人肺癌相关抗原基因ALT04-AG表达抑制对人肺癌细胞株生长及基因表达谱的影响

目的探讨抑制人肺癌相关抗原基因ALT04-AG表达对细胞生长特性及相关基因表达的影响。方法重组反义ALT04-AGRNA真核表达质粒,经脂质体介导转染人肺癌细胞株(L78),以MTT、FCM法分析转染细胞生长特性,以Northern b lot、免疫组化染色及基因芯片技术检测相关基因的表达。结果构建了重组表达反义ALT04-AGRNA的真核表达质粒[pALT04-AG(as)],经该质粒转染及二氟甲基鸟氨酸(d ifluorom ethylorn ith ine,DFMO)处理的L78细胞均引起ALT04-AG表达下调及细胞的增殖抑制,后者还导致细胞凋亡比例增加。结论pALT04-AG(as)转染L78细胞或用DFMO抑制多胺生物合成,可通过对相关基因表达的调控促使L78细胞恶性表型逆转,而后者的作用更为广泛。本结果为探索肿瘤的诊断与治疗提供了有意义的线索。     

Precursor IGF-II (proIGF-II) and mature IGF-II (mIGF-II) induce Bcl-2 And Bcl-X L expression through different signaling pathways in breast cancer cells

IGF-II plays a crucial role in fetal and cancer development by signaling through the IGF-I receptor. We have shown that inhibition of IGF-II by resveratrol (RSV) induced apoptosis and that proIGF-II (highly expressed in cancer) was more potent than mIGF-II in inhibiting this effect. Thus, we hypothesized that IGF-II differentially regulates the signaling cascade of the IGF-I receptor to stimulate the anti-apoptotic proteins Bcl-2 and Bcl-X(L) to prevent apoptosis. RSV treatment to breast cancer cells inhibited Bcl-2 and Bcl-X(L) expression and induced mitochondrial membrane depolarization. ProIGF-II was more potent than mIGF-II in: (1) activating the PI3/Akt pathway, (2) regulating Bcl-2 and Bcl-X(L) expression, and (3) inducing phosphorylation/nuclear translocation of Cyclic AMP-responsive element binding protein. Furthermore, IGF-II differentially regulated the intracellular translocation of Bcl-2 and Bcl-X(L), a critical process in breast cancer progression to hormone-independence. Our study provides a novel mechanism of how proIGF-II promotes progression and chemoresistance in breast cancer development.     

Gene therapy using an adenovirus vector for apoptosis-related genes is a highly effective therapeutic modality for killing glioma cells

Preclinical studies in animal models and human clinical trials have evaluated the safety and efficacy of adenoviral vectors for cancer gene therapy. These studies have indicated that gene delivery via adenoviral vectors, including p53 gene therapy, represents a promising therapeutic modality for many types of human cancers. This review focuses on novel strategies to induce apoptosis in glioma cells by transduction with adenoviral vectors carrying a variety of apoptosis-related genes, including Fas ligand, Fas, FADD, caspase-8, p53, p33ING1, p73alpha, Bax, Apaf-1, caspase-9, IkappaBdN, caspase-3, Bcl-2, and Bcl-X(L). We conclude that adenoviral vector-mediated delivery of apoptosis-related genes other than p53 is a potentially useful gene therapy approach toward the treatment of human brain tumors.     

The cancer-testis gene,NY-ESO-1, is expressed in normal fetal and adult testes and in spermatocytic seminomas and testicular carcinoma in situ

Cancer/testis genes are potential targets for therapeutic genetic and immunologic approaches, and are highly expressed in a large variety of human cancers. However, they are not expressed in normal tissues, with the exception of the testis. The NY-ESO-1 gene is the most recently identified member of the cancer/testis family and its product is one of the most immunogenic tumor antigens. We used immunohistochemistry to investigate the expression of NY-ESO-1 in healthy human prenatal and adult testes and in 59 human testicular tumors of different subtypes. We found that NY-ESO-1 was expressed from 18 weeks until birth in human fetal testes. In the adult testis, NY-ESO-1 was strongly expressed in spermatogonia and in primary spermatocytes, but not in post-meiotic cells or in testicular somatic cells. NY-ESO-1 was not expressed in the Sertoli cells, Leydig cells, classical seminomas, or nonseminomatous germ cells in the 59 testicular tumors. In contrast, NY-ESO-1 was expressed both in carcinomas in situ, which are the earliest stage of testicular tumors (7 of 15 cases), and in spermatocytic seminomas, which are believed to be derived from spermatogonia or primary spermatocytes (8 of 16 cases). We conclude that NY-ESO-1 is a marker that can be used to follow the early progression of testicular tumorigenesis when the tumors present a similar pattern of expression to the cells from which they originated, although the later tumors cease to express NY-ESO-1.     

