Inhibitory effect of Cyrtomium falcatum on melanogenesis in α-MSH-stimulated B16F10 murine melanoma cells

Objective:To explore the anti-melanogenic potential of Cyrtomium falcatum.Methods:The effects of Cyrtomium falcatum crude extract and its solvent fractions on tyrosinase activity,melanin content,and the expressions of melanogenesis-related genes and proteins were analyzed inα-melanocyte-stimulating hormone(α-MSH)-stimulated B16F10 cells.Results:α-MSH treatment significantly increased tyrosinase activity,and extracellular and intracellular melanin content,as well as the expression levels of tyrosinase,microphthalmia-associated transcription factor(MITF),tyrosinase-related protein(TRP)-1,and TRP-2 in B16F10 cells.Treatment with Cyrtomium falcatum crude extract and its solvent fractions reduced tyrosinase activity and extracellular and intracellular melanin content and downregulated the expression levels of tyrosinase,MITF,TRP-1,and TRP-2 in a dose-dependent manner.Conclusions:Cyrtomium falcatum has potential anti-melanogenesis effects and can be used as a potential source material in cosmeceutical industry for the research and development of novel lead molecules with whitening properties.

Three-Dimensional Analysis of Melanosomes Isolated from B16 Melanoma Cells by Using Ultra High Voltage Electron Microscopy

Melanosomes, isolated by centrifugal separation from culture broth of B16 melanoma cells derived from mouse, were observed by scanning electron microscopy (SEM), and by transmission electron microscopy (TEM). Some interesting structural features were found inside and outside of the melanosomes. By SEM observation, the melanosomes were ellipsoid shape, their surface was not smooth and was covered with rough substructure, 10 to 20 nm particles. By TEM, uneven structure and micro particles were observed in the melanosomes. Furthermore, three-dimensional analysis was tried by using the ultra-high voltage electron microscopy(UHVEM). Micrographs of the melanosomes were taken at various tilted angles by UHVEM, after preparing 500 nm thickness specimens stained with lead citrate. From the micrographs collected, the three-dimensional structures were reconstructed by using i-mode software. Melanin stained by lead and non stained parts was clearly observed in the reconstructed structure. Non stained parts were round, regular size, and distributed widely in the melanosomes.

Liaoqiao aqueous extract inhibits B16 melanoma growth involving MAPKs/Nrf2/HO-1 mediated anti-oxidation and anti-inflammation

Aim Forsythia suspensa （Thunb.） Vahl, Lianqiao in Chinese, is one of the most fundamental herbs in traditional Chinese medicine （TCM） with heat-clearing and detoxicating properties. In this study, we aimed to study the antitumor activity of Lianqiao aqueous extract against melanoma using cancer cell line-based in vitro and mouse allografl tumor in vivo models. Furthermore, we also investigated the underlying molecular mechanisms, par- ticularly the involvement of anti-inflammation and anti-oxidation properties in its antitumor activity. Methods The proliferation of cancer cells was measured by MTT assay. The transplanted B16-F10 melanoma in C57BL/6 mice were established and used for the evaluation of in vivo antitumor effect of LQ. Tumor growth was monitored twice a week. Ki67 and CD31 were used to detect cancer cell proliferation and angiogenesis in tumor, respectively. The anti-oxidative property of LQ was determined by measuring the levels of ROS, MDA and GSH. The anti-inflamma- tory effect of LQ was evaluated by measuring TNF-α and IL-6 using ELISA kits. Other protein expression was deter- mined by Western Blot. Results LQ strongly inhibited the growth of B16-F10 cells in vitro and the tumor growth in vivo. The survival time of tumor-bearing mice was significantly prolonged by LQ. LQ inhibited cancer cell prolif- eration and angiogenesis in tumor as evidenced by decreased expressions of Ki67 and CD31. Levels of ROS, MDA TNF-α and IL-6 decreased, while GSH increased in LQ treatment group, indicating a strong anti-oxidative and an- ti-inflammatory activity of LQ. The expression of antioxidant proteins Nff-2 and HO-1, tumor suppressors P53 and p-PTEN, and the MAPK pathways in tumor tissues were upregulated by LQ treatment. Conclusions LQ exhibited strong antitumor activity against B16-F10 murine melanoma both in vitro and in vivo. The antitumor effect of LQ in- volved the decreased oxidative stress and inflammation in tumor, which is closely related to the heat-clearing and detoxicating properties of LQ.

