Pentoxifylline impedes migration in B16F10 melanoma by modulating Rho GTPase activity and actin organisation

Cancer cell migration is a hallmark of metastatic cascade and compounds that can intervene in this process are clinically important. Pentoxifylline (PTX), a methyl xanthine derivative, inhibits B16F10 melanoma lung homing by inhibiting F10 invasion, MMP secretion and adhesion to matrix components. However, its effect on B16F10 migration remained unexamined, which we investigated in the present study. PTX significantly inhibits F10 migration in scratch wound assay. Elevation in cAMP levels inhibits F10 migration and PTX mediated inhibition of the process was found to be, in part, due to an increase in cellular cAMP levels. PTX induces Protein Kinase A (PKA) activity and PKA inhibitor partly reversed its effects on F10 motility. RhoA and Rac1 GTPases induce B16F10 motility and PTX was found to inhibit migration by affecting these molecules. Stress fibres and lamellipodial protrusions reduced significantly. This was accompanied with inhibition in RhoA and Rac1 membrane localisation. A stark inhibition in RhoA-GTP bound form was also observed. Taken together, the results indicate that PTX, through its phosphodiesterase action, inhibits RhoGTPases and associated actin organisation in B16F10 melanoma, thereby inhibiting cell motility.     

Suramin augments the antitumor and antimetastatic activity of pentoxifylline in B16F10 melanoma

Rapid tumor growth and metastasis are 2 major problems associated with treatment of malignant melanoma. Therefore, drugs that can intervene these processes are of clinical importance. Pentoxifylline (PTX), a methyl xanthine derivative, has been shown to inhibit B16F10 melanoma tumor growth and metastasis. We hypothesized that suramin when combined with PTX enhances its antineoplastic effects, which we have examined using the B16F10 mouse melanoma model. Suramin in simultaneous or sequential combination potentiated the cytotoxic effects of PTX on B16F10 cells. PTX arrested cells in the G0-G1 phase and suramin augmented the effects. Both the drugs inhibited F10 adhesion to laminin, matrigel and collagen type IV and showed enhanced inhibition in combination The combination also demonstrated significantly higher inhibition in cell motility (p = 0.002) and invasion through matrigel (p = 0.005) as compared to the single agents. Suramin synergized with PTX in its effects on secretion of MMP-9 gelatinase. DBA2/J mice implanted with intradermal B16F10 tumor were used as a model to study tumor growth. Animals were intratumorally treated with 50 mg/kg of PTX, 10 mg/kg of suramin and their combinations. Simultaneous administration of the drugs inhibited tumor growth by 5- to 6-folds. Tumor growth was completely blocked in sequential regimen with regression in some cases. The number and size of metastatic nodules on lung was also reduced significantly by the combination treatment. In conclusion, the novel combination of PTX and suramin has synergistic antitumor and antimetastatic activity in B16F10 melanoma and may be a promising approach in treatment of patients suffering from malignant melanoma.     

小鼠黑色素瘤B16F10-Red细胞株的建立与生物学特性分析

目的:构建表达香菇珊瑚红色荧光蛋白(discosomasp red fluorescent protein,DsRed)的小鼠黑色素瘤B16F10-Red细胞株,并检测其生物学特性.方法:用GenEscortTMⅡ转染试剂将pDsRed质粒导入小鼠黑色素瘤B16F10细胞,G418加压培养联合极限稀释法建立稳定、高水平表达DsRed的单克隆细胞系.FCM检测B16F10和B16F10-Red细胞的细胞周期.比较B16F10-Red和B16F10细胞的克隆球形成能力和小鼠体内致瘤能力.结果:稳定表达DsRed的小鼠黑色素瘤B16F10-Red细胞株基本保持了其亲代细胞的特征,能在C57BL/6小鼠腹部皮下形成肿瘤并继续生长和转移.结论:B16F10-Red细胞株构建成功,其移植瘤模型成瘤率和转移情况同B16F10肿瘤相比无明显差别.     

