside (alpha-arbutin) on melanogenesis in cultured human melanoma cells, HMV-II, and in a three-dimensional cultured human skin model. alpha-Arbutin showed no inhibitory effect on HMV-II cell growth at a concentration below 1.0 mM. Melanin synthesis in cells treated with alpha-arbutin at 0.5 mM decreased to 76% of that in non-treated cells. The cellular tyrosinase activity of HMV-II cells also significantly decreased, while the expression of its mRNA was not affected. Melanin synthesis in a human skin model was also evaluated by the macro- and microscopic observation of its pigmentation as well as by quantitative measurements of melanin. Treatment of the human skin model with 250 microg of alpha-arbutin did not inhibit cell viability, while melanin synthesis was reduced to 40% of that in the control. These results indicate that alpha-arbutin is an effective and safe ingredient for skin-lightening.     

紫草素对人黑素瘤细胞增殖及黑素合成的影响

目的探讨紫草素对人黑素瘤细胞增殖及黑素合成的影响。方法采用MTT法测定紫草素对人A375黑素瘤细胞增殖的影响,NaOH裂解法测定黑素合成。并分成实验组（紫草素组）、对照组（空白组）和阳性对照组[甲氧沙林（8-MOP）组],对其结果进行比较分析。结果紫草素浓度在0.25～1μmol/L对人A375黑素瘤细胞增殖无影响（P〉0.05）,2μmol/L和4μmol/L的紫草素对于A375黑素瘤细胞的增殖有一定的抑制作用（P〈0.05或P〈0.01）。紫草素对于人A375黑素瘤细胞中酪氨酸酶活性呈剂量依赖性激活作用。实验组与对照组比较,0.25、0.5及1μmol/L紫草素对人A375黑素瘤细胞酪氨酸酶活性有激活作用,差异有统计学意义（P〈0.01）;且阳性对照组紫草素作用优于对照组,差异有统计学意义（P〈0.05）;实验组浓度为0.25、0.5及1μmol/L紫草素溶液和阳性对照组与对照组比较,对于人A375黑素瘤细胞中黑素合成有激活作用,差异有统计学意义（P〈0.05或P〈0.01）。结论紫草素对于培养的人黑素瘤细胞酪氨酸酶活性及黑素生成有较强的剂量相关性激活作用。     

Selective effects of diallyl disulfide, a sulfane sulfur precursor, in the liver and Ehrlich ascites tumor cells

The present in vivo studies demonstrated that diallyl disulfide (DADS), occurring in garlic, elevated hepatic sulfane sulfur level and activities of gamma-cystathionase and 3-mercaptopyruvate sulfotransferase in healthy mice but did not affect the hepatic glutathione level. DADS efficiently corrected the concentrations of glutathione and sulfane sulfur, and ameliorated gamma-cystathionase activity that had been lowered in the livers of Ehrlich ascites tumor-bearing mice. In Ehrlich ascites tumor cells, diallyl disulfide did not alter bound sulfane sulfur level, sulfotransferases activity or glutathione level. These data indicate that this compound is capable of acting efficiently and selectively only in the liver and can be used for hepatoprotection during chemotherapy.     

Effects of chloramphenicol reduction products on hemopoietic precursor cells in vitro

This study demonstrates that the nitroso, hydroxamic acid, and acetoxyhydroxamate nitrogenous reduction metabolites of chloramphenicol are not directly toxic to myelopoietic and immunopoietic bone marrow precursor cells as assessed by the in vitro colony-forming cell and precursor plaque-forming cells assays, respectively.     

Selective cytotoxicity of oxysterols through structural modulation on rings A and B. Synthesis, in vitro evaluation, and SAR

Chemically diverse oxysterols were prepared and evaluated for cytotoxicity, aiming to push forward potency and selectivity. They were tested against seven cancer (HT-29, HepG2, A549, PC3, LAMA-84, MCF-7, and SH-SY5Y) and two noncancerous cell lines (ARPE-19 and BJ). The influence of the oxidation pattern on rings A and B was studied. Oxygen functionalities on ring B, such as oxo, oxime, acetamide, acetate, and alkoxy, were evaluated. Most oxysterols were cytotoxic in the low micromolar range, with emphasis to the tetrols 14 and 34, the 6β methoxy and acetoxy derivatives 21 and 45, and the oxime 28. In general, the oxysterols were more toxic to cancer cells and a set of compounds (9, 14, 21, 28, 45) with very high selectivity was identified. The cytotoxicity of 3β-acetates was lower than that of the parent alcohols, although incubation for a longer period rendered them equally cytotoxic, pointing them as potential prodrugs of oxysterols.     