<i>In-Vitro</i>Effect of Sex Steroids on Mouse Melanoma (B16F10) Cell Growth

A battery of sex steroids were used to check their effect on mouse melanoma (B16F10) cell growth in-vitro. Progesterone and its synthetic receptor antagonist RU-486 showed maximum inhibition on in-vitro melanoma cell growth [1]. Further research work showed that the inhibition by progesterone was not a toxic, spurious or non-specific effect on mouse melanoma cell growth and the inhibition by progesterone was not mediated through progesterone receptor.

Combined effects of oncolytic vaccinia virus and dendritic cells on the progression of melanoma B16-F10 in mice

Aim:We aimed to test the hypothesis that loading of dendritic cells(DCs)with both viral and tumor-specific antigens would enhance the efficacy antitumor DC-based therapy applied simultaneously with oncolytic virus.Methods:Vaccinia virus LIVP/GFP and melanoma B16-F10 were used in this study.DCs were pulsed with various combinations of viral and tumor-associated antigens.The maturation status of DCs was verified by expression of the markers CD80,CD86,and CCR7 and assessment of IL-6,TNF-α,and IL-12 secretion.The most efficient combination of antigens for DC loading was selected based on the analysis of the cytotoxic activity of T lymphocytes.Combination therapy using vaccinia virus LIVP/GFP and DCs pulsed with viral and tumor-specific antigens was administered to the B16-F10 melanoma/mouse C57Bl tumor model.Results:We found that loading of DCs with viral antigens,or with a combination of viral and tumor antigens,resulted in similar levels of expression of DC maturation markers.The maximal in vitro cytotoxicity against virus-infected and non-infected B16 melanoma cells exhibited T lymphocytes activated by DCs loaded with the heat inactivated lysate of vaccinia virus LIVP/GFP infected tumor cell.The results show that the combination of vaccinia virus LIVP/GFP and DCs loaded with both tumor and viral antigens inhibit tumor growth of B16-F10 murine melanoma by more than two-fold.Conclusions:Combination therapy with oncolytic vaccinia virus LIVP/GFP and tumor/virus antigen-loaded DCs limited the growth of established melanoma B16-F10,but no synergistic antitumor effects were observed.We propose that optimization of the therapy regimen could enhance the efficiency of combination therapy.

茯苓多糖对B16黑色素瘤人工肺转移模型的影响

[目的]探讨茯苓多糖对B16黑色素瘤小鼠人工肺转移的影响,并对其作用机制进行初步研究。[方法]C57BL/6小鼠接种B16黑色素瘤,分为茯苓多糖高、低剂量组、顺铂组、模型组,茯苓多糖高、低剂量组分别给予每只小鼠茯苓多糖0.5 mg、0.33 mg,顺铂组给予顺铂0.04 mg,模型组给予生理盐水,接种瘤细胞后第2天开始尾静脉注射给药,隔天给药1次。观察小鼠一般状态,造模后21 d取肺,计数肺表面转移灶个数、HE染色检测肺微小转移灶个数,并检测外周血白细胞数量及脾质量、脾指数。[结果]顺铂能够抑制肺转移,同时降低小鼠体质量、外周血白细胞数量、脾质量和脾指数。而茯苓多糖高、低剂量组对B16荷瘤鼠的体质量无明显影响,茯苓多糖高剂量可明显降低肺表面转移灶个数,茯苓多糖高、低剂量可减少肺微小转移灶个数,高剂量组外周血白细胞数量明显降低,低剂量组与模型组无统计学差异,低剂量可增加B16荷瘤鼠的脾质量和脾指数,高剂量对B16荷瘤鼠的脾质量和脾指数无明显影响。茯苓多糖高、低剂量组脾质量和脾指数均高于顺铂组。[结论]茯苓多糖能够抑制尾静脉注射B16荷瘤小鼠肺转移,增加其脾质量和脾指数,无明显增加其外周血白细胞数量的作用,推测活化外周血白细胞,增强免疫功能可能参与茯苓多糖抑制肿瘤转移的机制。

