小剂量依托泊甙对结肠癌细胞衰老的影响

目的:观察依托泊苷(etoposide, VP-16)对结肠癌细胞衰老的影响.方法:0～8 μg/mL VP-16作用结肠癌LS-174T细胞后,采用CCK-8(cell counting kit-8, CCK-8)法检测细胞增殖情况,FCM检测细胞凋亡及细胞周期改变,衰老相关β-半乳糖苷酶(senescence-associated β-galactosidase,SA-β-gal)染色检测细胞衰老情况,Western 印迹法检测p53、p16、RB、p21WAF-1/CIP1和E2F1蛋白的表达.结果:VP-16可以抑制结肠癌LS-174T细胞的增殖,且呈浓度依赖性;大剂量(2和4 μg/mL) VP-16可以促进结肠癌细胞凋亡;小剂量(1 μg/mL)VP-16作用后,结肠癌细胞呈现典型的大而扁平的衰老形态,SA-β-gal染色阳性,细胞周期阻滞于G2/M期.Western 印迹法检测结果显示,小剂量VP-16可以促进p21WAF-1/CIP1蛋白表达,抑制E2F1蛋白表达,而对p53、p16和RB蛋白表达无影响.结论:大剂量VP-16可促进结肠癌细胞凋亡;小剂量VP-16可促进结肠癌细胞衰老,其作用机制可能与VP-16促进细胞p21WAF-1/CIP1蛋白表达及抑制E2F1蛋白表达有关.     

p53β异构体在rmhTNF联合顺铂抑制胃癌细胞增殖中的作用

目的 探讨p53β异构体在rmhTNF联合顺铂干预人胃癌细胞MKN45和SGC7901生长实验中的生物学功能。方法 不同浓度rmhTNF单独或联合顺铂作用于人胃癌细胞MKN45和SGC7901,应用细胞增殖/毒性检测试剂盒（CCK-8试剂盒）检测抑制率;巢式反转录多聚酶链反应（RT-PCR）检测p53β和bcl-2 mRNA的表达变化情况。结果 rmhTNF单独或联合顺铂（4 μg/ml）作用MKN45细胞24 h,联合组抑制率大于单独组,且随rmhTNF浓度的增加而增加,组间差异有统计学意义（P<0.05）;在SGC7901细胞中,联合组抑制率虽有增加但差异无统计学意义（P>0.05）。rmhTNF单独使用对MKN45细胞中p53β和bcl-2表达无影响,差异无统计学意义（F=0.006,P>0.05;F=1.179, P>0.05）,而与顺铂联合作用可明显上调p53β和下调bcl-2的表达,并且随rmhTNF浓度的增加,p53β逐渐增加,bcl-2逐渐减少,差异有统计学意义（F=18.577,P<0.01;F=169.309, P<0.01）。在SGC7901细胞中未见p53β表达,但bcl-2高表达。rmhTNF和顺铂单独或联合作用时bcl-2虽有降低但差异无统计学意义（F=1.340, P>0.05）。p53β与bcl-2表达呈负相关（r=-0.897, P<0.01）,细胞抑制率与bcl-2的表达呈负相关（r=-0.906, P<0.01）。结论 rmhTNF和顺铂对p53β阳性的胃癌细胞MKN45表现出明显的抑制效应且rmhTNF和顺铂联合作用时可表现出协同抗肿瘤作用,其协同抗肿瘤效应可能是通过p53β调节下游分子bcl-2实现的。     

