ARF4通过EGFR通路调控非小细胞肺癌细胞增殖、迁移、侵袭和凋亡

目的:探究ADP核糖基化因子4(ADP-ribosylation factor 4,ARF4)在非小细胞肺癌(non-small cell lung cancer, NSCLC)发生发展中的生物学作用及作用机制。方法:采用蛋白质印迹法(Western blot, WB)和免疫组化技术检测ARF4在临床NSCLC组织和癌旁组织中的表达情况;使用TCGA数据库分析ARF4与肺癌患者预后的相关性;使用siARF4敲降人NSCLC细胞系A549和H1975细胞中ARF4的表达水平,并用WB和实时定量PCR(real-time quantitative PCR,RT-qPCR)法进行验证;通过CCK-8实验检测敲低ARF4以及敲低ARF4与顺铂联用对NSCLC细胞增殖的影响;利用Transwell实验检测敲低ARF4以及敲低ARF4与顺铂联用对NSCLC细胞迁移及侵袭的影响;通过流式细胞术检测敲低ARF4以及敲低ARF4与顺铂联用对NSCLC细胞凋亡的影响;通过WB实验探究敲低ARF4后NSCLC中EGFR通路的变化。结果:ARF4在NSCLC组织中的表达较癌旁组织显著升高,且和患者的较差预后显...     

野生型INK4a/ARF基因共转染对A549细胞生物学行为的影响

背景与目的INK4a／ARF基因是重要的细胞周期调控基因，其表达产物p16INK4a和p14ARF蛋白分别在Rb和p53通路中发挥重要调节作用。本研究将野生型INK4a／ARF基因同时转染到该基因位点缺失的人肺腺癌A549细胞中，研究其对该细胞生物学行为的影响。方法利用阳离子脂质体介导的基因转染技术，将含有人全长野生型INK4a／ARF基因的真核表达重组质粒pcDNA3-p16INK4a和pcDNA3-p14ARF同时导入A549细胞系中，确定所编码蛋白p16INK4a和p14ARF稳定表达；在设立空白组和空质粒转染组的情况下，进行了转染前后细胞生长曲线、克隆形成率、周期分布、凋亡指数等指标的检测和分析。结果转染INK4a／ARF基因后的细胞G0／G1期比例为59．9％，与未转染（51．2％）及空质粒转染（50．3％）细胞相比差异显著（P值分别为0．043和0．025），同时S期和G2／M期的比例下降；转染后细胞克隆形成率为63％，未转染细胞和空质粒转染细胞分别为85％和87％（P值均〈0．01），凋亡指数明显增加，转染INK4a／ARF基因后的细胞凋亡率为8．0％，另两种细胞均为2．7％，差异具有统计学意义（P〈0．01）。结论阳离子脂质体可以有效地将INK4a／ARF基因导入A549细胞，并可以抑制A549细胞的生长，促进凋亡作用的增强，为将来肺癌的基因治疗提供了实验依据。     

Twist ARF和E-cadher in在大肠癌组织中的表达及临床意义

目的:探讨Twist、ARF、E-cadherin蛋白在大肠癌组织中的表达与临床病理参数的关系.同时分析Twist、ARF、E-cad-herin与大肠癌生长,侵袭转移之间的关系.方法:采用免疫组织化学SP染色方法对60例大肠癌标本及60例正常大肠黏膜标本分别进行Twist、ARF、E-cadherin蛋白检测.结果:Twist在肿瘤组织中的阳性表达率明显高于正常大肠黏膜(P<0.05);ARF及E-cadherin在肿瘤组织中的阳性表达率明显低于正常大肠黏膜(P<0.05),差异有统计学意义.Twist的表达与大肠癌的病理学分级、肠壁浸润深度、有无淋巴结转移、TNM分期相关(P<0.05).ARF、E-cadherin的表达与大肠癌的病理学分级、肠壁浸润深度、有无淋巴结转移、TNM分期、有无远处转移相关(P<0.05).Twist与ARF共同阳性者lO例,共同阴性者8例,两者呈显著性负相关(r=0.806.P=0.004).Twist与E-cadherin共同阳性者3例,共同阴性者7例,两者呈显著性负相关(r=0.754,P=0.006).结论:Twist的过度表达,ARF、E-cadherin蛋白的表达缺失在大肠癌的发生及侵袭转移中起重要的作用,Twist通过调节ARF/MDM2/p53途径抑制细胞凋亡促进肿瘤细胞形成,同时影响E-cadherin的表达而促进上皮-间质转化现象,参与大肠癌的发生和转移.     

