Methotrexate reduces keratinocyte proliferation,migration and induces apoptosis in HaCaT keratinocytes in vitro and reduces wound closure in Skh1 mice in vivo

The aim of the present work was to evaluate MTX treatment (0.1, 1 and 10 μg mL^?1) in vitro in order to characterize its effects on cell proliferation alterations in cell cycle of HaCaT keratinocytes and wound healing in a Skh1 mice treated with MTX (low doses 30 mg kg^?1, high doses 200 mg kg^?1 and repeated doses at 1.5 mg kg^?1). We analyzed the cytotoxic effect of methotrexate by a resazurin assay. The effects in the proliferation, cell cycle and apoptosis of HaCaT cells were analyzed by flow cytometry. The effects of MTX on wound healing in vivo were also analyzed. A trend toward reduction in the resazurin assay was found (p > 0.05). Reduced proliferation was also identified in a clonogenic assay and a CFSE assay (p < 0.05) due to the MTX treatment. A reduction in the G₂/M and S phases was observed accompanied by apoptosis induction with increased sub G₀ phase and annexin V FITC staining. Effect of MTX was evidenced in vivo on the wound closure process after day 10 (p < 0.05) with alterations in tissue architecture and remodeling. There is a marked effect of MTX on wound healing in vivo in Skh1 mice with implications for long-term therapy and surgical interventions.     

Increased expression of MAP2 inhibits melanoma cell proliferation,invasion and tumor growth in vitro and in vivo

Please cite this paper as: Increased expression of MAP2 inhibits melanoma cell proliferation, invasion and tumor growth in vitro and in vivo. Experimental Dermatology 2010; 19 : 958–964. Abstract: Malignant melanoma (MM) is characterized by aggressive metastasis and high mortality rate. Microtubule‐associated proteins 2 (MAP2) is expressed abundantly in majority of melanocytic nevi and primary melanomas, but absent in metastatic melanomas. To determine whether MAP2 correlates with tumor progression of MM, we investigated the effects of MAP2 inhibition on the biological behaviour of metastatic melanoma in vitro and in vivo. Our results demonstrated that adenovirus‐mediated MAP2 induced apoptotic cell death and cell cycle arrest in metastatic human and mouse melanoma cell lines in vitro, and substantially inhibited the growth of melanomas in nude mice in vivo. In addition, intracellular expression of MAP2 was found to induce the morphologic alteration, suppress the migration and invasion and affect the assembly, stabilization and bundling of microtubules in melanoma cells. This is the first study that MAP2 expression significantly inhibits the growth of MM in vivo. Our results suggest that MAP2 may serve as a promising molecular target for therapy and chemoprevention of MM in humans.     

Endothelin-1 induces CXCL1 and CXCL8 secretion in human melanoma cells

The endothelin pathway plays a critical role in melanoma tumor progression by a variety of mechanisms that enhance tumor cell growth, invasion, and metastasis. Here, we investigate the effect of this pathway on CXC chemokine expression in human melanoma cells and melanocytes. As determined by ELISA, endothelin-1 (ET-1) induces CXCL1 and CXCL8 secretion in three human melanoma cell lines in a concentration-dependent fashion. These responses are mediated by the endothelin-B receptor and are sustained over a 40 h time course. ET-1 does not induce CXCL1 secretion in primary human melanocytes but ET-3, an endothelin isoform, induces a low level of CXCL1 secretion in certain cultures. Neither ET-1 nor ET-3 induces secretion of CXCL8 in primary human melanocytes; thus, this response may be specific for melanocytic cells that have undergone malignant transformation. We have previously demonstrated that ET-1 induces changes in the expression of adhesion molecules in melanoma cells such that invasion and metastasis are favored. This study demonstrates that ET-1 additionally induces secretion of CXC chemokines critical for melanoma metastasis and tumor progression.     

