表皮生长因子受体/丝氨酸/苏氨酸蛋白激酶通路对小鼠黑素瘤B16细胞迁移的影响

目的:研究表皮生长因子受体(EGFR)/丝氨酸/苏氨酸蛋白激酶(AKT)通路对小鼠黑素瘤B16细胞迁移的影响.方法:蛋白质免疫印迹方法分析各蛋白的表达;phagokinetic track motility法观察细胞的迁移.结果:表皮生长因子(EGF)能促进小鼠黑素瘤B16细胞迁移;AKT抑制剂(wortmannin)和水通道蛋白-3(aquaporins-3,AQP3)抑制剂(CuSO4)能抑制小鼠黑素瘤B16细胞迁移;EGF可诱导AKT磷酸化,EGF处理后5 min磷酸化AKT达到高峰.wortmannin和CuSO4可抑制细胞中AQP3的表达.结论:在小鼠黑素瘤B16细胞中,EGF通过磷酸化EGFR,激活AKT,进而使AQP3表达上调,促进B16细胞迁移.这一通路可被AKT和AQP3抑制剂阻断,这些涉及的信号通路可能成为潜在的治疗黑素瘤的靶点.     

依巴斯汀通过抑制AKT/mTOR通路诱导人黑素瘤细胞自噬

目的：研究依巴斯汀对人黑素瘤细胞自噬的影响及机制。方法：体外培养人黑素瘤细胞A375和M14,采用CCK-8增殖实验检测细胞活力,并计算IC50;利用mCherry-EGFP-LC3B双荧光指示系统检测自噬流;采用Western blot验证自噬相关蛋白LC3,Beclin1及信号通路蛋白的表达。结果：依巴斯汀可显著抑制人黑素瘤细胞的活力;依巴斯汀明显诱导人黑素瘤细胞中自噬小体和自噬溶酶体的产生;依巴斯汀显著上调人黑素瘤细胞中LC3-Ⅱ/Ⅰ的比值以及Beclin1的表达,同时抑制AKT/mTOR信号通路的活化,降低p-AKT/AKT和p-mTOR/mTOR。结论：依巴斯汀通过抑制AKT/mTOR通路诱导人黑素瘤细胞自噬的发生。     

PI3K/AKT信号通路及其与SLE的关系

PI3K/AKT信号通路是细胞内重要的信号传导通路之一,通过影响下游多种效应分子的活化调节细胞的基本功能,包括生存、生长、增殖和代谢.近年研究发现,T、B淋巴细胞PI3K/AKT信号通路的异常活化与SLE自身免疫的发生发展密切相关.概述PI3K/AKT信号通路的组成与功能,其与SLE发病机制的关系及其抑制剂在SLE治疗中的研究进展.     

CXCL12对人黑素瘤细胞株M14生物学行为的影响

目的:明确CXCL12对人黑素瘤细胞株M14体外增殖、迁移、侵袭的影响。方法:用不同浓度的CXCL12(0、10、50、100、200 ng/mL)刺激或诱导人黑素瘤细胞株M14,CCK8法检测细胞增殖,Transwell迁移和侵袭试验检测细胞迁移和侵袭能力。用100 ng/mL的CXCL12处理人黑素瘤细胞株M14,Western blot法检测处理后不同时间点(0、5、10、20、30、60 min)的ERK、p-ERK、AKT、p-AKT的表达。结果:CXCL12能够增强人黑素瘤细胞株M14细胞的体外增殖、迁移、侵袭能力,以及促进ERK、AKT信号通路的激活。结论:CXCL12可能与黑素瘤的进展过程相关。     

黑素瘤相关基因研究进展

利用二代高通量测序技术对黑素瘤基因进行测序分析,以找出关键突变位点进行靶向治疗是当前的研究热点.黑素瘤基因突变特点是:基因突变负荷大,丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号传导通路上关键基因突变和大量非编码基因突变,不同种族和组织学类型黑素瘤基因突变差异大....     