降脂药对肝癌细胞增殖、侵袭及中性粒细胞外诱捕网形成的影响

目的 探讨降脂药对肝癌细胞增殖、干性特征、迁移、侵袭和中性粒细胞外诱捕网（NETs）形成的作用。方法 下调ASPP2或HMGCR基因建立胆固醇合成增加或减弱的小鼠肝癌细胞Hepa1-6，分别用辛伐他汀和黄连素两种降脂药处理。CCK-8和平板克隆实验检测肝癌细胞增殖能力；低吸附板成球和qRT-PCR分析细胞干性特征和相关基因表达；划痕和Transwell实验分析细胞迁移、侵袭的能力。免疫荧光染色分析NETs形成。结果 降脂药可显著抑制Hepa1-6细胞贴壁和成球生长及干性基因表达（P<0.001）；显著抑制Hepa1-6细胞迁移和侵袭能力（P<0.001）；显著抑制中性粒细胞诱导的Hepa1-6细胞侵袭和NETs的生成（P<0.001）。结论 降脂药通过抑制肝癌细胞的干性特征和NETs形成，抑制肝癌细胞增殖和转移，是治疗肝癌转移的一种潜在方式。     

CD44V6和β-catenin与骨肉瘤关系的研究进展

CD44V6与β-catenin是两种重要的细胞黏附分子（cell adhesion molecules，CAMs），通过介导细胞与细胞、细胞与基质间的相互作用，参与多细胞生物的多种生理及病理过程，是近年来研究的热点。CD44V6主要分布于上皮源性细胞和肿瘤细胞，介导异质细胞间的黏附，与细胞的集聚、淋巴细胞的再循环、淋巴细胞的激活、细胞的迁移、细胞间的信号传导及肿瘤细胞的侵袭、转移密切相关；β-catenin介导同质细胞间的黏附，在脊椎动物的身体形成和发育及肿瘤的发生、发展及侵袭转移过程中起重要作用。已有研究证明，CD44V6、β-catenin在多种恶性肿瘤如骨肉瘤、乳腺癌、胃癌及结直肠癌中表达增高，其表达水平与肿瘤细胞的浸润、转移能力成正相关。因此，CD44V6与β—cattenin能反映肿瘤细胞的增殖活性及肿瘤的恶性程度，可以作为肿瘤恶性表型的标志，尤其可以作为肿瘤的辅助性诊断及预测转移的指标之一。     

CD44与大肠癌关系的研究进展

大肠癌是消化系统中常见的恶性肿瘤,其发病率呈逐年上升趋势。癌的侵袭和转移是引起癌症患者死亡的主要原因。肿瘤转移过程需要细胞与细胞外间质或与其他细胞的一系列相互作用,其中肿瘤细胞表面的黏附分子在肿瘤侵袭和转移中起着极为重要的作用,如CD44,该分子作为透明质酸的受体,直接影响到肿瘤细胞与细胞外基质的结合能力,进而影响肿瘤的侵袭和转移。通过选择性剪接形成可将CD44分为10种剪接变异体（CD44v）。研究认为,v6外显子的变异体-CD44v6与细胞的运动、大肠肿瘤的发生、发展和侵袭、转移行为密切相关。     

p38MAPK抑制剂SB203580抑制人绒癌JAR细胞的体外侵袭作用

目的：研究p38MAPK通路在人绒癌JAR细胞体外侵袭中的作用。方法：用细胞ELISA法测定JAR细胞中p38MAPK的活性变化，用Transwell细胞侵入系统检测细胞的侵袭作用。结果：PMA呈浓度依赖性地激活JAR细胞中p38MAPK,PMA能促进人绒癌JAR细胞的体外侵袭作用，而p38特异性抑制剂SB203580抑制了JAR细胞的侵袭能力。结论：p38MAPK通路在人滋养细胞的侵袭行为以及人绒癌的形成中具有重要作用，P38抑制剂可能会为人绒癌的防治提供新的途径。     

G6PD对大肠癌细胞生长侵袭的影响及其与HKⅡ的相关性

目的 探讨G6PD对大肠癌HCT116和SW480细胞生长侵袭的影响,以及G6PD与HKⅡ在大肠癌及其癌旁上皮组织中的表达和两者的相关性。方法 将体外培养的大肠癌HCT116和SW480细胞进行G6PD过表达和干扰处理。Western blot法检测细胞中G6PD和HKⅡ的蛋白表达水平;流式细胞术分析细胞周期进程;葡萄糖氧化酶法检测培养液中葡萄糖的含量;划痕实验检测细胞侵袭能力;免疫组织化学法检测大肠癌及癌旁组织中G6PD与HKⅡ蛋白的表达水平。结果 过表达实验中,与Flag转染组相比,Flag-G6PD转染组HCT116和SW480两种细胞培养液中葡萄糖浓度、S期所占比例及侵袭能力均无明显变化;干扰实验中,与阴性对照转染组相比,G6PD-Homo-1504转染组两种细胞的葡萄糖浓度显著升高,S期所占比例和侵袭能力显著降低。G6PD与HKⅡ表达水平呈正相关。结论 干扰G6PD减少了大肠癌细胞葡萄糖的消耗,抑制了细胞的增殖和侵袭能力;同时G6PD对HKⅡ可能存在调控作用。     