空间环境对黑色素瘤B16细胞致瘤基因和蛋白质表达的影响

初步探讨了空间环境对黑色素瘤B16细胞致瘤基因和蛋白质表达的影响。选择经第20颗返回式卫星搭载的B16细胞,进行体外和体内实验后,筛选出性状变异明显的空间诱变B16细胞株,检测空间诱变B16细胞基因和蛋白质表达的变化。将筛选出的空间诱变B16细胞接种于C57BL/6小鼠,2周后,处死荷瘤小鼠,取出移植瘤,利用免疫组化方法,检测接种空间诱变B16细胞的荷瘤小鼠移植瘤细胞Melan-A和VEGF蛋白的表达情况;利用RT-PCR方法,检测蛋白质水平上表达变化明显的空间诱变B16细胞melan-a,gp100,bag-3和l-pgds基因的表达情况。与对照细胞相比,1株空间诱变B16细胞荷瘤小鼠移植瘤细胞Melan-A和VEGF蛋白表达明显增加,其他细胞2种蛋白质的表达无明显变化。RT-PCR结果显示:此株细胞的melan-a,gp100和l-pgds基因表达增加,bag-3基因表达降低。空间环境的复合因素可诱导B16细胞基因和蛋白质表达产生变化。

丹参对小鼠黑色素瘤细胞株B16-BL6侵袭和转移能力的影响

目的研究活血化瘀中药丹参对小鼠黑色素瘤高转移细胞株B16-BL6生长、粘附、侵袭、运动、转移能力的影响,并初步探讨其作用机制。方法采用MTT法测定丹参对B16-BL6细胞增殖能力的影响;采用细胞粘附实验、重组基底膜侵袭实验、趋化性运动能力实验以及小鼠黑色素瘤自发性转移模型,观察丹参对B16-BL6细胞生长、粘附、侵袭、运动及转移能力的影响。结果丹参水提液对B16-BL6细胞的增殖具有双向调节作用;能显著促进B16-BL6细胞与基底膜成分纤维粘连蛋白粘附,显著抑制B16-BL6细胞侵袭基底膜能力及趋化性运动能力;能显著减小肺转移结节体积,但对结节数目没有明显作用。结论丹参提取液能够显著减轻小鼠黑色素瘤高转移细胞株B16-BL6的肺转移程度,可能与其能够抑制B16-BL6细胞对基底膜的侵袭能力以及降低B16-BL6的运动能力有关。

6种中药组方对小鼠B16黑素瘤细胞增殖和黑素生成的影响

目的研究6种中药组方水提物、醇提物对小鼠B16黑素瘤细胞增殖及黑素生成的影响。方法以MTT法测定各组方对B16细胞的增殖活性；NaOH裂解法测定各组方对B16细胞的黑素生成量。结果组方1、2、3水提物和组方2、4、6醇提物具有明显促细胞增殖作用（P〉0．05）；水提物中细胞增殖率最高的为组方2（0．625mg／ml），增殖率为22．60％；醇提物中细胞增殖率最高的为组方4（0．078mg／ml），增殖率为11．60％。组方1、2、4、5、6水提物和6种组方醇提物均具有促进黑色素生成的作用（P〈0．05）。水提物中黑素增率最高的为组方1（0.313mg／ml），增率为75．38％；醇提物中黑素生成增率最高的为组方1（1．25mg／ml），增率为88．01％。结论各组方对鼠黑素瘤B16细胞增殖及黑素生成均有一定的促进作用，但其作用强度与组方的组成密切相关。

复色1号方对小鼠B16黑素瘤细胞增殖及酪氨酸酶活性的影响

目的探讨复色1号方对小鼠B16黑素瘤细胞增殖及酪氨酸酶活性的影响。方法采用MTT法测定不同浓度的复色1号方对小鼠B16黑素瘤细胞增殖的影响,多巴速率氧化法测定对酪氨酸酶活性的影响,对其结果进行统计分析。结果复色1号方浓度在0.001～0.01g/l,范围内对小鼠B16黑素瘤细胞的增殖与对照组相比无统计学意义(P>0.05),0.05～0.1g/l浓度范围内对小鼠B16黑素瘤细胞的增殖与对照组相比有显著意义(P<0.05),呈现一定的抑制作用;复色1号方在0.001～0.1g/l浓度范围内对小鼠B16黑素瘤细胞中酪氨酸酶活性的影响与对照组相比,各浓度含药培养基组的A值均有显著性差异(P<0.05),呈剂量依赖性激活作用。在0.001～0.01g/l浓度范围内,复色1号方的最佳激活浓度为0.01g/l。结论复色1号方没有促进小鼠B16黑素瘤细胞增殖的作用;在一定浓度范围内(0.001～0.1g/l),对小鼠B16黑素瘤细胞酪氨酸酶的活性呈剂量依赖型激活作用。