IncRNA MAFG-AS1及miR-143-3p对宫颈癌细胞增殖、凋亡的影响及机制研究

目的 探讨lncRNA MAFG-AS1和miR-143-3p对宫颈癌细胞增殖和凋亡的影响及其潜在的作用机制。方法 MAFG-AS1和miR-143-3p表达载体转染至宫颈癌细胞Hela后,采用qRT-PCR检测宫颈癌组织及相应癌旁正常组织,宫颈癌细胞Hela和正常宫颈细胞株Ect1/E6E7中miR-143-3p和MAFG-AS1 mRNA的表达水平;Western blot检测增殖相关蛋白Bcl-2、Cyclin D1及凋亡相关蛋白Bax、cleaved-Caspase-3、p21、p27的表达;MTT法检测细胞增殖活性;流式细胞术检测细胞凋亡;双荧光素酶报告基因突变检测lncRNA MAFG-AS1与miR-143-3p的相互作用。结果 与癌旁正常组织相比,宫颈癌组织中MAFG-AS1 mRNA表达升高（0.905±0.115 vs 2.835±0.164,t=43.091,P<0.001）,miR-143-3p表达降低（0.944±0.075 vs 0.382±0.071,t=24.336,P<0.001）;相较于正常宫颈细胞株Ect1/E6E7,宫颈癌细胞Hela中MAFG-AS1 mRNA的表达显著升高（P<0.001）,miR-143-3p表达显著降低（P<0.001）。下调MAFG-AS1和过表达miR-143-3p均可抑制Hela细胞增殖活性,促进细胞凋亡;抑制Bcl-2和Cyclin D1蛋白表达,促进Bax、Caspase-3、p21、p27蛋白表达。MAFG-AS1可靶向调控miR-143-3p表达,且抑制miR-143-3p表达可逆转下调MAFG-AS1表达对Hela细胞抑制细胞增殖、促进细胞凋亡作用。结论 lncRNA MAFG-AS1可抑制宫颈癌细胞增殖,促进凋亡,其机制可能与靶向调控miR-143-3p有关。     

食管鳞状细胞癌HPV 16/18型感染与p53表达的相关性研究

目的 探讨人乳头状瘤病毒（HPV）16/18型在食管鳞状细胞癌中的感染情况及其与p53的关系。方法 采用聚合酶链式反应（polymerase chain reaction,PCR）检测56例食管鳞状细胞癌手术切除组织（鳞癌组）和24名健康者正常食管组织（对照组）中HPV l6/18 E6、E7基因的表达,并根据其判断HPV感染情况;采用免疫组化SP法检测p53蛋白的表达水平。 结果 鳞癌组HPV感染率和p53蛋白阳性表达率均高于对照组（44.6% vs 12.5%,χ²=7.630,P=0.006;41.1% vs 4.2%,χ²=10.896,P<0.001）。在食管鳞癌组织中,HPV 16/18感染与病理分级、TNM分期和淋巴结转移有关（P<0.05）;且HPV16/18感染与p53蛋白表达呈正相关（r=0.565,P<0.001）。Kaplan-Meier生存分析结果显示,HPV感染阳性、p53表达阳性患者中位OS均小于HPV感染阴性、p53表达阴性者（21个月 vs 39个月,χ²=4.306,P=0.038;17个月 vs 41个月,^χ2=5.868, P=0.015）。控制相关的潜在混杂因素后,Cox回归模型显示HPV感染（HR=1.834,95%CI：1.010~3.330,P=0.046）和p53阳性表达（HR=2.189,95%CI：1.182~4.054, P=0.013）均可增加食管鳞状细胞癌患者的死亡风险。结论 食管鳞状细胞癌中HPV感染和p53阳性表达均较高,可能共同促进食管鳞状细胞癌的发生、发展并影响其预后。     

野生型p53基因转染对卵巢癌细胞生长及药物敏感性影响

目的:探讨野生型p53基因转染对人卵巢癌细胞株SKOV-3的体外生长及裸鼠体内致瘤性抑制作用,并了解p53基因与SKOV-3细胞对顺铂敏感性之间的关系.方法:利用脂质体介导,将含有人野生型p53cDNA的真核表达质粒pCB6-p53导入SKOV-3细胞中,观察该细胞体外生长和裸鼠体内致瘤性改变;应用流式细胞仪检测细胞周期及凋亡情况,MTT法检测细胞药物敏感性改变.结果:免疫组化法证实外源性p53基因在阳性细胞克隆稳定存在;转染野生型p53基因的SKOV-3细胞体外生长速率下降,裸鼠体内致瘤性丧失;G1/G0期细胞百分比(81.5%)和凋亡细胞百分比(11%)增高;p53表达阳性的SKOV-3细胞对顺铂的敏感性明显增强.结论:野生型p53基因转染能使SKOV-3细胞出现生长抑制和对顺铂的化疗敏感性增强.     