敲低错配修复基因MSH6抑制结直肠癌细胞上皮间充质转化及其增殖

目的:探究错配修复基因MSH6对结直肠癌细胞的增殖、迁移和侵袭的影响及可能发生的机制。方法:根据美国国家生物信息技术中心(NCBI)中MSH6的基因序列构建靶向敲低MSH6的序列shMSH6-1、shMSH6-2和shMSH6-3,采用细胞转染技术敲低结直肠癌细胞中MSH6的表达,通过实时荧光定量 PCR 检测敲低效率并进行慢病毒包装,应用Western blot在细胞系中筛选MSH6高表达细胞系进行后续实验,应用CCK8检测细胞增殖能力,克隆形成实验检测细胞集落形成能力,伤口愈合和Transwell法检测细胞迁移和侵袭能力,通过Western blot方法检测上皮间充质相关蛋白的表达变化。结果:MSH6在结直肠癌细胞系中表达上调,其中RKO、SW620、LOVO细胞系上调明显,敲低MSH6明显限制了结直肠肿瘤细胞的增殖、迁移和侵袭,同时导致E-cadherin 蛋白水平增加,N-cadherin和Vimentin蛋白表达下降。结论:MSH6在结直肠癌中表达上调,敲低MSH6可抑制结直肠癌细胞的增殖、迁移和侵袭,诱导细胞发生凋亡并能够逆转EMT的发生。     

下调PRDX6对食管癌EC9706细胞增殖、迁移、侵袭及放射敏感性的影响

目的:探讨PRDX6表达水平对食管癌细胞增殖、迁移、侵袭及放射敏感性的影响。方法:应用GEPIA数据库分析食管癌组织PRDX6表达情况;将抑制基因PRDX6表达的“PRDX6-shRNA”质粒转染到EC9706细胞(sh-PRDX6组)。qRT-PCR和Western Blot检测转染效果。CCK8法检测增殖活力。划痕实验检测迁移能力。Transwell实验检测侵袭能力。克隆形成实验检测克隆形成能力。Western Blot检测AKT、p-AKT的蛋白表达量。结果:食管癌组织中PRDX6表达水平明显高于癌旁组织。qRT-PCR和Western Blot证实“PRDX6-shRNA”质粒有效抑制PRDX6基因表达。与对照组相比,sh-PRDX6组的细胞增殖、迁移、侵袭能力显著下降,放射敏感性增强。sh-PRDX6组细胞中AKT的表达水平无明显变化,而p-AKT表达水平明显下降。结论:PRDX6促进食管癌细胞EC9706的增殖、迁移、侵袭及放射抵抗。     

巨胞饮作用及其对脑胶质瘤的影响

巨胞饮作用是一种特殊的内吞作用。在肿瘤细胞中,巨胞饮作为一种营养递送的途径,使肿瘤细胞在营养缺乏的环境中存活。然而,在脑胶质瘤中,过度的刺激导致胶质瘤发生一种名为methuosis 的死亡方式。本综述就近几年关于巨胞饮的形成以及对脑胶质瘤作用的影响进行讨论。     

Inhibition of cell migration by PITENINs: the role of ARF6

We have reported previously the development of small-molecule phosphatidylinositol-3,4,5-trisphosphate (PIP3) antagonists (PITs) that block pleckstrin homology (PH) domain interaction, including activation of Akt, and show anti-tumor potential. Here we show that the same molecules inhibit growth factor-induced actin remodeling, lamellipodia formation and, ultimately, cell migration and invasion, consistent with an important role of PIP3 in these processes. In vivo, a PIT-1 analog displays significant inhibition on tumor angiogenesis and metastasis. ADP ribosylation factor 6 (ARF6) was recently identified as an important mediator of cytoskeleton and cell motility, which is regulated by PIP3-dependent membrane translocation of the guanine nucleotide exchange factors (GEFs), such as ADP-ribosylation factor nucleotide binding site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1). We demonstrate that PITs inhibit PIP3/ARNO or GRP1 PH domain binding and membrane localization, resulting in the inhibition of ARF6 activation. Importantly, we show that expression of the constitutively active mutant of ARF6 attenuates inhibition of lamellipodia formation and cell migration by PITs, confirming that inhibition of ARF6 contributes to inhibition of these processes by PITs. Overall, our studies demonstrate the feasibility of developing specific small-molecule targeting PIP3 binding by PH domains as potential anticancer agents that can simultaneously interfere with cancer development at multiple points.     