黑素瘤核因子-κB活性的研究进展

黑素瘤是一种侵袭性的皮肤肿瘤,人类黑素瘤中存在核因子-κB的上调,异常的核因子-κB信号通路参与黑素瘤增殖、凋亡及肿瘤转移、血管生成等病理生理进程,导致基因异常表达,加速病情的进展.核因子-κB信号转导通路、关键调节因子如TNFR/TRADD/TRAF、TRAIL/TRAILR、FasR/FasL/FAP-1及基因突变等在黑素瘤的发生发展中起重要作用.因此,核因子-κB信号通路的研究有可能为治疗黑素瘤提供新的思路和方法.     

p62通过调控PI3K/AKT通路促进黑素瘤增殖

目的探讨p62促进黑素瘤A375细胞生长增殖的作用及具体机制。方法取生长状态良好的黑素瘤A375细胞,采用干扰p62表达的siRNA片段转染A375细胞;EDU着色法检测干扰组和对照组的细胞分裂情况;采用Western blot检测干扰组和对照组PI3K/AKT/mTOR及下游因子的表达情况。结果干扰组的EDU着色数目较对照组明显减少;p62干扰后PI3K/AKT/mTOR及下游因子的表达明显受抑制。结论p62可能通过调控PI3K/AKT通路发挥促进黑素瘤增殖的作用。     

ERK1/2,JNK和P38MAPK在皮肤黑素瘤中的表达北大核心CSCD

目的检测ERK1/2,JNK和P38MAPK在皮肤黑素瘤中的表达情况,初步探讨它们与黑素瘤发生、发展的关系。方法分别采用免疫组化和Western印迹法检测磷酸化的ERK1/2,JNK及P38MAPK在皮肤黑素瘤、皮内痣组织中的表达情况;用实时荧光定量聚合酶链反应(Real time-PCR)方法定量分析皮内痣和皮肤黑素瘤中ERK1/2,JNK和P38MAPK mRNA的表达情况。结果免疫组化和Western印迹法结果均显示p-ERK1/2,p-JNK及p-P38MAPK在皮肤黑素瘤中的表达高于皮内痣组(P<0.05);Western印迹法显示p-ERK1/2,p-JNK及p-P38MAPK在皮内痣组中的表达高于正常对照组(P<0.05)。ERK1/2,JNK及P38MAPK mRNA在皮肤黑素瘤和皮内痣中的表达均高于正常皮肤组织(P<0.05);皮肤黑素瘤中ERK1/2,P38MAPKmRNA的表达均高于皮内痣(P<0.05);而皮肤黑素瘤中JNK mRNA的表达与皮内痣组相比,差异无统计学意义(P>0.05)。结论 ERK1/2,JNK和P38MAPK在皮肤黑素瘤表达增高,提示MAPK信号通路在皮肤黑素瘤发生、发展中起到重要作用,该信号通路有望成为预防和治疗皮肤黑素瘤的新靶点。     

ERK1/2,JNK和P38MAPK在皮肤黑素瘤中的表达

目的检测ERK1/2,JNK和P38MAPK在皮肤黑素瘤中的表达情况,初步探讨它们与黑素瘤发生、发展的关系。方法分别采用免疫组化和Western印迹法检测磷酸化的ERK1/2,JNK及P38MAPK在皮肤黑素瘤、皮内痣组织中的表达情况;用实时荧光定量聚合酶链反应(Real time-PCR)方法定量分析皮内痣和皮肤黑素瘤中ERK1/2,JNK和P38MAPK mRNA的表达情况。结果免疫组化和Western印迹法结果均显示p-ERK1/2,p-JNK及p-P38MAPK在皮肤黑素瘤中的表达高于皮内痣组(P0.05);Western印迹法显示p-ERK1/2,p-JNK及p-P38MAPK在皮内痣组中的表达高于正常对照组(P0.05)。ERK1/2,JNK及P38MAPK mRNA在皮肤黑素瘤和皮内痣中的表达均高于正常皮肤组织(P0.05);皮肤黑素瘤中ERK1/2,P38MAPKmRNA的表达均高于皮内痣(P0.05);而皮肤黑素瘤中JNK mRNA的表达与皮内痣组相比,差异无统计学意义(P0.05)。结论 ERK1/2,JNK和P38MAPK在皮肤黑素瘤表达增高,提示MAPK信号通路在皮肤黑素瘤发生、发展中起到重要作用,该信号通路有望成为预防和治疗皮肤黑素瘤的新靶点。     

MAPK/ERK信号传导通路在肿瘤治疗中作用机制的研究

丝裂原活化蛋白激酶/细胞外信号调节激酶(MAPK/ERK)信号传导通路在恶性肿瘤的发生和发展中有着重要的作用.其促进肿瘤生长的机制包括:促进肿瘤细胞增殖、抑制肿瘤细胞凋亡、促进肿瘤血管生成、诱导肿瘤组织侵袭等.MAPK/ERK信号传导通路有3个重要分子靶:Ras、Raf激酶及其MEK1/2和ERK1/2.理论上,干预Ras/Raf/MEK/ERK任何一种激酶都有可能阻止肿瘤的生长.因此,MAPK/ERK信号传导通路中某些关键信号元件的抑制剂成为近年来恶性肿瘤治疗中的一个新策略.同时,我们通过对近年相关文献的复习发现,某些刺激通过活化MAPK/ERK信号传导通路,可以激活下游的抑癌基因,进而导致肿瘤的生长抑制.所以,进一步完善MAPK/ERK信号通路在肿瘤治疗中的作用机制,将为肿瘤的治疗提供科学准确的思路.     

