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。     

氧化苦参碱抗B16黑色素瘤的实验研究

目的观察氧化苦参碱对B16黑色素瘤的作用。方法 B16细胞分别加入0.5、1.0、2.0、4.0、8.0 mg/mL氧化苦参碱,孵育24 h,噻唑蓝（MTT）比色法观察不同浓度氧化苦参碱对离体培养的B16黑色素瘤细胞增殖的影响;将B16荷瘤小鼠随机分为对照组及氧化苦参碱高、中、低剂量组,接种后第2 d开始给药,氧化苦参碱高、中、低剂量组分别灌胃给予120、60、30 mg/kg的氧化苦参碱,对照组给予等容积0.9%氯化钠注射液,连续10 d后,处死动物,测定肿瘤生长抑制率、脾脏指数和胸腺指数。结果 MTT比色法显示氧化苦参碱对B16黑色素瘤细胞具有明显的增殖抑制作用;氧化苦参碱能明显减轻小鼠瘤质量,升高脾脏指数、胸腺指数。结论氧化苦参碱可通过抑制肿瘤细胞增殖、调节荷瘤机体异常的免疫状态而发挥抗B16黑色素瘤作用。     

Effect of Withania somnifera on B16F-10 melanoma induced metastasis in mice

Withania somnifera, a plant with known immunopotentiating activity and its bioactive fraction-Withanolide D were studied for their anti-metastatic activity using B16F-10 melanoma cells in C57BL/6 mice. Simultaneous administration of Withania extract (122 +/- 10 tumour nodules) and Withanolide (126 +/- 9 lung tumour nodules) could significantly (p < 0.001) inhibit the metastatic colony formation of the melanoma in lungs. 72.58% by extract and 69.84% by Withanolide treated, as compared to the untreated control animals also increased the survival days. Lung collagen hydroxyproline content was highly elevated in the control animals (23.5 +/- 0.9 micro g/mg protein), which was reduced by the simultaneous administration of both the extract (16.3 +/- 2.0 micro g/mg protein) and Withanolide (15.3 +/- 1.8 micro g/mg protein). The level of lung hexosamines (4.85 +/- 0.20 mg/100 mg tissue) and uronic acids (330.1 +/- 23.7 micro g/100 mg tissue) content was also elevated in the control animals. The elevated level of hexosamine was significantly reduced by the treatment with extract (1.92 +/- 0.05) and Withanolide (1.85 +/- 0.05). Similarly, the uronic acid content was also been reduced by the simultaneous administration of both Withania extract (194.2 +/- 17.4) and Withanolide (183.2 +/- 8.8). The control animals had 35.3 +/- 3.8 U/L gamma-glutamyl transpeptidase (gamma-GT), which was reduced by 50% by the treatment of extract and Withanolide to 17.5 +/- 4.0 U/L and 16.3 +/- 4.4 U/L respectively. There was a significant reduction in the levels of sialic acid in the serum of Withania extract (60.7 +/- 7.7) and Withanolide (67.16 +/- 5.8) treated animals compared to the higher level (102.2 +/- 8.7) in the control animals. Histopathological analysis of the lung tissues also correlated with these findings. Prophylactic administrations of both extract as well as Withanolide were ineffective in inhibiting the metastasis of B16F-10 melanoma cells.     

Antiproliferative and proapoptotic actions of okra pectin on B16F10 melanoma cells