bcl-3基因通过cyclin D1及Bax蛋白影响人结肠癌RKO细胞的凋亡

目的:研究bcl-3基因对人结肠癌RKO细胞迁移及凋亡的影响及机制。方法:采用人bcl-3基因的RNA干扰慢病毒载体沉默人结肠癌RKO细胞bcl-3基因的表达后,划痕实验观察bcl-3基因沉默前后RKO细胞迁移能力的变化,Annexin V/PI双染色法检测bcl-3基因沉默前后RKO细胞凋亡率的变化,Western blot法检测bcl-3基因沉默前后细胞周期蛋白cyclin D1及凋亡相关蛋白Bax、Bcl-2的变化。结果:划痕实验显示,划痕后36 h,bcl-3基因沉默前后RKO细胞划痕愈合率分别为84.00%及40.00%,差异具有统计学意义(P0.05)。Annexin V/PI双染色法流式细胞术分析显示,bcl-3基因沉默前后的RKO细胞均经5μmol/L顺铂处理24 h后,沉默前后的RKO细胞凋亡率分别为12.89%及59.67%,差异具有统计学意义(P0.05)。Western blot法检测显示bcl-3基因沉默后cyclin D1蛋白表达显著下降(P0.05),Bax蛋白表达显著上升(P0.05),但Bcl-2表达无明显变化(P0.05)。结论:沉默bcl-3基因后,RKO细胞迁移能力下降,凋亡率增加,并伴细胞周期蛋白cyclin D1及凋亡相关蛋白Bax表达的变化。bcl-3基因可能通过改变cyclin D1及Bax蛋白的表达而影响RKO细胞的凋亡。     

Bcl-2 family gene expression during severe hyperoxia induced lung injury

Exposure of the lung to severe hyperoxia induces terminal transferase dUTP end-labeling (TUNEL) indicative of DNA damage or apoptosis and increases expression of the tumor suppressor p53 and of members of the Bcl-2 gene family. Because cell survival and apoptosis are regulated, in part, by the relative abundance of proteins of the Bcl-2 family, we hypothesized that lung cells dying during exposure would show increased expression of pro-apoptotic members, such as Bax, whereas surviving cells would have increased expression of anti-apoptotic members, such as Bcl-X(L). The hypothesis is tested in the current study by determining which Bcl-2 genes are regulated by hyperoxia, with specific focus on correlating expression of Bax and Bcl-X(L) with morphologic evidence of apoptosis or necrosis. Adult mice exposed to greater than 95% oxygen concentrations for 48 to 88 hours had increased whole-lung mRNA levels of Bax and Bcl-X(L), no change in Bak, Bad, or Bcl-2, and decreased levels of Bcl-w and Bfl-1. In situ hybridization revealed that hyperoxia induced Bax and Bcl-X(L) mRNA in uniform and overlapping patterns of expression throughout terminal bronchioles and parenchyma, coinciding with TUNEL staining. Electron microscopy and DNA electrophoresis, however, suggested relatively little classical apoptosis. Unexpectedly, Western analysis demonstrated increased Bcl-X(L), but not Bax, protein in response to hyperoxia. Bax and Bfl-1 were not altered by hyperoxia in p53 null mice; however, oxygen toxicity was not lessened by p53 deficiency. These findings suggest that oxygen-induced lung injury does not depend on the relative expression of these Bcl-2 members.     