GLS／IL-12对黑色素瘤细胞B16的增殖及凋亡的影响

目的:体外检测人GLS活性肽和小鼠IL-12基因的共表达产物对黑色素瘤细胞B16的增殖及凋亡的影响。方法:用脂质体将含GLS活性肽和小鼠IL-12基因的共表达质粒转染黑色素瘤细胞B16并用RT-PCR检测其表达,再用MTT试验、Hoechst33258染色、AO/EB染色和AnnexinⅤ—FITC流式细胞分析检测基因表达产物对B16细胞的增殖抑制与促凋亡作用。结果:GLS活性肽和小鼠IL-12基因能在B16细胞中表达,MTT法观察到表达产物对B16有明显的细胞增殖抑制作用,经Hoechst33258染色、AO/EB染色和流式细胞分析观察到细胞核浓缩、深染、细胞膜改变等凋亡特征,凋亡指数明显高于对照组。结论:GLS活性肽和小鼠IL-12基因的共表达产物对黑色素瘤细胞B16具有明显的增殖抑制作用及促凋亡作用。

IL-17对黑色素瘤B16F10细胞增殖和侵袭的影响

目的:探讨白细胞介素-17(IL-17)对小鼠黑色素瘤B16F10细胞增殖和侵袭的影响。方法:体外培养小鼠黑色素瘤B16细胞,选择对数生长期的细胞,在培养基中加入一定浓度的IL-17,并设空白对照组,MTT法检测IL-17对B16细胞增殖能力的影响;transwell侵袭实验检测IL-17对B16细胞侵袭能力的影响;明胶酶谱法分析IL-17处理后细胞MMP-2和MMP-9的表达活性。结果:IL-17处理组与对照组相比,B16细胞的增殖能力无变化(P>0.05)。transwell侵袭实验显示,IL-17处理组B16细胞穿膜细胞数明显增多(P<0.01)。明胶酶谱实验显示,IL-17处理后细胞MMP-2和MMP-9的表达活性增强(P<0.01)。结论:IL-17对黑色素瘤B16F10细胞的增殖能力无影响,但可促进细胞侵袭,可能通过增强MMP-2和MMP-9的活性而发挥作用。

沉默pim-3基因对黑色素瘤细胞B16的抑制作用

目的:观察靶向沉默原癌基因pim-3对小鼠黑色素瘤细胞B16的抑制作用。方法:用脂质体将特异性靶向沉默pim-3载体(pim-3-shRNA)和pim-3过表达载体(pim-3-MIGR1)转染到黑色素瘤细胞B16中,荧光定量PCR和Western blotting法检测pim-3 mRNA和蛋白表达的变化,Annexin V/PI染色和TUNEL染色检测细胞凋亡情况,荧光定量PCR和Western blotting法检测pBad、Bcl-2、Bcl-xl、Bax、caspase-3等凋亡相关分子表达的变化,MTT法检测细胞增殖情况,流式细胞术检测细胞周期,RTCAxCELLigence系统检测细胞迁移情况,Transwell侵袭实验检测细胞侵袭的变化。结果:转染pim-3-sh RNA组B16细胞的pim-3mRNA及蛋白表达较对照组明显下降(P<0.05),转染pim-3过表达质粒组B16细胞的pim-3 mRNA及蛋白表达较对照组显著增加(P<0.05)。转染pim-3-shRNA后,B16细胞的凋亡明显增加,在此基础上共转pim-3过表达质粒后则明显逆转沉默pim-3所引起的凋亡(P<0.05);进一步检测发现沉默pim-3明显下调凋亡相关分子p-Bad、Bcl-2、Bcl-xl的表达,上调Bax的表达,最终引起caspase-3活性增加(P<0.05)。沉默pim-3后B16细胞增殖和迁移受到抑制(P<0.05),而细胞周期无明显变化。结论:靶向沉默pim-3基因能促进黑色素瘤细胞的凋亡,抑制细胞的增殖和迁移,提示pim-3基因可以作为黑色素瘤基因治疗的新靶点。