顺铂、得力生对胰腺癌细胞株生长和凋亡的作用

目的:体外研究胰腺癌细胞株p1,p2,p3,p4,p7的Survivin、Bc l-2和p53蛋白的表达情况,顺铂、中药得力生对胰腺癌细胞株的细胞增殖和凋亡的作用,研究细胞毒性药物与中药对胰腺癌细胞的联合作用,以及相关蛋白表达对上述作用的影响。方法:免疫组化检测上述蛋白在细胞中的表达;MTT方法检测细胞的增殖;TUNEL原位检测细胞的凋亡。结果:5种细胞株中均有不同程度Survivin和p53蛋白的表达,均无Bc l-2的表达。Survivin在p1、p3表达最强,在p2表达最弱;p3主要表达在细胞浆,其余4种细胞均表达在细胞核及细胞浆;p53在p2和p4细胞中表达率最高,为60%,但是在p2细胞中表达较弱。顺铂、中药得力生对胰腺癌细胞株的细胞增殖均具有抑制作用,且有协同趋势;Survivin和p53的表达程度与顺铂对胰腺癌细胞增殖的作用呈一定的反向关系;顺铂和中药得力生诱导下均检测到凋亡细胞。结论:Survivin和p53的表达可能调节胰腺癌细胞对顺铂的药物敏感性;中西医结合治疗胰腺癌可能具有很好的应用前景。     

野生型p53基因转染对卵巢癌细胞生长及药物敏感性影响

目的：探讨野生型p53基因转染对人卵巢癌细胞株SKOV-3的体外生长及裸鼠体内致瘤性抑制作用。并了解p53基因与SKOV-3细胞对顺铂敏感性之间的关系。方法：利用脂质体介导,将含有人野生型p53cDNA的真核表达质粒pCB6-p53导入SKOV-3细胞中,观察该细胞体外生长和裸鼠体内致瘤性改变;应用流式细胞仪检测细胞周期及凋亡情况,MTT法检测细胞药物敏感性改变。结果：免疫组化法证实外源性p53基因在阳性细胞克隆稳定存在;转染野生型p53基因的SKOV-3细胞体外生长速率下降,裸鼠体内致瘤性丧失;G1/G0期细胞百分比（81.5%)和凋亡细胞百分比（11%）增高;p53表达阳性的SKOV-3细胞对顺铂的敏感性明显增强。结论：野生型p53基因转染能使SKOV-3细胞出现生长抑制和对顺铂的化疗敏感性增强。     

宫颈及宫颈癌间质成纤维细胞的分离、鉴定与衰老检测研究

背景与目的：宫颈癌作为最常见的妇科恶性肿瘤之一,近年来发病呈年轻化趋势。目前认为人类乳头瘤病毒(human papillomavirus,HPV)的感染,特别是高危型HPV感染是其发生的主要因素。已有研究证实,在肿瘤发生的微环境中,间质成纤维细胞衰老可能促进上皮性肿瘤的发生,但宫颈癌的发生是否也伴有间质成纤维细胞的衰老鲜见报道。本研究拟对正常宫颈和宫颈癌间质成纤维细胞进行特异性研究,以揭示慢性炎症诱导宫颈癌发生的机制,旨在为宫颈癌的诊治提供新的理论依据。方法：分离纯化正常宫颈成纤维细胞(normal fibroblasts,NFs)和宫颈癌相关成纤维细胞(cancer-associated fibroblasts,CAFs),通过ELISA法检测细胞因子白介素-6(interleukin-6,IL-6)和血管内皮生长因子(vascular endothelial growth factor,VEGF)的分泌水平;并用衰老相关β-半乳糖甘酶染色(senescence-associated β-galactosidase,SA-β-gal)、细胞计数和蛋白质印记法(Western blot)分别鉴定细胞衰老、绘制细胞生长曲线和检测NFs与CAFs中p16蛋白表达。结果：CAFs分泌的细胞因子IL-6和VEGF比NFs高2倍以上(P<0.05);CAFs中SA-β-gal的活性比NFs高;而CAFs中p16水平的表达水平比NFs中高。结论：间质成纤维细胞衰老可能与宫颈癌的发生和HPV密切相关,研究细胞因子介导的宫颈癌间质衰老可能有助于宫颈癌的预防、诊断和治疗。     