沉默/增强Stat1对胰腺癌细胞增殖和迁移的影响

目的研究沉默/增强Stat1对胰腺癌细胞增殖和迁移能力的影响。方法将胰腺癌细胞(Bx PC-3)分为:对照(control)组、Stat1组和si Stat1组,分别转染Stat1质粒和特异性干扰Stat1表达的siRNA;Western blot法检测Bx PC-3细胞中Stat1、VEGF、MMP-2和MMP-9蛋白的表达;MTT法绘制细胞增殖曲线;Transwell法检测Bx PC-3细胞侵袭能力。结果沉默Stat1表达后,Bx PC-3细胞增殖及迁移能力明显增加,且VEGF、MMP-2和MMP-9表达明显增加。而增强Stat1表达后,Bx PC-3细胞增殖及迁移能力明显降低(P0.05)。结论 Stat1可以抑制Bx PC-3细胞增殖及迁移能力。且VEGF、MMP-2和MMP-9可能在Stat1抑制肿瘤过程中起一定作用。     

Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells

Polycomb-repressive complex 2 (PRC2)-mediated histone methylation plays an important role in aberrant cancer gene silencing and is a potential target for cancer therapy. Here we show that S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) induces efficient apoptotic cell death in cancer cells but not in normal cells. We found that DZNep effectively depleted cellular levels of PRC2 components EZH2, SUZ12, and EED and inhibited associated histone H3 Lys 27 methylation (but not H3 Lys 9 methylation). By integrating RNA interference (RNAi), genome-wide expression analysis, and chromatin immunoprecipitation (ChIP) studies, we have identified a prominent set of genes selectively repressed by PRC2 in breast cancer that can be reactivated by DZNep. We further demonstrate that the preferential reactivation of a set of these genes by DZNep, including a novel apoptosis affector, FBXO32, contributes to DZNep-induced apoptosis in breast cancer cells. Our results demonstrate the unique feature of DZNep as a novel chromatin remodeling compound and suggest that pharmacologic reversal of PRC2-mediated gene repression by DZNep may constitute a novel approach for cancer therapy.     

小干扰RNA抑制RUNX2对人乳腺癌MCF-7细胞增殖、凋亡与侵袭的影响

目的探讨小干扰RNA抑制人RUNT相关转录因子2(RUNX2)对人乳腺癌MCF-7细胞增殖、凋亡与侵袭的影响。方法应用化学合成小干扰RNA技术沉默人乳腺癌MCF-7细胞中RUNX2基因的表达,用逆转录-聚合酶链反应(RT-PCR)和Western blot实验验证基因沉默效率。应用MTT方法检测细胞增殖能力变化,流式细胞仪检测细胞凋亡的变化,Transwell实验检测细胞侵袭能力变化。结果设计的siRNA能够明显抑制人乳腺癌MCF-7细胞RUNX2的表达。RUNX2基因沉默后,人乳腺癌MCF-7细胞的增殖能力降低,细胞凋亡率明显提高,侵袭能力显著降低(P<0.01)。结论RUNX2基因是治疗人乳腺癌MCF-7细胞的潜在靶点。     

应用"decoy"技术靶向性阻断Stat3对乳腺癌细胞系MDA-MB-231增殖抑制的研究

目的：应用针对核转录信号子和激活子3（Stat3）的诱骗寡核苷酸序列（decoy—ODN）阻断乳腺癌细胞系的增殖及其机制研究。方法：阳离子聚合物介导ODN转染乳腺癌细胞系MDA—MB-231，通过细胞计数检测转染Stat3decoy-ODN及随机序列核苷酸（scrambleODN）后的细胞系增殖能力；流式细胞术（FCM）检测转染后细胞周期变化；荧光显微镜观察荧光标记的decoy—ODN转染效率；RT—PCR及Westernblot法分别检测Stat3下游抗凋亡基因在转录水平和蛋白水平的变化。结果：转染Stat3Decoy—ODN序列后MDA—MB-231细胞的增殖受到了明显抑制（P〈0．05）；Stat3下游基因Bcl—xl、c—myc和CylinD1在转录水平和蛋白表达水平均明显降低。结论：Stat3decoy—ODN通过抑制乳腺癌细胞系JAKs／STAT3通路从而抑制了细胞增殖，提示可以通过诱骗技术阻断肿瘤生长而达到基因治疗目的。     