Punarnavine Induces Apoptosis in B16F-10 Melanoma Cells by Inhibiting NF-kB Signaling

The objective of this study was to assess the effect of Punarnavine, an alkaloid isolated from Boerhaaviadiffusa, on apoptosis in B16F-10 melanoma cells. Treatment of B16F-10 melanoma cells with nontoxicconcentrations of Punarnvine resulted in the presence of apoptotic bodies and DNA fragmentation in a dosedependent manner. Cell cycle analysis and TUNEL assays also confirmed the observation. The apoptotic genesp53 and caspase-3 were found upregulated in Punarnavine treated cells, whereas the antiapoptotic gene Bcl-2was downregulated. The inhibited nuclear translocation of NF-κBp65, NF-κBp50, NF-κB-c-Rel, c-Fos, ATF-2and CREB-1 in Punarnavine treated B16 F-10 cells pointed to suppression of NF-κB signaling by Punarnavine.All these results demonstrate that Punarnavine induces apoptosis via activation of p53 induced caspase-3 mediatedpro-apoptotic signaling and suppression of NF-κB induced Bcl-2 mediated survival signaling.     

输血对局部B16黑色素瘤生长的影响

用小鼠的输血模型，探讨输血对局部Ｂ１６黑色素瘤生长的影响，结果发现，同种异品系间的输血，可降低血鼠的ＮＫ细胞活性和淋巴细胞转化率，并能促进肿瘤的生长，肿瘤的体积及重量均明显高于输生理盐水组，而同种同品系间的输血则无此不良影响，本研究提示，Ｈ－２不相容性输血可促进肿瘤的生长，免疫功能下降可能是输血促进肿瘤生长的重要因素。     

Nitric oxide-mediated regulation of hypoxia-induced B16F10 melanoma metastasis

Tumour hypoxia is associated with resistance to therapy and with increased invasion and metastatic potential. Recent studies in our laboratory have shown that the hypoxic up-regulation of tumour cell invasiveness and chemoresistance is in part due to reduced nitric oxide (NO) signaling. Using B16F10 murine melanoma cells, we demonstrate here that the increased metastatic potential associated with exposure to hypoxia is mediated by a reduction in cGMP-dependent NO-signaling. Pre-incubation of B16F10 cells in hypoxia (1% vs. 20% O(2)) for 12 hr increased lung colonization ability by over 4-fold. This effect of hypoxia on metastasis was inhibited by co-incubation with low concentrations of the NO-mimetic drugs glyceryl trinitrate (GTN) and diethylenetriamine NO adduct (DETA/NO). In a manner similar to hypoxia, pharmacological inhibition of NO synthesis resulted in a significant increase in lung nodule formation, an effect that was prevented by co-incubation with GTN. An important NO-signaling pathway involves the activation of soluble guanylyl cyclase and the consequential generation of cGMP. Culture in the presence of a non-hydrolysable cGMP analogue (8-Br-cGMP) abrogated the hypoxia-induced lung nodule formation, suggesting that the effects of NO on metastasis are mediated via a cGMP-dependent pathway. These findings suggest that a novel mechanism whereby hypoxia regulates metastatic potential involves a downstream inhibition of cGMP-dependent NO signaling.     

Paclitaxel Combined with Ticagrelor Inhibits B16F10 and Lewis Lung Carcinoma Cell Metastasis

It is widely accepted that tumor metastasis is the dominant factor leading to cancer-related death. Tumor metastasis is mediated by cell invasion, blood circulation and lymphatic circulation. Paclitaxel, as a common anti-tumor drug and a mitotic inhibitor, promotes microtubule assembly and inhibits microtubule depolymerization. In addition, ticagrelor, an anti-platelet drug, is used to treat acute coronary syndrome. Increasing numbers of studies have reported that platelets can facilitate tumor metastasis. Therefore, inhibiting the effects of platelets can serve as a novel therapeutic strategy for cancer. To explore the effect of anti-tumor and anti-platelet drugs on tumor progression, we chose paclitaxel and ticagrelor. Interestingly, the results demonstrated that paclitaxel and ticagrelor could not only suppress the proliferation, migration and invasion of B16F10 and LLC cells, but they could also prevent tumor metastasis to the lungs. Furthermore, the inhibitory effect of paclitaxel and ticagrelor was more apparent when both drugs were used in combination. Collectively, the current study demonstrated that the combination of paclitaxel and ticagrelor could be considered as a potential anti-tumor therapy approach.     