Thymol,a monoterpene phenolic derivative of cymene,abrogates mercury‐induced oxidative stress resultant cytotoxicity and genotoxicity in hepatocarcinoma cells

Thymol (TOH) was investigated for its ability to protect against mercuric chloride (HgCl₂)‐induced cytotoxicity and genotoxicity using human hepatocarcinoma (HepG2) cell line. 3‐(4,5‐Dimethylthiazol‐2‐yl)?2,5‐diphenyl tetrazolium bromide assay confirmed the efficacy of TOH pretreatment in attenuating HgCl₂‐induced cytotoxicity. Pretreatment with TOH inhibited HgCl₂‐induced genotoxicity, depolarization of mitochondrial membrane, oxidative stress, and mitochondrial superoxide levels. Interestingly, TOH (100 µM) alone elevated the intracellular basal glutathione S‐transferase (GST) levels and TOH pretreatment abrogated the decrease in glutathione, GST, superoxide dismutase, and catalase levels even after HgCl₂ intoxication. Furthermore, TOH was also capable of inhibiting HgCl₂‐induced apoptotic as well as necrotic cell death analyzed by flowcytometric analysis of cells dual stained with Annexin‐FITC/propidium iodide. The present findings clearly indicate the cytoprotective potential of TOH against HgCl₂‐induced toxicity, which may be attributed to its free radical scavenging ability which facilitated in reducing oxidative stress and mitochondrial damage thereby inhibiting cell death. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 968–980, 2015.     

新二萜醌化合物杀尔威辛的体外细胞毒作用(英文)

目的:研究杀尔威辛体外抗肿瘤活性及对人肿瘤细胞周期分布的影响,方法:四唑鎓盐还原法(MTT)测定体外抗肿瘤活性;流式细胞术检测细胞周期时相分布,结果:杀尔威辛对3株人白血病细胞的活性与鬼臼乙叉甙(VP-16)相同,但弱于长春新碱(VCR),对12株人实体瘤细胞的作用分别是VCR和VP-16的5.41和4.15倍以上,对人胃癌和肺癌有相对高的活性,分别是VCR的10.81和7.68倍,VP-16的9.03和3.24倍.杀尔威辛阻滞增殖的K562细胞于G_1期,细胞DNA断裂增加和S期细胞明显减少,结论:杀尔威辛对人肿瘤细胞尤其是实体瘤细胞有较强的生长抑制效应,阻滞增殖的人白血病K562细胞于G_1期,S期可能是其作用的敏感期。     

Synthesis and actions of a melanotropin conjugate, Ac-[Nle4, Glu(gamma-4'-hydroxyanilide)5, D-Phe7]alpha-MSH4-10-NH2, on melanocytes and melanoma cells in vitro

L-Glutamic acid (gamma-4'-hydroxyanilide) (GHB) is oxidized by tyrosinase to a quinone which inhibits DNA polymerase, RNA polymerase, and mitochondrial energy production within mushrooms. It was previously shown that GHB can kill B16 melanoma cells in culture, but lacks cytotoxicity for nontyrosinase-containing cells. We have conjugated this drug to a superpotent melanotropic peptide and examined the bioactivity of this conjugate to melanoma cells. 4'-Hydroxyaniline was attached to glutamic acid at position 5 in the superpotent melanotropin fragment analogue, Ac-[Nle4, D-Phe7]alpha-MSH4-10-NH2. The melanotropin:anilide conjugate, Ac-[Nle4, Glu(gamma-4'-hydroxyanilide)5, D-Phe7]alpha-MSH4-10-NH2, was not cytotoxic to B16 or Cloudman S91 mouse melanoma cells in culture, as determined by cell counts and protein assays. Interestingly, we also found that GHB stimulated melanoma cell tyrosinase above control levels in both melanoma cell lines. In our study, GHB itself also was found not to be cytotoxic to B16 or S91 melanoma cells in culture. In the frog skin bioassay, the melanotropin conjugate was more potent than alpha-MSH or Ac-[Nle4, D-Phe7]alpha-MSH4-10 in stimulating melanosome dispersion. These results demonstrate that putative chemotherapeutic ligands can be incorporated into active-site fragment analogues of MSH without loss of biological activity.     