TP与PF方案同期放疗治疗食管癌的疗效对比

目的观察紫杉醇加顺铂化疗同步放疗与顺铂加氟尿嘧啶化疗同步放疗治疗中晚期食管癌的疗效和不良反应。方法将72例食管癌患者随机分成紫杉醇加顺铂化疗(TP组)同步放疗组和顺铂加氟尿嘧啶化疗(PF组)同步放疗组各36例。TP组采用紫杉醇:135～165 mg/m²,d1,顺铂：20～30 mg/m²,d1～3,21天一个周期,共3～4周期。PF组采用顺铂：20～30 mg/m²,d1～3,氟尿嘧啶：500 mg/m²,d1～5,21天一个周期,共3～4周期。两组放疗方法相同,总剂量GTV：(60～66)Gy/(30～33)F。结果两组近期疗效比较差异无统计学意义(P=0.691)。TP组和PF组的1年和3年生存率：TP组分别为86%和53%;PF组分别为83%和50%。两组比较差异无统计学意义(χ²=0.148,P=0.701)。不良反应的比较差异无统计学意义。结论紫杉醇加顺铂化疗同步放疗与顺铂加氟尿嘧啶化疗同步放疗食管癌相比疗效相当且不增加不良反应。     

MCUR1表达对卵巢癌细胞生长的影响及作用机制

 目的 探讨线粒体钙单向转运体调节分子1（mitochondrial calcium uniporter regulator 1，MCUR1）与卵巢癌患者预后的关系，及其对卵巢癌细胞增殖和凋亡的影响和可能的作用机制。方法 从TCGA（the cancer genome atlas）数据库下载372例卵巢癌患者数据集，采用Kaplan-Meier法分析MCUR1与卵巢癌患者预后的关系。采用免疫组织化学法检测MCUR1在卵巢癌组织中的表达水平。转染MCUR1干扰片段及过表达质粒至卵巢癌Ａ2780细胞（siMCUR1组和MCUR1组），分别设置相应阴性对照（siCtrl组和EV组）。采用qRT-PCR和Western blot实验检测转染效果。采用MTS法、克隆形成实验检测细胞增殖情况。裸鼠皮下荷瘤后采用免疫组织化学法和组织TUNEL法检测增殖和凋亡情况，Western blot检测细胞增殖和凋亡相关分子的表达情况。结果 MCUR1高表达组中位生存期高于低表达组（53.7 个月 vs 41.3 个月，P=0.003）。与siCtrl组相比，siMCUR1组细胞增殖能力升高（P<0.01），裸鼠皮下荷瘤生长速度加快（P<0.01），Ki67表达升高（P<0.01），细胞凋亡能力减弱（P<0.05），p53、Caspase-3和Bax蛋白表达下调，而Bcl2、Cyclin D1和Cyclin E蛋白表达上调；与EV组相比，MCUR1组细胞增殖能力下降（P<0.01），裸鼠皮下荷瘤生长速度减缓（P<0.01），Ki67表达降低（P<0.01），细胞凋亡能力增强（P<0.05），p53、Ｃaspase-3和Bax蛋白表达上调，而Bcl2、Cyclin D1和Cyclin E蛋白表达下调。结论 MCUR1通过调控p53通路进而调控卵巢癌细胞的增殖与凋亡，MCUR1可能作为抑癌因子在卵巢癌中发挥作用，有望成为卵巢癌诊断及治疗靶点。     