Combined targeting of MAPK and AKT signalling pathways is a promising strategy for melanoma treatment

BACKGROUND: In melanoma, several signalling pathways are constitutively activated. Among them, the RAS/RAF/MEK/ERK (MAPK) and PI3K/AKT (AKT) signalling pathways are activated through multiple mechanisms and appear to play a major role in melanoma development and progression. OBJECTIVES: In this study, we examined whether targeting the MAPK and/or AKT signalling pathways would have therapeutic effects against melanoma. METHODS: Using a panel of pharmacological inhibitors (BAY 43-9006, PD98059, U0126, wortmannin, LY294002) we inhibited the MAPK and AKT signalling pathways at different levels and evaluated the effects on growth, survival and invasion of melanoma cells in monolayer and organotypic skin culture. RESULTS: Antiproliferative and proapoptotic effects of inhibitors alone in monolayer culture were disappointing and varied among the different cell lines. In contrast, combined targeting of the MAPK and AKT signalling pathways significantly inhibited growth and enhanced apoptosis in monolayer culture. To verify our data in a more physiological context we incorporated melanoma cells into regenerated human skin mimicking the microenvironment of human melanoma. Combinations of MAPK and AKT inhibitors completely suppressed invasive tumour growth of melanoma cells in regenerated human skin. CONCLUSIONS: Combined targeting of MAPK and AKT signalling pathways is a promising strategy for melanoma treatment and should encourage further in-depth investigations.     

Sorafenib induces pigmentation via the regulation of β-catenin signalling pathway in melanoma cells

We conducted large-scale screening test on drugs that were already approved for other diseases to find pigmentation-modulating agents. Among drugs with potential for pigmentation control, we selected sorafenib and further investigated the effect on pigmentation using HM3KO melanoma cells. As a result of treating melanoma cells with sorafenib, pigmentation was promoted in terms of melanin content and tyrosinase activity. Sorafenib increased mRNA and protein levels of pigmentation-related genes such as MITF, tyrosinase and TRP1. To uncover the action mechanism, we investigated the effect of sorafenib on the intracellular signalling pathways. Sorafenib reduced phosphorylation of AKT and ERK, suggesting that sorafenib induces pigmentation through inhibition of the AKT and ERK pathways. In addition, sorafenib significantly increased the level of active β-catenin, together with activation of β-catenin signalling. Mechanistic study revealed that sorafenib decreased phosphorylation of serine 9 (S9) of GSK3β, while it increased phosphorylation of tyrosine 216 (Y216) of GSK3β. These results suggest that sorafenib activates the β-catenin signalling through the regulation of GSK3β phosphorylation, thereby affecting the pigmentation process.     

Activation of the PI3K/AKT signalling pathway in non‐melanoma skin cancer is not mediated by oncogenic PIK3CA and AKT1 hotspot mutations

Please cite this paper as: Activation of the PI3K/AKT signalling pathway in non‐melanoma skin cancer is not mediated by oncogenic PIK3CA and AKT1 hotspot mutations. Experimental Dermatology 2010; 19 : e222–e227. Abstract: Non‐melanoma skin cancer represents the most frequent human cancer entity. Activation of the PI3K/AKT signalling pathway has been reported both in squamous cell carcinoma (SCC) of the skin and in basal cell carcinoma (BCC). In many cancers, including SCC of the head and neck, the oesophagus and the penis, activation of this pathway is mediated by oncogenic PIK3CA and AKT1 mutations. We therefore screened 61 non‐melanoma skin cancer samples (30 SCC and 31 BCC) for the presence of activating PIK3CA and AKT1 mutations. PIK3CA hotspot mutations were analysed using a highly sensitive SNaPshot assay, and exon 4 of AKT1 was sequenced directly. In addition, immunohistochemistry was performed for phosphorylated AKT protein. Immunohistochemical expression of pAkt was observed both in SCC and in BCC samples, indicating an activation of the PI3K/AKT pathway. Although SCC showed higher expression levels than BCC, this difference was not significant. However, none of the 61 non‐melanoma skin cancer samples revealed any PIK3CA and AKT1 hotspot mutations at the investigated loci. We conclude that PIK3CA and AKT1 hotspot mutations do not contribute to the activation of the PI3K/AKT signalling pathway in non‐melanoma skin cancer. The distinct PIK3CA mutation spectrum between SCC of the skin and SCC of other tissues may reflect the different carcinogens which are involved into the mutagenesis of these cancers. PIK3CA and AKT1 hotspot mutations are obviously not caused by UV light exposure, the main risk factor in non‐melanoma skin cancer.     