BAD和Bcl-X/L基因在小鼠胸腺细胞凋亡时的表达及其意义

目的检测BAD和Bcl-X/L基因在小鼠胸腺细胞凋亡的表达,探讨细胞凋亡调控基因在小鼠胸腺细胞凋亡时的作用。方法应用免疫组织化学染色法,观察不同剂量糖皮质激素诱导的小鼠胸腺细胞凋亡时BAD和Bcl-X/L基因的表达。结果免疫组织化学结果表明,正常对照组胸腺内BAD呈低表达,地塞米松组随剂量的增加BAD的表达成递增趋势,地塞米松各组与对照组比较差异有有统计学意义(P<0.05);正常对照组胸腺内Bcl-X/L呈高表达,地塞米松组随剂量的增加Bcl-X/L的表达成递减趋势,地塞米松各组与对照组比较差异有统计学意义(P<0.05)。结论促凋亡蛋白BAD和抗凋亡蛋白Bcl-X/L在糖皮质激素诱导引起的胸腺细胞凋亡调控中起重要作用。     

shRNA介导的hWAPL表达沉默对人宫颈癌CaSki细胞增殖与凋亡的影响

 目的： 为探讨人半翼(hWAPL)蛋白在细胞增殖与凋亡中的作用及其作为分子靶点用于肿瘤治疗的潜在价值,本研究用RNA干扰技术抑制hWAPL基因的表达,并观察了其对人宫颈癌细胞CaSki细胞增殖和细胞凋亡的影响。方法：实时荧光定量PCR和Western blotting 检测hWAPL shRNA的干扰效率;MTT检测细胞增殖;Annexin V-PE和 Hoechst 33258染色检测细胞凋亡;蛋白质印迹分析凋亡蛋白cleaved caspase-3和细胞周期抑制蛋白p21、p27的表达。裸鼠荷瘤模型体内研究hWAPL沉默对CaSki细胞增殖的影响。结果：实时荧光定量PCR和Western blotting检测结果表明,筛选到的hWAPL shRNA能有效抑制内源hWAPL的表达。MTT实验表明 shRNA介导的hWAPL表达沉默显著抑制CaSki细胞的增殖。Annexin V-PE分析结果表明, hWAPL 沉默增加了凋亡细胞数目。细胞核经Hoechst　33258染色出现浓染致密的固缩形态和颗粒状荧光; 蛋白质印迹结果显示cleaved caspase-3、 p21和p27表达增加;裸鼠成瘤实验表明,hWAPL 沉默的肿瘤体积减小,增殖细胞核抗原(PCNA)蛋白表达增加。结论：hWAPL沉默抑制CaSki细胞增殖,促进细胞凋亡,是一个潜在的肿瘤治疗新靶点。     

Nucleic acid modulation of gene expression: approaches for nucleic acid therapeutics against cancer

Most cancers are characterized by abnormal gene expression, which is thought to contribute to the pathogenesis and maintenance of the malignant phenotype; abnormal proliferation, maturation, and apoptosis. Silencing such genes would appear to be a rational approach to the therapy of cancer, and some preliminary clinical studies support this concept. Of the strategies available, the anti-mRNA gene silencing approach has attracted much attention and is the focus of this review. This strategy includes three types of agents: (1) single-stranded antisense oligonucleotides; (2) catalytically active oligonucleotides, such as ribozymes, and DNAzymes that possess inherent RNA cleaving activity; and (3) small interfering RNA (siRNA) molecules that induce RNA interference (RNAi). Among these agents, antisense oligonucleotides, especially phosphorothioate (PS) oligonucleotides, have been the most frequently used in clinical trials. In this article, we provide an overview of anti-mRNA gene silencing agents and their development for use as cancer therapeutics.     

Immunohistochemical analysis of bcl-2, bax, bcl-X, and mcl-1 expression in prostate cancers.