B16黑色素瘤皮内移植瘤模型的建立

背景与目的： 建立B16黑色素瘤细胞(简称B16细胞)皮内移植瘤模型。 材料与方法： 制备商品源B16细胞悬液,按每只C57BL鼠接种0.1 ml (3×105个)B16细胞于后肢外侧皮内;分离、培养移植瘤源B16细胞,倒置显微镜下观察细胞形态。 结果： 移植瘤出现时间为(9.6±1.2)d,致瘤率为100％;皮内注射B16细胞18 d内,肿瘤形态呈圆形或类圆形,边缘清楚;移植瘤源原代B16细胞多为圆形、类圆形或短梭形,商品源B16细胞为梭形或不规则形;HE染色表明移植瘤内有大量B16细胞。 结论： B16细胞皮内移植瘤模型易于对肿瘤生长的观察和测量,是早、中期肿瘤研究的合适模型之一。     

Ethanol Extract of Hizikia fusiforme Induces Apoptosis in B16F10 Mouse Melanoma Cells through ROS-Dependent Inhibition of the PI3K/Akt Signaling Pathway

Background: Previous studies have reported that Hizikia fusiforme, an edible brown seaweed, has diverse health-promoting effects; however, evidence for its anti-cancer potential is still lacking. In this study, we examined the effect of ethanol extract of H. fusiforme (EHF) on the proliferation of B16F10 mouse melanoma cells. Methods: Analyses of cell viability and apoptosis were performed to study the actions of EHF on B16F10 cells. Cellular reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were measured using a flow cytometer. Western blot analysis was carried out to measure apoptosis and phosphoinositide 3-kinase (PI3K)/Akt signaling related proteins. Results: EHF treatment significantly decreased B16F10 cell viability, which was associated with induction of apoptosis. EHF activated caspase-8 and caspase-9, which are involved in the initiation of extrinsic and intrinsic apoptosis pathways, respectively, and also increased caspase-3 activity, a typical effect caspase, subsequently leading to poly (ADP-ribose) polymerase cleavage. In addition, EHF destroyed the integrity of mitochondria and increased Bax/Bcl-2 ratio, which contributed to cytosolic release of cytochrome c. EHF further enhanced intracellular levels of ROS and the addition of N-acetyl cysteine (NAC), a ROS inhibitor, significantly diminished EHF-induced mitochondrial dysfunction and growth inhibition. Moreover, EHF inactivated the PI3K/Akt signaling pathway and LY294002, a PI3K/Akt inhibitor, increased the apoptosis-inducing effect of EHF. However, increased apoptosis and reduced cell viability by simultaneous treatment of EHF and LY294002 were significantly attenuated in the presence of NAC. Conclusion: These results indicate that EHF induces apoptosis through activation of extrinsic and intrinsic apoptotic pathways and ROS-dependent inactivation of PI3K/Akt signaling in B16F10 cells.     