Human leukocyte elastase induces keratinocyte proliferation in vitro and in vivo

Neutrophil infiltration and epidermal hyperproliferation are major histopathologic changes observed in psoriasis. Neutrophils contain human leukocyte elastase, which is thought to be released during neutrophil infiltration of the epidermis. As active human leukocyte elastase is known to be present in psoriatic lesions we were interested whether human leukocyte elastase induces hyperproliferation in keratinocytes in vitro and in vivo. In the cultured murine keratinocyte cell line PAM-212 concentrations of human leukocyte elastase from 1 to 30 nM induced significant proliferation as determined by 5-bromo-2'-deoxy-uridine-incorporation. Daily topical application of 0.043-434.8 pmol human leukocyte elastase per cm2 skin on hairless mice induced a concentration-dependent epidermal hyperproliferation and an increase in 5-bromo-2'-deoxy-uridine incorporation of up to 5-fold in basal keratinocytes within 3 d. Hyperproliferation resulted in a up to 2-fold increase of keratinocyte layers. Histologic analysis revealed marked vasodilatation but no inflammatory infiltrate. Application of porcine pancreatic elastase (3-300 pmol per cm2 skin) resulted in similar epidermal changes as observed for human leukocyte elastase. Hyperproliferative effects of human leukocyte elastase in vitro and in vivo were abolished by the addition of elastase inhibitors, such as elafin, anti-leukoprotease, and alpha1-protease inhibitor. In summary, human leukocyte elastase induces proliferation in murine keratinocytes in concentrations, which can be found on the skin surface of psoriatic lesions. These results may provide an explanation for the epidermal hyperproliferation observed in psoriasis.     

苦参碱抑制人恶性黑素瘤A375细胞株的侵袭

目的 探讨苦参碱对恶性黑素瘤细胞A375转移能力的影响及其作用机制。方法 采用MTT法和膜联蛋白-V-FITC/PI双染色法分别检测不同浓度苦参碱对A375细胞增殖和凋亡的影响,半定量RT-PCR分析经苦参碱干预后A375细胞乙酰肝素酶mRNA的表达变化,细胞黏附实验检测细胞黏附能力的改变,基质胶侵袭实验观察细胞侵袭能力的变化。结果 苦参碱浓度≥0.5mg/mL时可抑制A375细胞增殖并诱导细胞凋亡,其作用呈剂量依赖性;苦参碱浓度在0.125～0.5mg/mL时,能明显下调A375细胞乙酰肝素酶mRNA的表达,并能抑制细胞的黏附、侵袭能力(P<0.01),其抑制效应呈剂量依赖性,在不同浓度组间比较差异均有统计学意义(P<0.01)。结论 苦参碱在体外能抑制A375细胞的黏附、侵袭能力。苦参碱抗肿瘤侵袭可能与抑制肿瘤细胞增殖、诱导细胞凋亡以及下调乙酰肝素酶的表达有关。     

白藜芦醇对恶性黑素瘤细胞株增殖的影响

目的 研究白藜芦醇对恶性黑素瘤体外抗癌作用,探讨其抗癌作用的分子机制.方法 采用MTT法检测白藜芦醇对人恶性黑素瘤细胞系A375及小鼠恶性黑素瘤细胞系B16-F1增殖的影响;Annexin-V/PI双标流式细胞术检测白藜芦醇对A375、B16-F1细胞凋亡的影响,PI单标流式细胞术测定白藜芦醇对A375、B16-F1细胞周期的影响;Western印迹法检测白藜芦醇对A375、B16-F1细胞Bcl-2、Bax蛋白表达的影响.结果 白藜芦醇对A375、B16-F1细胞增殖具有明显的抑制作用,且呈量效及时效关系.25 μmol/L白藜芦醇作用24 h即可诱导A375细胞凋亡,其细胞凋亡率为16.7%±2.1%.100μmol/L白藜芦醇作用24 h时B16-F1细胞凋亡率可达39.6%±3.3%.100 μmol/L白藜芦醇作用12 h时A375细胞凋亡率为17.2%±1.7%,作用72 h时达52.3%±4.1%;相同浓度白藜芦醇作用12 h时B16-F1细胞凋亡率为18.4%±1.6%,作用72 h时达56.7%±4.5%.白藜芦醇处理组A375及B16-F1细胞周期均发生变化,细胞周期被阻滞于G1期,此阻滞效应随白藜芦醇浓度的增加而增加,25 μmol/L、100μmol/L白藜芦醇作用24 h时,A375 G1期比例分别为40.51%±3.97%和55.64%±4.95%.B16-F1细胞分别为41.34%±3.12%和53.93%±5.12%.白藜芦醇明显下调A375及B16-F1细胞抗凋亡蛋白Bcl-2的表达,同时上调促凋亡蛋白Bax的表达水平.结论 白藜芦醇可通过调控细胞周期进程、诱导细胞凋亡而有效抑制A375及B16-F1的增殖,其机制与调节Bel-2、Bax的表达有关.     