The proliferation and apoptosis of metastatic melanoma cells are often abnormal. We have evaluated the action of a pectic rhamnogalacturonan obtained by hot buffer extraction of okra pods (okra RG‐I) on melanoma cell growth and survival in vitro. We added okra RG‐I containing an almost pure RG‐I carrying very short galactan side chains to 2D (on tissue culture polystyrene, tPS) and 3D (on poly(2‐hydroxyethylmethacrylate), polyHEMA) cultures of highly metastatic B16F10 mouse melanoma cells. We then analyzed cell morphology, proliferation index, apoptosis, cell cycle progression and the expression of adhesion molecules. Immunostaining and western blotting were used to assay galectin‐3 (Gal‐3) protein. Incubation with okra RG‐I altered the morphology of B16F10 cells and significantly reduced their proliferation on both tPS and polyHEMA. The cell cycle was arrested in G2/M, and apoptosis was induced, particularly in cells on polyHEMA. The expression of N‐cadherin and α5 integrin subunit was reduced and that of the multifunctional carbohydrate‐binding protein, Gal‐3, at the cell membrane increased. These findings suggest that okra RG‐I induces apoptosis in melanoma cells by interacting with Gal‐3. As these interactions might open the way to new melanoma therapies, the next step will be to determine just how they occur. Copyright © 2009 John Wiley & Sons, Ltd.     

Stellettin A induces endoplasmic reticulum stress in murine B16 melanoma cells

Isomalabaricanes are a small class of rearranged triterpenoids obtained from marine sponges. Most of these are cytotoxic to tumor cells, but the underlying mechanism is not clear. In this study, it was demonstrated that stellettin A (1), obtained from Geodia japonica, inhibited the growth of B16F10 murine melanoma cells by the induction of endoplasmic reticulum stress and accumulation of unfolded proteins. Immunoblotting analysis revealed abnormal glycosylation patterns of two melanoma marker proteins, tyrosinase and tyrosinase-related protein 1, and the retention of these proteins in the endoplasmic reticulum. Compound 1 induced the upregulation of the unfolded protein chaperone, glucose-regulated protein 78, in a dose-dependent manner. Increase of autophagosome-associated protein light chain 3 (LC3) in a membrane-bound form (LC3II) and its immunofluorescence co-localization with tyrosinase suggest the possible removal of deglycosylated and unfolded proteins by autophagy of the cells. There was no change in either the expression of the apoptosis marker protein Bcl-2 or the appearance of apoptotic nuclei in 1-treated cells. Taken together, 1 is an endoplasmic reticulum stressor that inhibits the growth of B16 melanoma cells by induction of abnormal protein glycosylation and autophagy.     

A potent tyrosinase activator from Radix Polygoni multiflori and its melanogenesis stimulatory effect in B16 melanoma cells

Tyrosinase is a key enzyme in melanin biosynthesis. Activators of tyrosinase with stimulatory effects on melanogenesis are beneficial for the treatment of hypopigmentation diseases. In the present study, mushroom tyrosinase activity assay was performed to screen tyrosinase activators from traditional Chinese herbs. Four components from Radix Polygoni multiflori were tested. The most active compound, 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside (THSG), was found to be a significant tyrosinase activator. The maximal activation was 126% at a concentration of 75.0 microg/mL. The three anthraquinones slightly activated tyrosinase with effects in the range 7-31%. All the compounds were tested in B16 melanoma cells, the anthraquinones were found to inhibit cell proliferation at a concentration of 0.1-2.5 microg/mL, and THSG was found to be non-cytotoxic at a concentration of 0.1-12.5 microg/mL. THSG significantly increased the activity of murine tyrosinase and stimulated melanin biosynthesis in B16 melanoma cells. In conclusion, THSG is a potent tyrosinase activator and stimulator of melanogenesis with potential for the treatment of hypopigmentation disease.     

Effects of phenolic compounds isolated from Rabdosia japonica on B16-F10 melanoma cells

Pedalitin isolated from the aerial part of Rabdosia japonica (Labiatae), exhibited cytotoxicity against the murine B16-F10 melanoma cell line with an IC(50) of 30 microm (9.5 microg/mL). As the cells were cultured with this flavone, melanin production was not suppressed, but rather enhanced. Quercetin isolated from the same source exhibited similar activities, but rutin showed neither activity.     

Atractylenolide II induces G1 cell-cycle arrest and apoptosis in B16 melanoma cells

Ethnopharmacological relevance

Atractylenolide II (AT-II) is a sesquiterpene compound isolated from the dried rhizome of Atractylodes macrocephala (Baizhu in Chinese), which is traditionally prescribed for melanoma treatment by Chinese medicine practitioners. Our previous study showed that AT-II can inhibit B16 cells proliferation. Here we investigate the mechanistic basis for the anti-proliferative activity of AT-II in B16 melanoma cells.