The role of PI3'-lipid signalling in melanoma initiation,progression and maintenance

Phosphatidylinositol-3'-kinases (PI3Ks) are a family of lipid kinases that phosphorylate the 3' hydroxyl (OH) of the inositol ring of phosphatidylinositides (PI). Through their downstream effectors, PI3K generated lipids (PI3K-lipids hereafter) such as PI(3,4,5)P₃ and PI(3,4)P₂ regulate myriad biochemical and biological processes in both normal and cancer cells including responses to growth hormones and cytokines; the cell division cycle; cell death; cellular growth; angiogenesis; membrane dynamics; and autophagy and many aspects of cellular metabolism. Engagement of receptor tyrosine kinase by their cognate ligands leads to activation of members of the Class I family of PI3'-kinases (PI3Kα, β, δ & γ) leading to accumulation of PI3K-lipids. Importantly, PI3K-lipid accumulation is antagonized by the hydrolytic action of a number of PI3K-lipid phosphatases, most notably the melanoma suppressor PTEN (lipid phosphatase and tensin homologue). Downstream of PI3K-lipid production, the protein kinases AKT1-3 are believed to be key effectors of PI3'-kinase signalling in cells. Indeed, in preclinical models, activation of the PI3K→AKT signalling axis cooperates with alterations such as expression of the BRAF^V600E oncoprotein kinase to promote melanoma progression and metastasis. In this review, we describe the different classes of PI3K-lipid effectors, and how they may promote melanomagenesis, influence the tumour microenvironment, melanoma maintenance and progression to metastatic disease. We also provide an update on both FDA-approved or experimental inhibitors of the PI3K→AKT pathway that are currently being evaluated for the treatment of melanoma either in preclinical models or in clinical trials.     

Melanocyte receptors: clinical implications and therapeutic relevance

The activation or the inhibition of melanocyte-specific receptors offers novel means of augmenting normal melanocyte function, skin color, and photoprotection, or treating melanocytic disorders, namely at this time, metastatic melanoma. Melanocyte-specific receptors include melanocortin-1 (MCR1) and melatonin receptors. Other receptors that play an important role in melanoma progression are G-protein couple receptors such as Frizzled 5 and receptor tyrosine kinases such as c-Kit and hepatocyte growth factor (HGF) receptor. These receptors activate two crucial cell-signaling pathways, RAS/RAF/MEK/ERK and PI3K/AKT, integral to melanoma cell survival, and can serve as targets for therapy of disseminated melanoma. Activation of death receptors is another pathway that can be exploited with targeted therapeutics to control advanced melanoma. This article reviews the current understanding of melanocyte receptors, their agonists and inhibitors, and their potential to treat the melanocytic pathology.     

Suppression of mTOR complex 2-dependent AKT phosphorylation in melanoma cells by combined treatment with rapamycin and LY294002

Background  Inhibition of mTOR complex 1 (mTORC1) with rapamycin leads to phosphorylation of AKT in some cancer cells, with unknown biological consequences. The role of this phosphorylation in melanoma is unknown, although preliminary clinical data indicate poor activity of rapalogues in melanoma.
Objectives  We aimed at elucidating the role of AKT phosphorylation after mTORC1 inhibition in melanoma cells.
Methods  Western blotting, apoptosis assays, cell cycle analyses and viability assays were performed to analyse the effects of rapamycin and LY294002 treatment on melanoma cells. For suppression of mTOR complex 2 (mTORC2) an siRNA directed against rictor was used.
Results  Rapamycin showed limited effects on cell viability but resulted in strong and lasting AKT phosphorylation in melanoma cells. Combined PI3K/mTOR inhibition with LY294002 had pronounced effects on viability but also led to increased AKT phosphorylation after prolonged treatment. In contrast, combination of rapamycin plus LY294002 suppressed AKT phosphorylation. Suppression of AKT phosphorylation did not correlate with decreases in cell viability. Inhibition of mTORC2 led to reduced levels of phosphorylated AKT.
Conclusions  mTORC1 inhibition with rapamycin and with LY294002 can lead to AKT phosphorylation in melanoma cells via mTORC2. Combination of rapamycin and LY294002 suppresses AKT phosphorylation but without significant effect on treatment efficacy.