腺病毒介导的p16基因表达对体外细胞增殖和衰老的影响

Objective To investigate the influence of exogenous p16 expression on cell proliferation and senescence in normal and tumor cells. Method Genes encoding p16 and GFP were subcloned into an adenovirus vector,then recombinant adenovirus was produced in 293 cells and purified for subsequent infection into MEF and SMMC7721 cells. Cell proliferation was examined by CCK8 assay and senescence by SA-β-gal staining. Results The number of senescent MEF cells was much higher in cultures after infection with adenovirus expressing p16 than after infection with control adenovirus （15±5 vs 0 senescent cells/field）, and p16-expressing cultures showed significantly lower proliferation on days 1-4［（6.8±0.25）%,（10.6±0.68）%,（12.4±0.93）% and（45.7±1.13）%;P<0.01］. In contrast, no significant differences in proliferation or senescence were observed between control SMMC-7721 cultures or cultures expressing p16. Conclusion Expression of p16 alone is insufficient to induce cell growth arrest and senescence in tumor cells.     

A2M介导Fas信号传导途径提高卵巢癌对铂类化疗的敏感性研究

目的 探讨A2M基因的表达与卵巢癌患者耐药及预后的关系及其对Fas信号传导通路的影响。方法 利用癌症基因图集（The Cancer  Genome Atlas,TCGA）数据库中铂类敏感和耐药的卵巢癌患者的基因表达谱,分析A2M基因在铂类敏感和耐药患者中的表达差异。利用已构建的带有绿色荧光标记的卵巢癌SKOV3敏感细胞（SKOV3-GFP）和顺铂耐药细胞（SKOV3-GFP/DDPⅡ）进行裸鼠皮下种植,成瘤后分次予以顺铂体内干预,以实时荧光定量PCR（real-time quantitative PCR,qRT-PCR）技术检测不同次数顺铂干预后的肿瘤组织中A2M mRNA与Fas信号传导通路上重要节点基因的表达水平。用双变量线性回归分析A2M mRNA与所检测基因的表达量的相关性。结果 相对于铂类敏感病例,A2M mRNA在铂类耐药患者中的表达下降（P<0.05）,A2M mRNA的表达与耐药相关（P=0.02）,与卵巢癌患者总生存期、化疗间隔生存期和无疾病进展生存期高度相关（P均<0.001）,其表达量越高,患者生存期越长。A2M mRNA以及细胞凋亡相关的Fas信号传导通路上重要节点基因Fas、FADD、caspase10、caspase9 和caspase3随顺铂注射次数的增加,在耐药的移植瘤组织中相对低表达（P<0.001）;而其下游与DNA修复相关的基因PARP1随顺铂注射次数的增加,在耐药的移植瘤组织中相对高表达（P<0.05）。A2M mRNA的表达与Fas信号传导通路上节点基因的表达存在线性相关（P<0.05）。结论 A2M可能是卵巢癌顺铂耐药的潜在信号分子,它可能维持肿瘤细胞对铂类药物的敏感性。其在耐药细胞中的低表达可能通过某种机制抑制下游的Fas信号传导通路,使其传导失调,最终引起PARP1的表达上调,促进DNA修复,抑制细胞凋亡,诱发卵巢癌细胞对顺铂的耐药。     

Molecular signature of oncogenic ras-induced senescence

Senescence irreversibly arrests the proliferation of cells that have sustained significant cellular stress. Replicative senescence, due to the shortening and dysfunction of telomeres, appears to provide a barrier to the immortalization of cells and development of cancer. In normal human fibroblasts, senescence induced by oncogenic H-ras displays a nearly identical cellular phenotype to that of replicative senescence, suggesting the activation of a common senescence mechanism. In this study, we investigated the gene expression profile of oncogenic H-ras-induced senescent human diploid fibroblasts. We found altered gene expression of various cell cycle regulators in both oncogenic H-ras-induced senescent cells and replicative senescent cells. Similar to replicative senescent cells, H-ras-induced senescent cells exhibited specific downregulation of genes involved in G2/M checkpoint control and contained tetraploid cells that were arrested in a G1 state. This observation suggests that the inactivation of G2/M checkpoints may be involved in senescence and may play a role in the generation of senescent G1 tetraploid cells. Lastly, we have identified two genes, topoisomerase IIalpha and HDAC9, whose expression was specifically altered under several conditions associated with senescence, suggesting that these two molecules may be novel biomarkers for senescent human fibroblasts.     