Knockdown of Skp2 by siRNA inhibits melanoma cell growth in vitro and in vivo

BACKGROUND: Low levels of p27Kip1 expression are associated with poor prognosis in various malignancies including malignant melanoma. Recently, it has been reported that S phase kinase-interacting protein 2 (Skp2), the specific ubiquitin ligase subunit that targets p27Kip1 for degradation, was overexpressed and was inversely related to p27Kip1 levels in malignant melanoma with poor prognosis. OBJECTIVE: We investigated whether small interfering RNA (siRNA)-mediated gene silencing of Skp2 can be employed in order to inhibit p27Kip1 down-regulation and suppress melanoma cell growth as a consequence in vitro and in vivo. METHODS: We constructed a plasmid vector, which synthesizes siRNAs to determine the effects of decreasing the high constitutive levels of Skp2 protein in melanoma cells. Western blot and real-time RT-PCR were performed to examine the decreases of Skp2 protein and mRNA in vitro. Furthermore, melanoma cells were injected into the back of nude mice subcutaneously to examine the suppression of tumorigenicity targeting Skp2 gene silencing in vivo. RESULTS: Skp2 protein was decreased and the p27Kip1 protein was accumulated in Skp2 siRNA transfected melanoma cells. Skp2 siRNA inhibited the cell growth of melanoma cells in vitro. Moreover, Skp2 siRNA also suppressed tumor proliferation in vivo. CONCLUSION: Our results suggest that siRNA-mediated gene silencing of Skp2 can be a potent tool of cancer gene therapy for suppression of p27Kip1 degradation in malignant melanoma.     

白花丹素对黑素瘤A375细胞体外增殖及凋亡的影响

目的: 评价白花丹素对黑素瘤细胞体外增殖及凋亡的影响.方法: 体外培养黑素瘤细胞A375细胞株,应用不同浓度的白花丹素进行处理,培养24 h后,MTT法测定对细胞增殖的影响;用流式细胞术检测细胞的凋亡率;Western blot检测bcl-2的表达.结果: 浓度为1～13 μmol/L的白花丹素能显著抑制A375细胞株的增殖,且随浓度增加抑制作用递增;白花丹素作用24 h的半数抑制浓度约为10 μmol/L;当浓度为2.5 μmol/L、5.0 μmol/L和10.0 μmol/L作用24 h,细胞的凋亡率分别为8.52%±0.96%、14.83%±1.34%和19.56%±1.85%,与空白对照组(4.58%±0.46%)比较差异有统计学意义(P<0.05);细胞内bcl-2蛋白表达量随药物浓度升高而递减,与对照组比较差异有统计学意义(P<0.05).结论: 白花丹素体外能抑制黑素瘤细胞增殖,并诱导细胞凋亡及下调黑素瘤细胞bcl-2蛋白的表达.     

多西紫杉醇对黑素瘤B16F10细胞增殖及凋亡的影响

目的:探讨多西紫杉醇对黑素瘤B16F10细胞增殖及凋亡的体外作用.方法:以不同浓度多西紫杉醇处理B16F10细胞,MIT法检测细胞增殖速度,倒置显微镜观察细胞形态变化,流式细胞仪检测细胞周期分布,TUNEL法检测原位细胞凋亡.结果:多西紫杉醇呈剂量和时间依赖性抑制细胞增殖.以多西紫杉醇10μmol/L处理B16F10细胞,24h即可出现细胞形态改变,G<,2/M期阻滞,但凋亡细胞数目增多不明显(P<0.05);作用48h凋亡细胞数目明显增多(P0.05).结论:多西紫杉醇具有抑制B16F10细胞增殖、诱导细胞凋亡等作用,细胞凋亡的发生迟于周期阻滞.     