Materials and methods

Cell viability was examined by MTT assay. Cell cycle distribution and apoptosis were determined by flow cytometry. Protein expression was determined by Western blotting.

Results

AT-II treatment for 48 h dose-dependently inhibited cell proliferation with an IC₅₀ of 82.3 μM, and induced G1 phase cell cycle arrest. Moreover, treatment with 75 μM AT-II induced apoptosis. These observations were associated with the decrease of the expression of Cdk2, phosphorylated-Akt, phosphorylated-ERK and Bcl-2, the increase of the expression of phosphorylated-p38, phosphorylated-p53, p21, p27, and activation of caspases-8, -9 and -3. In addition, a chemical inhibitor of p53, PFTα, significantly decreased AT-II-mediated growth inhibition and apoptosis.

Conclusions

We demonstrated that the G1-arresting and apoptotic effects of AT-II in B16 cells involve p38 activation as well as ERK and Akt inactivation, and the cytotoxic/apoptotic effects of AT-II are potentially p53 dependent. These findings provided chemical and pharmacological basis for the traditional application of Baizhu in melanoma treatment.     

B16黑色素瘤动物模型制备方法探讨

目的利用C57BL/6小鼠制备黑色素瘤动物模型,选择合适的造模条件。方法将黑色素瘤细胞株B16制备成1×108L-1、1×109L-1、5×109L-1、1×1010L-1浓度的细胞悬液,分别注射于小鼠背部,观察小鼠的出瘤时间、生存期及小鼠生活习性的改变。结果小鼠出瘤率为100%。浓度越高出瘤时间、生存期越短,生活习性改变越明显。结论选择背部为注射部位较方便,细胞浓度为1×109L-1制备的动物模型,出瘤时间、生存时间更适于实际工作。     

冬虫夏草繁育品对恶性黑色素瘤B16细胞增殖及迁移能力的影响

目的探讨冬虫夏草Cordyceps sinensis繁育品对恶性黑色素瘤B16细胞增殖及迁移能力的影响。方法通过MTT法、平板克隆实验检测冬虫夏草繁育品对B16细胞增殖能力的影响;采用划痕实验、transwell小室实验检测其对B16细胞迁移能力的影响;通过黏附实验检测其对B16细胞黏附能力的影响;采用流式细胞仪检测其对B16细胞周期分布的影响;采用Western blotting法检测其对B16细胞中MMP-2、MMP-9、Bax、Bcl-2、Cyclin D1、CDK2、CDK4、P21及p-Akt蛋白表达的影响。结果冬虫夏草繁育品能够将B16细胞阻滞在G1/S期,从而显著抑制B16细胞的增殖,并显著降低B16细胞的迁移能力。同时可导致B16细胞MMP-9、MMP-2、Bcl-2、Cyclin D1、CDK2、CDK4、p-Akt蛋白表达减少和Bax、P21蛋白表达增加。结论冬虫夏草繁育品能显著抑制B16细胞的增殖及迁移能力,其作用机制可能与调控细胞周期相关蛋白、MMPs家族蛋白及p-Akt蛋白的表达有关。     