Temporally distinct roles for tumor suppressor pathways in cell cycle arrest and cellular senescence in Cyclin D1-driven tumor

ABSTRACT: BACKGROUND: Cellular senescence represents a tumor suppressive response to a variety of aberrant and oncogenic insults. We have previously described a transgenic mouse model of Cyclin D1-driven senescence in pineal cells that opposes tumor progression. We now attempted to define the molecular mechanisms leading to p53 activation in this model, and to identify effectors of Cyclin D1-induced senescence. Results: Senescence evolved over a period of weeks, with initial hyperproliferation followed by cell cycle arrest due to ROS production leading to activation of a DNA damage response and the p53 pathway. Interestingly, cell cycle exit was associated with repression of the Cyclin-dependent kinase Cdk2. This was followed days later by formation of heterochromatin foci correlating with RB protein hypophosphorylation at Cdk4-dependent sites. In the absence of the Cdk4-inhibitor p18Ink4c, cell cycle exit was delayed but most cells eventually showed a senescent phenotype. However, tumors later arose from this premalignant, largely senescent lesion. We found that the p53 pathway was intact in tumors arising in a p18Ink4c-/- background, indicating that the two genes represent distinct tumor suppressor pathways. Upon tumor progression, both p18Ink4c-/- and p53-/- tumors showed increased Cdk2 expression. Inhibition of Cdk2 in cultured pre-tumorigenic and tumor cells of both backgrounds resulted in decreased proliferation and evidence of senescence. Conclusion: Our findings indicate that the p53 and the RB pathways play temporally distinct roles in senescence induction in Cyclin D1-expressing cells, and that Cdk2 inhibition plays a role in tumor suppression, and may be a useful therapeutic target.     

Cyclin D1、MMR、FIB与结肠癌预后的相关性分析

目的 分析细胞周期蛋白D1（Cyclin D1）、错配修复蛋白（mis-match repair，MMR）、纤维蛋白原（fibrinogen，FIB）与结肠癌预后的关系。方法 回顾性分析210例结肠癌根治术患者的临床病理资料。采用免疫组化法检测Cyclin D1、MMR的表达，凝固法检测静脉血中FIB的浓度，并分析三者与结肠癌患者术后生存的关系。结果 Cyclin D1低表达的结肠癌患者1年、3年、5年生存率均高于高表达患者（Log-rank χ²=9.878，P=0.002）；错配修复基因缺失（mis-match repair genes deletion，dMMR）的患者1年、3年、5年生存率亦较错配修复基因完整（mis-match repair genes complete，pMMR）患者高（Log-rank χ²=5.138，P=0.023）；术前FIB正常的患者1年、3年、5年生存率高于FIB异常患者（Log-rank χ²=9.490，P=0.002）。Cyclin D1高表达、FIB异常为结肠癌预后的独立危险因素（HR=1.931， 95%CI：1.131~3.296；HR=2.029，95%CI：1.170~3.519）。结论 Cyclin D1高表达、pMMR及术前FIB异常的结肠癌患者预后不良。     

ESE-3, an Ets family transcription factor, is up-regulated in cellular senescence