B16-F10-luc-G5黑色素瘤细胞生物学特性的研究

目的：探讨B16-F10-luc-G5黑色素瘤细胞的生物学特性.方法：倒置荧光显微镜下连续观察B16-F10-luc-G5细胞生长动态.流式细胞术检测冻存复苏后、培养基漂浮细胞、胰酶消化损伤后的细胞死亡率以评价其对实验损伤的耐受性.1×104/孔B16-F10-luc-G5细胞按1∶2梯度稀释至0.78×102/孔,加入底物荧光素后检测其生物发光特性.Babl/C和C57 bl/6雄性小鼠各10只接种该细胞,目测肿瘤生长速度和活体成像监测Babl/C小鼠移植瘤生长动态以评价其成瘤特性.结果：B16-Fl0-luc-G5细胞生长动态符合典型B16-F10细胞特性,无自发及激发荧光.B16-F10-luc-G5细胞冻存、漂浮细胞及对数生长期细胞消化后死亡率分别为23.8％、35.8％和4.8％.B16-F10-luc-G5细胞数与实测平均光子数之间存在线性回归关系,检测光子数可反映细胞数目.两组小鼠移植成瘤率均为100％,平均肿瘤出现时间差异显著（t =9.05,P＜0.05）,移植瘤病理符合B16黑色素瘤典型生长特点;活体成像监测可灵敏监测移植瘤生长动态.结论：B16-F10-luc-G5细胞具有生长快、对各种实验操作耐受性好、标记生物发光基因易追踪等优点,且其一般特性与B16-F10细胞基本相同,是肿瘤学研究良好的实验材料.     

莲房原花青素对黑色素瘤B16细胞的诱导分化作用

背景与目的:探讨莲房原花青素(LSPC)对荷瘤小鼠体内黑色素瘤B16细胞的诱导分化作用.材料与方法:以荷黑色素瘤B16小鼠为模型,随机分LSPC 3个剂量(60、120、240 mg/kg)组和阴性对照组,每组10只,实验组用LSPC、阴性对照组用等体积溶剂分别灌胃13 d后取材,检测黑色素瘤B16细胞中酪氨酸酶活力、黑色素含量、S-100蛋白表达,并用光学显微镜和透射电镜观察其细胞形态的变化.结果:60～120 mg/kg的LSPC能抑制黑色素瘤B16细胞在小鼠体内生长,且呈浓度依赖关系.其中,120 mg/kg LSPC组作用最显著(P＜0.01),抑瘤率达55.3%,并使B16细胞内酪氨酸酶活力和黑色素含量分别提高28.5%和23.8%,与阴性对照比较,差异有统计学意义(P＜0.05),并使S-100蛋白表达下降.结论:LSPC显著抑制B16细胞在小鼠体内的生长,并从形态上和功能上诱导B16细胞分化.     

An Ester Extract of Cochinchina Momordica Seeds Induces Differentiation of Melanoma B16 F1 Cells via MAPKs Signaling

Cochinchina momordica seeds (CMS) have been widely used due to antitumor activity by Mongolian tribesof China. However, the details of the underlying mechanisms remain unknown. In the present study, we foundthat an EtOAc (ethyl ester) extract of CMS (CMSEE) induced differentiation and caused growth inhibition ofmelanoma B16 F1 cells. CMSEE at the concentration of 5-200 μg/ml exhibited strongest anti-proliferative effectson B16 F1 cells among other CMS fractions (water or petroleum ether). Moreover, CMSEE induced melanomaB16 F1 cell differentiation, characterized by dendrite-like outgrowth, increasing melanogenesis production, aswell as enhancing tyrosinase activity. Western blot analysis showed that sustained phosphorylation of p38 MAPaccompanied by decrease in ERK1/2 and JNK dephosphorylation were involved in CMSEE-induced B16 F1cell differentiation. Notably, 6 compounds that were isolated and identified may be responsible for inducingdifferentiation of CMSEE. These results indicated that CMSEE contributes to the differentiation of B16 F1 cellsthrough modulating MAPKs activity, which may throw some light on the development of potentially therapeuticstrategies for melanoma treatment.     