Polypoid melanoma and superficial spreading melanoma different subtypes in the same lesion

Melanoma is a malignant melanocytic neoplasm with high mortality rate, andsteadily and universally increasing incidence rates. Polypoid melanoma isconsidered an exophytic variant of the nodular subtype. The incidence ofpolypoid melanoma is extremely variable, most likely because of the differentcriteria used for its characterization. We presented a rare case of polypoidmelanoma and superficial spreading melanoma in the same lesion.     

载芬维A铵脂质体体外对恶性黑素瘤细胞增殖、凋亡和迁移的影响

目的 探讨载芬维A铵脂质体（4-HPR-L）对A375及B16F10黑素瘤细胞增殖、凋亡和迁移的影响。方法 采用薄膜-超声分散法制备载4-HPR-L。体外分别培养A375及B16F10黑素瘤细胞,分为3组,空白对照组仅加入细胞和新鲜培养基,4-HPR组和4-HPR-L组分别加入同浓度4-HPR和4-HPR-L。细胞增殖抑制实验（CCK-8法）检测各组细胞增殖情况;Hoechst33258染色法和流式细胞仪检测细胞凋亡情况;细胞划痕实验检测细胞迁移能力;通过激光共聚焦显微镜观察4-HPR-L入胞情况。采用SPSS22.0软件进行统计分析,多组间数据比较采用单因素方差分析,两组间比较采用t检验。结果 4-HPR与4-HPR-L对A375和B16F10的增殖抑制作用均表现出浓度依赖,0.1、1、15、30、50、70 mg/L 4-HPR和4-HPR-L处理A375和B16F10细胞48 h后,4-HPR组A375细胞存活率分别为（94.3 ± 1.4）%、（91.7 ± 2.5）%、（84.4 ± 2.5%）、（78.8 ± 2.1）%、（59.0 ± 1.1）%、（42.8 ± 2.0）%,4-HPR-L组分别为（86.0 ± 0.2）%、（76.5 ± 0.6）%、（60.9 ± 1.5）%、（49.0 ± 0.5）%、（32.9 ± 0.2）%、（18.9 ± 0.5）%,同浓度两组间比较,t值分别为8.019、8.298、11.455、19.978、33.672、16.314,均P < 0.01;4-HPR组B16F10细胞存活率分别是（95.4 ± 1.9）%、（90.5 ± 2.6）%、（77.0 ± 0.8%）、（64.4 ± 3.5）%、（59.1 ± 2.9）%、（49.9 ± 1.9）%,4-HPR-L组分别是（88.4 ± 2.0）%、（80.9 ± 3.4）%、（60.9 ± 2.2）%、（51.5 ± 2.9）%、（41.1 ± 1.2）%、（33.5 ± 2.4）%,同浓度两组间比较,均P < 0.05。相同浓度下,4-HPR同浓度组A375和B16F10细胞存活率均高于4-HPR-L组。Hoechst33258染色显示,对照组、4-HPR组细胞无明显变化,而4-HPR-L组细胞体积变小,细胞质浓缩,细胞核裂解为碎块,产生凋亡小体。流式细胞仪检测显示,4-HPR-L组A375和B16F10细胞凋亡率均显著高于4-HPR组,均P < 0.01。细胞划痕实验显示,4-HPR-L较4-HPR能更好地抑制细胞移行,显著降低划痕的愈合程度。激光共聚焦显微镜观察显示,C6脂质体入胞迅速。结论 4-HPR-L能更好地进入A375细胞、B16F10细胞,且能有效抑制A375、B16F10细胞的增殖和迁移,并诱导其凋亡。     