莪术醇诱导小鼠黑色素瘤B16-F10细胞凋亡作用研究

目的:研究莪术醇对小鼠皮肤黑色素瘤B16-F10细胞凋亡的影响。方法:采用四氮唑盐(MTT)法检测不同浓度的莪术醇对B16-F10细胞增殖的影响;并用光学显微镜观察不同浓度的莪术醇对B16-F10细胞状态的影响;通过流式FITC-Annexin V/PI双染法,检测不同浓度的莪术醇对细胞凋亡率的影响;通过间接荧光标记法检测不同浓度的莪术醇对细胞内活性氧水平的影响;通过罗丹明123染色,检测不同浓度的莪术醇对细胞内线粒体膜电位的影响。结果:不同浓度莪术醇处理B16-F10细胞24、48、72h,均可呈浓度、时间依赖性的抑制细胞增殖;显微镜观察到莪术醇可以引起细胞形态变圆,漂浮等细胞死亡现象。12.5、25、50、100μg/ml莪术醇作用48h后,诱导的细胞凋亡率分别为(19.5±3.4)%、(35.1±2.8)%、(45.9±4.1)%、(60.5±1.9)%,与对照组具有显著性统计学差异;诱导的细胞内活性氧水平的百分率分别为(6.9±1.8)%、(12.4±2.1)%、(20.9±3.1)%、(29.1±3.5)%,与对照组相比具有显著统计学差异。引起的线粒体膜电位的百分比分别为(40±1.1)%、(33.7±4.2)%、(27.3±2.5)%、(17.9±2.9)%,与空白对照组相比具有统计学差异。结论:莪术醇可以抑制B16-F10的增殖并促进细胞凋亡,其凋亡机制可能与细胞内活性氧组分的产生以及线粒体膜电位的变化有关。     

Inhibition of melanogenesis in murine B16/F10 melanoma cells by Ligusticum sinensis Oliv

Ligusticum sinensis Oliv. (LSO) is an herbal drug commonly used as a topical treatment of epidermal hyperdepigmentation in Chinese medicine. However, the mechanism underlying the depigmentation by LSO is still unclear. The purpose of this study was to investigate the effects of LSO on the process of melanogenesis and its possible underlying mechanism. Suppressed DOPA oxidase activity of mushroom tyrosinase was first noted when incubated with aqueous extracts of LSO, demonstrating the direct inhibitory effect of LSO on mushroom tyrosinase. Further experiments were carried out in murine B16/F10 melanoma cells and the effects of LSO extract on melanin formation, tyrosinase activity and tyrosinase gene expression were tested. Under conditions without affecting the viability of murine B16/F10 melanoma cells, LSO extract significantly reduced the cellular melanin content in a dose-dependent manner. The DOPA oxidase activity of tyrosinase in B16/F10 cells was dose-dependently inhibited by LSO treatment, possibly mediated by the suppressed tyrosinase mRNA expression in LSO-treated B16/F10 cells. In conclusion, the inhibitory effect of LSO on melanogenesis is likely associated with decreased DOPA oxidase activity of tyrosinase that is most likely the result of the down-regulation of tyrosinase mRNA expression.     

Inhibitory effects of Sargassum polycystum on tyrosinase activity and melanin formation in B16F10 murine melanoma cells

Ethnopharmacological relevance

Sargassum polycystum, a type of brown seaweed, has been used for the treatment of skin-related disorders in traditional medicine.

Aim of the study

The aim of the present study is to investigate the antimelanogenesis effect of Sargassum polycystum extracts by cell-free mushroom tyrosinase assay followed by cell viability assay, cellular tyrosinase assay and melanin content assay using B16F10 murine melanoma cells.

Materials and methods

Sargassum polycystum was extracted with 95% ethanol and further fractionated with hexane, ethyl acetate and water. The ethanolic crude extract and its fractionated extracts were tested for their potential to act as antimelanogenesis or skin-whitening agents by their abilities to inhibit tyrosinase activity in the cell-free mushroom tyrosinase assay and cellular tyrosinase derived from melanin-forming B16F10 murine melanoma cells. The tyrosinase inhibitory activity was correlated to the inhibition of melanin production in α-MSH-stimulated and unstimulated B16F10 cells.

Results

Sargassum polycystum ethanolic extract and its fractions had little or no inhibitory effect on mushroom tyrosinase activity. However, when tested on cellular tyrosinase, the ethanolic extract and its non-polar fraction, hexane fraction (SPHF), showed significant inhibition of cellular tyrosinase activity. In parallel to its cellular tyrosinase inhibitory activity, SPHF was also able to inhibit basal and α-MSH-stimulated melanin production in B16F10 cells.