Blue Light Inhibits the Growth of B16 Melanoma Cells

Although a number of studies have been carried out to examine the biological effects of radiation and ultraviolet radiation (UV), little is known concerning the effects of visible light. In the present study, exposure of B16 melanoma cells to blue light (wavelength 470 nm, irradiance 5.7 mW/cm²) from a light-emitting diode (LED) inhibited cell growth in proportion to the period of exposure, with no increase observed in the number of dead cells. The number of B16 melanoma colonies that formed after exposure to blue light for 20 min was only slightly less than that in non-exposed controls, but the colony size as assessed by the area covered by colonies and cell counts per colony were markedly decreased. The percentages of G0/G1 and G2/M phase cells were markedly increased, with a reduction in S phase cells as determined by flow cytometry after exposure to blue light. Furthermore, analysis of the incorporation of 5–bromo–2'–deoxyuridine (BrdU) into DNA also showed a reduction in the percentage of S phase cells after exposure. These results indicate that blue light exerts cytostatic effects, but not a cytocidal action, on B16 melanoma cells.     

三羟异黄酮对黑色素瘤B16细胞的诱导分化作用

背景与目的评价三羟异黄酮对黑色素瘤B_(16)细胞的诱导分化作用。材料与方法以4',5,7三羟异黄酮处理黑色素瘤B_(16)细胞1~4d后,以生长曲线反映其增殖活力,以B_(16)细胞形态、黑色素含量、及流式细胞仪检测细胞周期变化等观察三羟异黄酮对黑色素瘤B16细胞的诱导分化作用。结果用10、30、90μmol/L的三羟异黄酮作用肿瘤细胞,有不同程度的抑瘤作用。表现为黑色素生成能力增加,细胞生长缓慢。可使B16细胞阻断在S期。结论三羟异黄酮不同剂量(10、30、90μmol/L)对B_(16)细胞均有明显的分化诱导作用,对体外黑色素瘤增殖有明显的抑制作用。     

三氧化二砷抑制恶性黑色素瘤B16细胞生长及对端粒酶活性的影响

背景与目的： 研究三氧化二砷(As2O3)对恶性黑色素瘤B16细胞生长抑制作用及对端粒酶活性表达的影响,探讨砷及其化合物治疗恶性黑色素瘤的作用机制,为临床治疗恶性黑色素瘤提供新的理论和实验依据。 材料与方法：四甲基噻唑氮蓝(Methyl thiazolyl tetrazolium,MTT)比色法检测As2O3对B16 细胞的生长抑制作用：端粒重序列扩增酶联免吸附实验(Telomeric repeat amplification protocol enzyme-linked immunosorbant assay,TRAR-ELISA)和聚内烯酰胺凝胶电泳银染(Telomeric repeat amplification protocol,poly acryl amide gel electrophoresis silver-staining,TRAP-PAGE)法检测B16细胞的端粒酶活性。 结果： As2O3可显著抑制B16细胞的生长并可下调端粒酶活性,其抑制作用有显著的时间和剂量依赖关系。 结论： As2O3对恶性黑色素瘤B16细胞生长抑制作用及对端粒酶活性表达的抑制作用随药物浓度的增加和作用时间的延长而增强。     

mB7－1基因转染小鼠黑色素瘤细胞诱导的抗瘤效应研究

目的：研究共刺激Ｂ７－１（ＣＤ６０）在诱导有效的抗肿瘤免疫反应反应中所起的作用。方法：在体外,三种肿瘤细胞与同上鼠脾淋巴细胞混合培养（ＭＬＴＣＳ）后,测定淋巴细胞增殖指数（ＭＴＴ法）和特异杀伤活性（ＬＤＨ释放改良法）;ＭＴＴＩ地测定肿瘤细胞体外增殖能力后,将Ｂ１６－ｎｅｏ和Ｂ１６－ｍＢ７－１妆种于Ｃ５７ＢＬ／６小鼠皮下后观察成瘤期、荷瘤小鼠存活以及肿瘤结节生长速度。结果：与野生型Ｂ１６细胞和模拟转     

Noggin blocks invasive growth of murine B16-F1 melanoma cells in the optic cup of the chick embryo