Involvement of a novel ADP-ribosylation factor GTPase-activating protein, SMAP, in membrane trafficking: implications in cancer cell biology

The endocytosis of cell membrane proteins is initiated by the binding of activated Arf6, a member of Ras-related GTPases, to the PM. A GAP specific for Arf6 triggers the budding of endocytotic vesicles from the PM by inactivating GTP-bound Arf6. We recently identified the SMAP gene that encodes an ArfGAP and is involved in the endocytosis of TfnR and possibly E-cadherin. In this review, we summarize the process of intracellular membrane trafficking, highlighting the roles played by the SMAP gene. Progression of cancer to malignancy occurs in parallel with the disappearance of E-cadherin, a central component of the adherens junction in epithelial cells. Therefore, elucidation of the molecular mechanism of E-cadherin endocytosis should be one of the key elements in tumor cell biology.     

非小细胞肺癌组织中Pokemon、P14ARF及E2F1的表达及其临床意义

目的:检测Pokemon、P14ARF及E2F1蛋白在非小细胞肺癌组织中的表达情况,探讨其在NSCLC发病中的作用和机制。方法:应用免疫组化方法检测NSCLC组和双正常对照组（即支气管对照组和肺泡对照组）中Pokemon、P14ARF及E2F1蛋白的表达情况,分析其与临床病理资料的关系及各指标间的相关性。结果:Pokemon蛋白在NSCLC组、肺鳞癌组、肺腺癌组中的表达均显著高于双对照组（P<0.05）,Pokemon蛋白表达与NSCLC的TNM分期相关（P<0.05）;E2F1蛋白在NSCLC组、肺鳞癌组、肺腺癌组中的表达均显著高于双对照组（P<0.05）,E2F1蛋白表达与NSCLC的TNM分期和淋巴结转移均有关（P<0.05）;P14ARF蛋白在NSCLC组、肺鳞癌组、肺腺癌组中的表达均显著低于双对照组（P<0.05）,P14ARF蛋白的表达水平与临床病理资料均无关（P>0.05）;Pokemon与P14ARF表达水平呈负相关（r=-0.57,P<0.01）,与E2F1表达水平呈正相关（r=0.37,P<0.05）,P14ARF与E2F1表达水平呈负相关（r=-0.48,P<0.01）。结论:非小细胞肺癌发病过程中,Pokemon通过拮抗P14ARF调节E2F1表达水平来发挥促癌作用。     

CDKN2A基因座在膀胱癌中的表达及临床意义

目的:探讨CDKN2A基因座两种编码产物p16INK4A和p14ARF在膀胱癌组织中的表达及其临床意义。方法:采用免疫组织化学技术SP法观察13例正常膀胱组织和52例膀胱尿路上皮癌组织中p16INK4A和p14ARF的表达情况。结果:p16INK4A在正常组织、膀胱癌中阳性表达率分别为84.62%(11/13)、40.38%(21/52),两组间差异显著(P<0.01)。p14ARF在正常膀胱组织、膀胱癌中阳性表达率分别为92.31%(12/13)、23.08%(12/52),两组间差异显著(P<0.01)。p16INK4A和p14ARF的阳性表达率与肿瘤病理分级、临床分期呈负相关(P<0.05);两者在膀胱癌中的表达没有相关性(P>0.05),但他们均与患者的预后正相关(P<0.05)。结论:p16INK4A和p14ARF在膀胱癌中的表达异常与膀胱癌的发生发展密切相关,但它们所起的生物学作用不尽相同,联合检测两者的表达可能为膀胱癌的早期诊断及预后判断提供帮助。     

The ARF protein in tumor suppression: lessons from mouse models and human tumors

The ARF protein is a key mediator of the activation of the p53 tumor suppressor in response to excessive mitogenic signals. ARF is encoded in the INK4a/ARF locus, together with the cell-cycle inhibitor p16INK4a. Mice genetically deficient for ARF show a marked predisposition to tumor formation, supporting an important role for ARF in tumor protection. Alterations of the INK4a/ARF locus are a highly frequent event in human tumors. In some instances, the alterations in the locus result in specific inactivation of ARF. This review will summarise the current knowledge about the biological function and regulation of ARF, and will discuss the evidence from genetically modified murine models, and human tumors which supports a relevant role for ARF in tumor suppression.     