Proteins encoded by bcl-2 family genes are important regulators of programmed cell death and apoptosis. Alterations in the expression of these apoptosis-regulating genes can contribute to the origins of cancer, as well as adversely influence tumor responses to chemo- and radiotherapy. Using antibodies specific for the Bcl-2, Bax, Bcl-X, and Mcl-1 proteins in combination with immunohistochemical methods, we examined for the first time the expression of these bcl-2 family genes in 64 cases of adenocarcinoma of the prostate, including 10 Gleason grade 2 to 4 tumors, 21 grade 5 to 7 tumors, 17 grade 8 to 10 tumors, 8 lymph node metastases, and 8 bone metastases. In addition, 24 cases of prostatic intraepithelial neoplasia (PIN) or PIN coexisting with carcinoma were also evaluated. All immunostaining results were scored with regard to approximate percentage of positive tumor cells and relative immunostaining intensity. Expression of the anti-apoptotic protein Bcl-2 was present in 16 of 64 (25%) adenocarcinomas and tended to be more frequent in high grade tumors (Gleason grade 8 to 10; 41%) and nodal metastases (38%) than in lower grade (Gleason 2 to 7) primary tumors (16%; P < 0.05). Bcl-X was expressed in all 64 (100%) tumors evaluated. Bcl-X immunointensity was generally stronger in high grade primary tumors (grade 8 to 10) and metastases compared with PIN and low grade neoplasms (P < 0.0001). In addition, the proportion of specimens with > 50% Bcl-X-immunopositive tumor cells also was higher in advanced grade primary tumors (Gleason 8 to 10) and metastases than in PIN and low grade tumors (Gleason 2 to 7; P < 0.005). The anti-apoptotic protein Mcl-1 was expressed in 52 of 64 (81%) tumors, compared with only 9 of 24 (38%) cases of PIN (P < 0.001). In addition, the percentage of Mcl-1-positive cells was typically higher in Gleason grade 8 to 10 tumors and metastases than in PIN or lower grade tumors (P = 0.025). In contrast, the pro-apoptotic protein Bax was expressed in all prostate cancers evaluated, with high percentages of immunopositive cells and strong immunointensity typically occurring regardless of tumor grade. The findings suggest that expression of several anti-apoptotic members of the bcl-2 gene family, including bcl-2, bcl-X, and mcl-1 increases during progression of prostate cancers, a finding that may be relevant to the hormone-insensitive, metastatic phenotype of most advanced adenocarcinomas of the prostate.     

Stretch-induced alveolar type II cell apoptosis: role of endogenous bradykinin and PI3K-Akt signaling

Apoptosis of alveolar type II (ATII) cells in response to high-amplitude mechanical stretch represents an important mechanism of ventilation-induced lung injury. Previously, it was demonstrated in an in vitro model that stretch-induced ATII cell apoptosis was prevented by angiotensin-converting enzyme (ACE) inhibitors. This study investigates the mechanism by which ACE inhibitors prevent stretch-induced apoptosis and elucidates the role of bradykinin as an endogenous anti-apoptotic factor. Rat ATII cells cultured on flexible membranes were subjected to cyclic stretch (40 cycles/min; 30% increase in surface area) and compared with static controls. Angiotensinogen, the bradykinin precursor T-kininogen, and bradykinin receptor expression were measured by RT-PCR; Angiotensin II and phosphoinositol 3 OH-kinase (PI3K) activity (as phospho-Akt) were measured by enzyme-linked immunosorbent assay; and Bcl-2 and Bcl-X(L) were measured by Western blot. Stretch did not influence angiotensinogen expression or induce angiotensin II generation. The angiotensin II receptor antagonist saralasin did not prevent stretch-induced apoptosis, whereas ACE inhibitors did. Stretch reduced ATII cell bradykinin release (T-kininogen expression and bradykinin supernatant concentration), and subsequently led to reduced PI3K activity and decreased concentrations of the anti-apoptotic proteins Bcl-2/Bcl-X(L). Bradykinin substitution or addition of keratinocyte or hepatocyte growth factor prevented stretch-induced decrease in PI3K activity and Bcl-2/Bcl-X(L) and reduced stretch-induced apoptosis. Mechanical stretch impairs a constitutively expressed, autocrine anti-apoptotic ATII cell survival signal involving bradykinin-mediated stimulation of the PI3K-Akt-Bcl-2/Bcl-X(L) pathway. Restoration of this pathway prevents stretch-induced apoptosis. This may be beneficial when mechanical ventilation cannot completely avoid alveolar overdistension to maintain oxygenation.