Normal cells irreversibly stop dividing after being exposed to a variety of stresses. This state, called cellular senescence, has recently been demonstrated to act as a tumor-suppressing mechanism in vivo. A common set of features are exhibited by senescent cells, but the molecular mechanism leading to the state is poorly understood. It has been shown that p38, a stress-induced mitogen-activated protein kinase (MAPK), plays a pivotal role in inducing cellular senescence in diverse settings. To better understand the senescence-inducing pathway, microarray analyses of normal human fibroblasts that ectopically activated p38 were performed. It was found that five genes encoding ESE-3, inhibin betaA, RGS5, SSAT and DIO2 were up-regulated in senescent cells induced by RasV12, H(2)O(2) and telomere shortening, but not in quiescent or actively growing cells, suggesting that these genes serve as molecular markers for various types of cellular senescence. The ectopic expression of ESE-3 resulted in retarded growth, up-regulation of p16(INK4a) but not of p21, and increased levels of SA-beta-gal activity. In contrast, RGS5, SSAT and the constitutive active form of the inhibin betaA receptor gene did not induce such senescence phenotypes when ectopically expressed. ESE-3 expression increased the activity of the p16(INK4a) promoter in a reporter assay, and recombinant ESE-3 protein bound to the Ets-binding sequences present in the promoter. These results suggest that ESE-3 plays a role in the induction of cellular senescence as a downstream molecule of p38.     

Effect of p16INK4a on chemosensitivity in nasopharyngeal carcinoma cells

The p16INK4a tumor suppressor gene is frequently inactivated in nasopharyngeal carcinoma (NPC) and hence it may play an important role in the suppression of this tumor. In order to study the effect of p16INK4a restoration in NPC cells, full-length human p16INK4a gene was transfected into a NPC cell line, CNE1. Four individual clones with differential levels of p16INK4a protein expression, were selected for further studies. The introduction of p16INK4a into CNE1 cells induced growth suppression through G0/G1 cell cycle arrest; however, the cell growth rate was not correlated to the levels of p16INK4a protein expression. To study whether transfection of p16INK4a could protect NPC cells from radiation, cisplatin and 5-fluorouracil (5FU), the cellular sensitivity of p16INK4a transfectants and vector control were investigated. An increase in sensitivity to 5FU was observed (2-fold compared to IC50) in all 4 clones compared to vector-transfected control. P16INK4a transfection also resulted in increased sensitivity to cisplatin (1.5-1.8-fold) in 3 out of 4 cell lines. However, no difference in radiosensitivity was found between the p16INK4a transfectants and the control. These findings indicate that p16INK4a suppresses NPC cell growth through G0/G1 arrest and modulating cellular response to chemotherapeutic drugs in NPC cells. Therefore, restoration of p16INK4a may have a therapeutic purpose in the treatment of NPC.     

Knockdown of AGR2 induces cellular senescence in prostate cancer cells

Anterior-gradient 2 (AGR2), overexpressed in many tumors including prostate cancer (PCa), is implicated in stimulation of cell proliferation, adhesion, anti-apoptosis and cell cycle regulation. Here, a potential role of AGR2 in cellular senescence was investigated. We first observed that AGR2 was overexpressed in Chinese Han PCa tissues and had a positive correlation with cyclin D1 and p-Rb but not with p16(INK4a). AGR2 expression profiles varied among cell lines, with PC3 cells being the highest level, LNCaP and DU145 relatively less. The expression of cyclin D1 showed similar pattern to the AGR2 in cell lines. Knockdown of AGR2 caused a decrease in cell viability in PC3 cells, whereas forced expression of AGR2 led to an increased cell proliferation of LNCaP and DU145 cells. Importantly, AGR2 depletion resulted in accumulation of cells at the G(0)/G(1) phase and induction of cellular senescence in all three PCa cell lines as indicated by an increase of flat, enlarged and senescence-associated β-galactosidase (SA-β-Gal) positive cells. Senescent response to AGR2 silencing was also evidenced by elevated γH2AX and fluorescent punctuate formation of tri-methyl-histone H3 in AGR2-depleted cells. Further studies indicated that LNCaP underwent a p21(CIP1)-dependent cellular senescence in response to AGR2 depletion that requires inactivation of ERK signaling, whereas PC-3 was also p21(CIP1) dependent but involved in suppression of PI3K/Akt. Unlike LNCaP and PC-3, senescent response of DU145 was found to be mainly p27(KIP1) dependent that may require upregulation of PTEN and inhibition of PI3K/Akt signaling. Thus, these findings suggest a novel role of AGR2 in regulation of cellular senescence.