头花蓼对胃黏膜上皮细胞GES-1细胞毒性的研究

目的：探讨头花蓼对胃黏膜上皮细胞株GES‐1增殖的影响。方法体外培养人胃黏膜上皮细胞株GES‐1,采用MTT法连续监测7d,以存活细胞数OD值对培养时间（h）作图,即得生长曲线。不同浓度头花蓼（1024μg／mL、512μg／mL、256μg／mL、128μg／mL、64μg／mL、32μg／mL、16μg／mL、8μg／mL,）与对数期GES‐1细胞共同培养48h,采用MTT法检测细胞增殖。结果（1）体外培养胃黏膜上皮细胞株GES‐1,生长曲线显示：细胞于培养的48h进入对数生长期,120h进入平台期。本实验选96h加药进行后续实验;（2）MTT实验表明：低于128μg／mL时,活细胞数量没有明显变化,细胞增殖无影响。结论头花蓼浓度高于128μg／mL时,对胃黏膜上皮细胞株GES‐1的生长有明显的抑制作用,头花蓼因细胞毒性较低具有潜在的临床应用前景,为头花蓼对H．pylori相关性胃炎的抗菌、抗炎机制奠定了实验基础。     

In vitro cytotoxicity of nanoparticles in mammalian germline stem cells.

Gametogenesis is a complex biological process that is particularly sensitive to environmental insults such as chemicals. Many chemicals have a negative impact on the germline, either by directly affecting the germ cells, or indirectly through their action on the somatic nursing cells. Ultimately, these effects can inhibit fertility, and they may have negative consequences for the development of the offspring. Recently, nanomaterials such as nanotubes, nanowires, fullerene derivatives (buckyballs), and quantum dots have received enormous national attention in the creation of new types of analytical tools for biotechnology and the life sciences. Despite the wide application of nanomaterials, there is a serious lack of information concerning their impact on human health and the environment. Thus, there are limited studies available on toxicity of nanoparticles for risk assessment of nanomaterials. The purpose of this study was to assess the suitability of a mouse spermatogonial stem cell line as a model to assess nanotoxicity in the male germline in vitro. The effects of different types of nanoparticles on these cells were evaluated by light microscopy, and by cell proliferation and standard cytotoxicity assays. Our results demonstrate a concentration-dependent toxicity for all types of particles tested, whereas the corresponding soluble salts had no significant effect. Silver nanoparticles were the most toxic while molybdenum trioxide (MoO(3)) nanoparticles were the least toxic. Our results suggest that this cell line provides a valuable model with which to assess the cytotoxicity of nanoparticles in the germ line in vitro.     

In vitro cytotoxicity of mitoxantrone-incorporated albumin microspheres on acute promyelocytic leukaemia cells

In the present study, the preparation and characterization of bovine serum albumin (BSA) microspheres and the evaluation of the in vitro cytotoxicity of these microspheres on acute promyelocytic leukaemia (HL-60) cells were described. Mitoxantrone (MTZ)-incorporated microspheres were evaluated for particle size, drug loading, release characteristics and surface morphology. The biological effect of MTZ released from BSA microspheres was determined on an in vitro cultured HL-60 cell line, showing that, after encapsulation, MTZ still retains cytotoxic activity. For this purpose, methyl-thiazol-tetrazolium (MTT) assay was used to evaluate the in vitro cytotoxicity of MTZ-loaded microspheres. Particle size of BSA microspheres was determined between 17.61-20.38 microm and they were smooth and spherical in shape. Encapsulation efficiency of the drug-loaded microspheres was between 22.26-60.50%. For MTZ-containing microspheres, the cell death ratios were greater than 80% for all formulations. This study demonstrate that BSA microspheres were well suited for the controlled release of MTZ and were promising for anti-cancer chemotherapy.