Conclusions

Our findings showed that (i) cellular tyrosinase assay is more reliable than mushroom tyrosinase assay in the initial testing of potential antimelanogenesis agents and, (ii) SPHF inhibited melanogenesis by inhibiting cellular tyrosinase activity. SPHF may be useful for treating hyperpigmentation and as a skin-whitening agent in cosmetics industry.     

miR-7介导加味四君子汤含药血清抑制B16恶性黑色素瘤细胞增殖和促进凋亡的研究

目的:研究miR-7是否介导加味四君子汤含药血清抑制B16恶性黑色素瘤细胞增殖和促进B16恶性黑色素瘤细胞凋亡的过程。方法:应用血清药理学方法给药,37℃、5%CO2的恒温培养箱中培养B16恶性黑色素瘤细胞,收集对数期细胞,终浓度10%的小鼠含药血清,作用24 h。实验分为空白对照组、血清对照组、4.61 g/kg、9.22 g/kg和18.44 g/kg加味四君子汤含药血清组,每组含5个样本数,应用Real-time PCR法检测miR-7的内源性表达;分别转染miR-7模拟物和miR-7抑制物到B16恶性黑色素瘤细胞,应用Real-time PCR法检测miR-7模拟物和miR-7抑制物的转染效率、应用MTT法、流式细胞仪Annexin V-FITC/PI法检测miR-7对B16恶性黑色素瘤细胞增殖和凋亡的影响。结果:加味四君子汤含药血清作用24h,与空白对照组和血清对照组相比,9.22g/kg和18.44 g/kg加味四君子汤含药血清组B16恶性黑色素瘤细胞中miR-7表达升高;miR-7模拟物和miR-7抑制物成功转染到B16恶性黑色素瘤细胞中,其Relative Conc值分别是(6.47±0.18)和(0.20±0.01),与空白对照组、血清对照组和阴性对照组相比分别显著升高和显著降低;与空白对照组和血清对照组及阴性对照组相比,miR-7模拟物组的抑制率显著升高(53.61±2.11)%,miR-7抑制物组抑制率显著降低(6.76±0.48)%;与空白对照组和血清对照组及阴性对照组相比,miR-7模拟物组的凋亡率显著升高(34.30±2.13)%,miR-7抑制物组凋亡率显著降低(5.80±0.78)%。结论:miR-7介导加味四君子汤含药血清抑制B16恶性黑色素瘤细胞增殖和促进B16恶性黑色素瘤细胞凋亡的过程,miR-7可能是加味四君子汤抗恶性黑色素瘤的重要分子之一。     

葡萄籽原花青素抑制小鼠B16-F0黑色素瘤细胞黑色素生成的影响

目的:研究葡萄籽原花青素对小鼠B16-F0黑色素瘤细胞黑色素生成的抑制作用,并对其机理进行初步探究。方法:采用四甲基偶氮唑蓝(MTT)检测小鼠B16黑色素瘤细胞增值率;左旋多巴氧化法测定酪氨酸酶活;Na OH裂解法测定黑色素合成;分别采用RT-PCR法和Western-blotting法检测B16-F0细胞中TYR、TRP-1、TRP-2mRNA和蛋白表达水平。结果:葡萄籽原花青素显著抑制B16黑色素瘤细胞的增殖,且与浓度和作用时间相关;12.5~200μg/ml浓度范围的葡萄籽原花青素显著抑制细胞酪氨酸酶活性及黑色素生成。当葡萄籽原花青素浓度为200μg/ml作用时间为48 h时,对B16-F0细胞内酪氨酸酶活性及黑色素合成抑制效果最佳。此外,与对照组相比,经浓度为100μg/ml和200μg/ml,作用时间为48 h的葡萄籽原花青素处理,B16-F0细胞内TYR、TRP-1、TRP-2的mRNA和蛋白表达水平显著降低。结论:葡萄籽原花青素抑制B16黑色素瘤细胞的增殖及黑色素的合成,其机制可能是通过抑制TYR、TRP-1、TRP-2的表达,进而抑制酪氨酸酶活性实现的。