Melanoma cells originate from the neural crest and are characterized by high migratory potential and invasive growth. After transplantation into the neural tube of the chick embryo, melanoma cells spontaneously emigrate along the neural crest pathways without tumor formation or malignant growth. This emigration depends on the constitutive over-expression of bone morphogenetic protein-2 (BMP-2) and can be ablated by the BMP-antagonist noggin. When transplanted into the embryonic optic cup, melanoma cells invade the host tissue and form malignant tumors. Here, we asked if the invasive growth of melanoma cells in the optic cup could be influenced by BMP-2 or noggin. Mouse B16-F1 cells were grown as aggregates, treated with BMP-2 or noggin during aggregation and transplanted into the optic cup of 3-day chick embryos. After 3 days of subsequent incubation, embryos were evaluated for melanoma cell invasiveness. Immunohistochemical examination revealed that untreated and BMP-2-treated melanoma cells had grown malignantly into the host tissue. However, noggin pretreatment of the aggregates had blocked melanoma cell invasiveness and tumor formation. We conclude that invasive growth of melanoma cells in vivo is BMP-dependent and can be ablated by noggin, thus rendering noggin a promising agent for the treatment of BMP-over-expressing melanoma.     

Inhibition of metastasis by a dialysable factor in fetal bovine serum in B16 melanoma cells

Fetal bovine serum (FBS) supplemented to the culture medium inhibited the metastasis of B16BL6 melanoma cells in a dose-dependent manner. This metastasis-inhibiting activity was accompanied by growth-promoting activity, up to the concentration of 10% FBS. However, among the clones isolated from B16BL6 cells, there were clones in which FBS significantly inhibited the metastasis without promoting their growth. Dialysis (cutoff M.W. 8000 Da) removed the metastasis-inhibiting activity but not the growth-promoting activity from FBS. These results indicate that FBS has metastasis-inhibiting activity which is independent of growth-promoting activity, and that the metastasis-inhibiting activity is carried by molecules removed by dialysis.     

Growth Inhibition and Differentiation of Murine Melanoma B16-BL6 Cells Caused by the Combination of Cisplatin and Caffeine

We preliminarily investigated the combined effects of cisplatin and caffeine on murine melanoma B16-BL6 cells in vitro . When caffeine was added before or simultaneously with cisplatin, there was little growth inhibition. The addition of 2.0 mM caffeine after 1 h of exposure to cisplatin inhibited growth and induced cell differentiation. This treatment resulted in fewer cells, and the numbers of melanosomes and mitochondria and the amount of Golgi's complex and endoplasmic reticulum were increased. DNA histograms obtained by flow cytometry showed that cells treated with cisplatin alone accumulated in the G₂/M phase, with a partial G₂ block. The addition of 2.0 m M caffeine after 1 h of treatment with cisplatin reduced this block. Caffeine caused murine melanoma B16-BL6 cells treated with cisplatin to differentiate, and this inhibited growth.     

苯乙酸钠对小鼠B16黑色素瘤细胞的分化诱导作用

目的　评价苯乙酸钠对黑色素瘤 B1 6 的抑瘤作用。方法　以 MTT法测定细胞增殖的抑制率。B1 6 胞形态 ,黑色素含量 ,细胞周期变化及体内致瘤能力的测定作为观察指标。结果　用 7.5 m M～ 17.5 m M的苯乙酸钠作用于肿瘤细胞 ,见有不同程度的抑瘤作用 P<0 .0 1。 MTT法测得IC5 0 为 2 .6 7～ 3.6 3m M。在 7.5～ 17.5 m M浓度下 ,对 B1 6 细胞都有较强的分化诱导作用 ,表现为黑色素颗粒生成能力明显增多 ,生长缓慢。细胞动力学研究表明 ,苯乙酸钠可使 B1 6 细胞阻断在 G1 期因而 S期明显减少 ,体内致瘤表明成瘤能力明显降低。结论　苯乙酸钠对 B1 6 细胞有强力分化诱导作用 ,而对体内外黑色素瘤增殖有明显抑制。