知母皂苷AⅢ对黑色素瘤B16细胞生长和Bax,Bcl-2 mRNA的影响

目的观察知母皂苷AⅢ对体外培养黑色素瘤B16细胞增殖和凋亡的影响。方法体外培养B16细胞,分别以浓度为1,2,4,8,16μmol·L-1的知母皂苷AⅢ作用24 h,通过MTT比色法检测对细胞生长的影响;倒置显微镜观察对细胞形态的影响;Annexin V-FITC/PI双染流式检测对细胞凋亡的影响;逆转录PCR检测对细胞凋亡相关基因Bax,Bcl-2的影响。结果 8,16μmol·L-1的知母皂苷AⅢ明显抑制细胞增殖,24 h的半数抑制浓度(IC50)为10.16μmol·L-1;细胞形态显示16μmol·L-1的知母皂苷AⅢ使细胞体积缩小变圆,细胞减少。与对照组比较,10μmol·L-1知母皂苷AⅢ细胞凋亡率为(4.83±0.25)%(P<0.01),而Bax/Bcl-2比值升高(P<0.05)。结论体外实验显示知母皂苷AⅢ能明显抑制B16细胞生长,并能诱导部分细胞发生凋亡。由于诱导细胞凋亡率与24 h的IC50有一定差距,因此知母皂苷AⅢ对B16细胞生长的影响可能不仅仅与激活凋亡信号通路有关,还与其他细胞生长信号通路有关。

炎症因子IL-23对黑色素瘤B16F10细胞增殖和侵袭能力的影响

目的:探讨白细胞介素-23(IL-23)对黑色素瘤B16F10细胞增殖和侵袭能力的影响。方法:体外培养小鼠黑色素瘤B16F10细胞,采用MTT法检测IL-23对B16细胞增殖能力的影响;Transwell侵袭实验检测IL-23对B16细胞侵袭能力的影响;明胶酶谱法分析IL-23处理后肿瘤细胞B16细胞分泌MMP-2和MMP-9的活性表达情况。免疫印迹杂交法检测IL-23处理组肿瘤细胞核转录因子κB P65(NF-κB P65)的表达水平。结果:与对照组相比,IL-23处理组B16F10细胞穿膜数明显增多(P<0.01);肿瘤细胞MMP-2和MMP-9的表达活性增强(P<0.01);核转录因子NF-κB P65的表达水平显著提高(P<0.01);B16细胞的增殖能力未见明显变化(P>0.05)。结论:IL-23对黑色素瘤B16F10细胞的增殖能力无影响,可通过上调MMP-2和MMP-9的活性促进肿瘤细胞侵袭,其分子机制可能与NF-κB信号通路的激活相关。

ENHANCED ANTITUMOR EFFECTS OF INTERLEUKIN-2 LIPOSOMES IN C57BL/6N MICE WITH B-16 MELANOMA PULMONARY METASTASES

为研究白细胞介素－２（ＩＬ－２）脂质体对小鼠Ｂ－１６黑色素瘤肺转移瘤的抑制效应和对荷瘤鼠脾淋巴细胞增殖的影响，应用改良超声－薄膜法制备ＩＬ－２脂质体；正常及免疫受抑Ｃ５７ＢＬ／６Ｎ小鼠建立Ｂ－１６黑色素瘤肺转移瘤模型，腹腔注射游离ＩＬ－２（０．２×１０４～５×１０４ｕｋｇ－１ｄ－１×１０ｄ）或脂质体包封的ＩＬ－２（０．０４×１０４～１．０×１０４ｕｋｇ－１ｄ－１×１０ｄ）。结果表明：改良超声－薄膜法制备的ＩＬ－２脂质体为直径２００～２５００ｎｍ的大单层脂质体，包封率４８．２％，具有良好的稳定性。ＩＬ－２脂质体可使荷瘤鼠肺湿重下降０．４％～１３．１％，肺转移瘤结节降低２．７％～４３．５％，脾细胞３Ｈ－ＴｄＲ掺入量增加１．０５～１．９０倍，其作用效应相当于５倍量的游离ＩＬ－２，且对免疫受抑荷瘤鼠的作用更强。提示ＩＬ－２经脂质体包封后，其抑瘤活性和促进淋巴细胞增殖的活性提高约５倍，是一种有希望应用于肿瘤生物治疗的新型制剂。