Abrogation of ARF6 in promoting erastin-induced ferroptosis and mitigating capecitabine resistance in gastric cancer cells

BackgroundADP ribosylation factor 6 (ARF6) is a member of the Rat sarcoma virus (RAS) superfamily that is involved in the regulation of vesicular trafficking, membrane lipid remodeling, and signaling pathways. Our earlier work discovered that ARF6, as a downstream effector of the Kirsten rat sarcoma viral oncogene (Kras)/extracellular signal-regulated kinases (ERK) signaling pathway, may increase proliferation and induce the Warburg effect in gastric cancer (GC) cells. Additionally, ARF6 appears to be a potential biomarker for predicting the prognosis of GC. Ferroptosis has recently been described as a type of nonapoptotic iron-dependent cell death that is strongly associated with the Kras mutation. Therefore, it is critical to continue investigating the link between ARF6 and ferroptosis.MethodsWe first created ARF6 silenced cancer cell lines with lentivirus transfection. The knockdown efficiency was confirmed through quantitative polymerase chain reaction (qPCR) and western blotting. Subsequently, we used Cell Counting Kit-8 (CCK-8) and malondialdehyde (MDA) assay for lipid peroxidation measurement. Following this, qPCR and western blotting were conducted to clarify the mechanism involved. Finally, immunohistochemistry was used to stain human GC samples.ResultsOur findings established that, whereas ARF6 did not directly regulate lipid peroxidation, it did render GC cells susceptible to oxidative stress, particularly erastin-induced lipid peroxidation. Additionally, our research demonstrated that ARF6 may control capecitabine resistance via several routes.ConclusionsARF6 may play a critical role in the development of GC.     

Growth suppression by a p14(ARF) exon 1beta adenovirus in human tumor cell lines of varying p53 and Rb status

We have analyzed the ability of an adenoviral vector encoding the exon 1beta region of the p14(ARF) tumor suppressor (ARF) to suppress the growth and viability of an array of tumor cell lines of various origins and varying p53 and Rb status, in order to establish the clinical potential of ARF. An important activity of ARF is regulation of p53 stability and function through binding to the mdm2 protein. By sequestering mdm2, ARF may promote growth suppression through the Rb pathway as well because mdm2 can bind to Rb and attenuate its function. Whereas the high frequency of ARF gene deletion in human cancers, accounting for some 40% of cancers overall, suggests that ARF would be a strong candidate for therapeutic application, the possible dependence of ARF activity on p53 and Rb function presents a potential limitation to its application, as these functions are often impaired in cancer. We show here that a replication-defective adenovirus, Ad1beta, encoding the exon 1beta region of ARF is most effective in tumor cells expressing endogenous wild-type p53. Nevertheless, Ad1beta suppresses tumor cell growth and viability in vitro and in vivo, inducing G1 or G2 cell cycle arrest and cell death even in tumor cells lacking both functional Rb and p53 pathways, and independently of induction of the p53 downstream targets, p21, bax, and mdm2. These results point to an activity of ARF in human tumor cells that is independent of Rb or p53, and suggest that therapeutic applications based on ARF would have a broad clinical application in cancer.     

Tumor spectrum in ARF-deficient mice

The p19ARF product of the INK4a/ARF locus is induced in response to potentially oncogenic hyperproliferative signals and activates p53 by interfering with its negative regulator, Mdm2. Mice lacking ARF are highly prone to tumor development, and in this study, 80% of these animals spontaneously developed tumors and died within their first year of life. Mice that were heterozygous for ARF also developed tumors after a longer latency, whereas their wild-type littermates did not. In heterozygotes, tumor formation was accompanied by loss of the residual ARF allele and/or lack of ARF mRNA expression, implying that ARF can act as a canonical "two-hit" tumor suppressor gene. Tumors occurred earlier in life in ARF-null animals that were neonatally irradiated or given dimethylbenzanthrene, and several animals treated with carcinogen simultaneously developed multiple forms of malignancy arising from distinct cell lineages. Although p53-null mice primarily develop lymphomas and fibrosarcomas, the frequency of these two tumor types was inverted in ARF-null animals, with undifferentiated sarcomas predominating in a 3:2 ratio; 28% of ARF-null animals developed carcinomas and tumors of the nervous system, which have been rarely observed in untreated p53-null mice. The longer latency of tumor formation in ARF-null versus p53-null mice, therefore, appears to enable a broader spectrum of tumors to emerge.