短发卡RNA抑制黑素瘤细胞BRAFV600E突变基因的研究

目的 探讨BRAF^V600E突变基因在黑素瘤发展过程中的作用。方法 用构建成功的质粒载体转染人黑素瘤细胞株A375,并建立动物模型来检测其在体内的干扰作用。分别采用RT-PCR和蛋白质印迹法检测体内外BRAF基因的mRNA和蛋白表达。结果 特异性短发卡RNA在体内外均可抑制人黑素瘤细胞BRAF基因mRNA和蛋白的表达,使其蛋白表达量分别减少62%和90%。在动物模型中使黑素瘤细胞的成瘤性下降,可以抑制肿瘤增长缓慢,抑瘤率达到62%。结论 BRAF^V600E突变基因在黑素瘤的发展中有一定的作用。     

白藜芦醇对人A375及鼠B16F10黑素瘤细胞增殖及凋亡的影响

目的:探讨白藜芦醇(resveratrol,Res)对人A375及鼠B16F10黑素瘤细胞增殖及凋亡的调节作用.方法:利用体外细胞培养技术、MTT法、流式细胞仪、光学显微镜技术,检测不同浓度的Res对人A375及鼠B16F10细胞增殖及凋亡的影响.结果:Res对人A375及鼠B16F10细胞均有显著的增殖抑制作用,呈剂量效应依赖性关系.结论:Res在体外可抑制恶性黑素瘤(MM)细胞的增殖.     

乙酰肝素酶-siRNA表达载体的构建及其抑制黑素瘤细胞株体外侵袭能力的观察

目的 构建针对人乙酰肝素酶（HPSE）基因的小干扰RNA（siRNA）及其表达载体，观察其对A375细胞HPSE基因的干扰作用及其对肿瘤细胞体外侵袭的抑制作用。方法 设计并构建了重组质粒pRNATU6.1/HPSE-siRNA，转染A375细胞，用实时荧光定量PCR法和蛋白质印迹法分别测定HPSE基因RNA和蛋白水平的表达变化，Matrigel侵袭实验观察A375细胞体外侵袭能力的改变。结果 将针对HPSE基因的siRNA的双链寡核苷酸片段正确克隆到pRNATU6.1载体；转染A375细胞，与对照组比较，HPSE-siRNA组HPSE基因和蛋白的表达均明显降低。转染后肿瘤细胞的体外侵袭能力与对照组相比受到明显抑制。结论 成功构建了针对HPSE基因的siRNA载体，HPSE-siRNA转染A375细胞可以显著降低细胞HPSE基因和蛋白的表达。     

内皮素受体B在黑素瘤中的表达及内皮素3对黑素瘤细胞A375的体外促生长效应

目的 研究内皮素受体B（ETR－B）在人恶性黑素瘤中的表达及内皮素3对黑素瘤细胞A375的体外促生长效应。方法 采用免疫组化SP法检测ETR－B在黑素瘤组织中的表达，用RT-PCR法检测ETR－B基因在人恶性黑素瘤细胞A375和SK－mel－1中的表达，用MTT比色法检测不同浓度内皮素3对黑素瘤细胞A375的体外促增殖活性。结果 ETR-B在41例恶性黑素瘤和23例色素痣中的阳性率分别为78.05%和8.69%，两组间差异有统计学意义，P < 0.05。15例原位和26例Ⅰ ~ Ⅳ期恶性黑素瘤ETR-B阳性表达率分别为53.33%和92.31%，两组比较，P < 0.05。13例转移性恶性黑素瘤（Ⅲ ~ Ⅳ期）中ETR-B阳性表达率100%，28例未转移者（0 ~ Ⅱ期）的阳性表达率为67.86%，两组比较，P < 0.05。ETR－B基因在人恶性黑素瘤细胞A375和SK－mel－1中均有表达；内皮素3对黑素瘤细胞A375在体外具有很强的促增殖作用，且促增殖能力呈内皮素3浓度依赖性。结论 内皮素3/ETR－B在促黑素瘤细胞生长中有重要作用