Dietary crocin reverses melanoma metastasis

Crocus sativus and its bioactive constituent crocin are well known for anti-tumor potential in different models.However, the efficacy of crocin on in-vivo melanoma metastasis is not yet reported. In this study, melanoma metastatic model was developed by tail vein injection of B16 F-10 cells in to C57 BL/6 mice. Metastatic mice treated with two different doses of crocin（250 and 500 μg/kg of bodyweight） for 10 days and parameters such as lung metastasis inhibition, mean survival time, lung hydroxyproline, uronic acid and hexosamine levels were analyzed after 21 days of treatment. Then blood was collected and serum gamma glutamyl transpeptidase（γ-GGT）, sialic acid,tumor necrosis factor alpha（TNF-a）, interleukin 10（IL-10）, IL-6, IL-2, and TIMP-1 levels were measured. Further, a lung histological examination was done in crocin treated metastatic mice. Subsequently hallmark metastatic parameters such as matrix metalloproteinases（MMPs）, extracellular regulated kinase 2（ERK2）, vascular endothelial growth factor（VEGF）, and K-ras gene expression were investigated in the lungs of crocin treated metastatic mice.Further, in-vitro adhesion, invasion and migration of B16 F-10 cells were examined after 24 hours of crocin（5 and 10μg/mL） treatment. Administration of crocin to tumor bearing C57 BL/6 mice reduced the lung metastasis by 85%.Elevated levels of hydroxyproline, uronic acid, hexosamine, serum sialic acid and y-GGT in metastatic control were found to be significantly reduced in crocin treated mice. Crocin also inhibited expression of MMP-2, MMP-9, ERK-2,K-ras, and VEGF. Crocin reduced the ability of B16 F-10 cells invasion（P〈0.05）, migration（P〈0.05） and adhesion by upregulating E-cadherin expression. In conclusion, crocin elicited marked anti-metastatic potential by regulating the metastasis induced biomarkers.

One extraction tool for in vitro-in vivo extrapolation?SPME-based metabolomics of in vitro 2D,3D,and in vivo mouse melanoma models

Solid phase microextraction(SPME)in combination with high-resolution mass spectrometry was employed for the determination of metabolomic profile of mouse melanoma growth within in vitro 2D,in vitro 3D,and in vivo models.Such multi-model approach had never been investigated before.Due to the low-invasiveness of SPME,it was possible to perform time-course analysis,which allowed building time profile of biochemical reactions in the studied material.Such approach does not require the multiplication of samples as subsequent analyses are performed from the very same cell culture or from the same individual.SPME already reduces the number of animals required for experiment;therefore,it is with good concordance with the 3Rs rule(replacement,reduction,and refinement).Among tested models,the largest number of compounds was found within the in vitro 2D cell culture model,while in vivo and in vitro 3D models had the lowest number of detected compounds.These results may be connected with a higher metabolic rate,as well as lower integrity of the in vitro 2D model compared to the in vitro 3D model resulting in a lower number of compounds released into medium in the latter model.In terms of in vitro-in vivo extrapolation,the in vitro 2D model performed more similar to in vivo model compared to in vitro 3D model;however,it might have been due to the fact that only compounds secreted to medium were investigated.Thus,in further experiments to obtain full metabolome information,the intraspheroidal assessment or spheroid dissociation would be necessary.

Inhibitory Effect of Cigarette Smoke Extract on Experimental Lung Metastasis of Mouse Melanoma by Suppressing Tumor Invasion

We investigated the effect of a nicotine-and tar-free cigarette smoke extract (CSE) using an experimental metastasis mouse model which was intravenously injected with B16-BL6 mouse melanoma cells. Three-hour pretreatment of cells with various concentrations of CSE (0, 0.1, 0.3, and 1%) dose-dependently reduced the number of lung metastatic nodules 14 days after tumor injection. To elucidate the mechanism of this anti-metastatic effect of CSE, we examined the invasion and migration activities of B16-BL6 cells pretreated with CSE for three hours in vitro. CSE significantly reduced the invasion of cells at 1% and the migration at 0.3% and 1%. Under the same pretreatment conditions, CSE had no effect on the proliferation of cells. These findings suggest that CSE contains some ingredients that suppress hematogenic lung metastasis via inhibition of the invasion and migration activities of mouse melanoma cells.

Influence of Ozonized Oil Nanoemulsions on B-16 Melanoma Cells: An <i>in Vitro Study</i>

In this study, we aimed to use a novel approach to overcome the current limitations of ozone therapy in medicine through ozonized oil nanoemulsions (OZNEs). We evaluated dose-dependency on the cellular activities of B-16 melanoma cell line which were incubated with various OZNE doses (v/v). Antitumor effects of OZNE against cancer cell lines were evaluated by cellular morphology, apoptosis and cell cycle analysis. Flow cytometry results showed that OZNE induced DNA damage, apoptosis, and arrested cell cycle in G0-1 phase in B-16 melanoma cells. Thus, OZNE treatment could pose an effective way to act as a potential therapeutic for patients with tumors in the future.