The Significance of Epithelial-to-Mesenchymal Transition for Circulating Tumor Cells

Epithelial to mesenchymal transition (EMT) is a process involved in embryonic development, but it also plays a role in remote metastasis formation in tumor diseases. During this process cells lose their epithelial features and adopt characteristics of mesenchymal cells. Thereby single tumor cells, which dissolve from the primary tumor, are enabled to invade the blood vessels and travel throughout the body as so called “circulating tumor cells” (CTCs). After leaving the blood stream the reverse process of EMT, the mesenchymal to epithelial transition (MET) helps the cells to seed in different tissues, thereby generating the bud of metastasis formation. As metastasis is the main reason for tumor-associated death, CTCs and the EMT process are in the focus of research in recent years. This review summarizes what was already found out about the molecular mechanisms driving EMT, the consequences of EMT for tumor cell detection, and suitable markers for the detection of CTCs which underwent EMT. The research work done in this field could open new roads towards combating cancer.     

The Role of MicroRNAs in Breast Cancer Stem Cells

The concept of the existence of a subset of cancer cells with stem cell-like properties, which are thought to play a significant role in tumor formation, metastasis, resistance to anticancer therapies and cancer recurrence, has gained tremendous attraction within the last decade. These cancer stem cells (CSCs) are relatively rare and have been described by different molecular markers and cellular features in different types of cancers. Ten years ago, a novel class of molecules, small non-protein-coding RNAs, was found to be involved in carcinogenesis. These small RNAs, which are called microRNAs (miRNAs), act as endogenous suppressors of gene expression that exert their effect by binding to the 3′-untranslated region (UTR) of large target messenger RNAs (mRNAs). MicroRNAs trigger either translational repression or mRNA cleavage of target mRNAs. Some studies have shown that putative breast cancer stem cells (BCSCs) exhibit a distinct miRNA expression profile compared to non-tumorigenic breast cancer cells. The deregulated miRNAs may contribute to carcinogenesis and self-renewal of BCSCs via several different pathways and can act either as oncomirs or as tumor suppressive miRNAs. It has also been demonstrated that certain miRNAs play an essential role in regulating the stem cell-like phenotype of BCSCs. Some miRNAs control clonal expansion or maintain the self-renewal and anti-apoptotic features of BCSCs. Others are targeting the specific mRNA of their target genes and thereby contribute to the formation and self-renewal process of BCSCs. Several miRNAs are involved in epithelial to mesenchymal transition, which is often implicated in the process of formation of CSCs. Other miRNAs were shown to be involved in the increased chemotherapeutic resistance of BCSCs. This review highlights the recent findings and crucial role of miRNAs in the maintenance, growth and behavior of BCSCs, thus indicating the potential for novel diagnostic, prognostic and therapeutic miRNA-based strategies.     

Mechanisms and Αpplications of Ιnterleukins in Cancer Immunotherapy

Over the past years, advances in cancer immunotherapy have resulted in innovative and novel approaches in molecular cancer diagnostics and cancer therapeutic procedures. However, due to tumor heterogeneity and inter-tumoral discrepancy in tumor immunity, the clinical benefits are quite restricted. The goal of this review is to evaluate the major cytokines-interleukins involved in cancer immunotherapy and project their basic biochemical and clinical applications. Emphasis will be given to new cytokines in pre-clinical development, and potential directions for future investigation using cytokines. Furthermore, current interleukin-based approaches and clinical trial data from combination cancer immunotherapies will also be discussed. It appears that continuously increasing comprehension of cytokine-induced effects, cancer stemness, immunoediting, immune-surveillance as well as understanding of molecular interactions emerging in the tumor microenvironment and involving microRNAs, autophagy, epithelial-mesenchymal transition (EMT), inflammation, and DNA methylation processes may hold much promise in improving anti-tumor immunity. To this end, the emerging in-depth knowledge supports further studies on optimal synergistic combinations and additional adjuvant therapies to realize the full potential of cytokines as immunotherapeutic agents.     

人核糖核苷酸酶抑制因子表达下调对膀胱癌T24细胞在体内外发生上皮间质转化的影响

目的探讨人核糖核苷酸酶抑制因子(human ribonuclease inhibitor,hRI)表达下调对膀胱癌T24细胞在体内外发生上皮间质转化(epithelial-mesenchymal transition,EMT)的影响。方法干涉质粒pGensil-1-RI稳定转染T24细胞,经G418筛选阳性克隆,并设实验组、空质粒对照组及未转染对照组,RT-PCR检测各组细胞中RI基因mRNA的转录水平、细胞免疫荧光和Western blot检测RI蛋白的表达水平;HE染色检测细胞形态的变化;Western blot检测各组细胞中E-cadherin、Twist、Slug和Vimentin等相关蛋白表达水平;将3组细胞注入BALB/c裸鼠皮下,35 d后取瘤并称重;免疫组织化学检测瘤组织中RI、E-cadherin和Vimentin蛋白的表达水平。结果荧光显微镜下观察可见,干涉质粒pGensil-1-RI转染成功;实验组RI基因mRNA的转录水平和蛋白的表达水平较两对照组分别降低了63.31%和64.11%、49.6%和49.5%(P<0.001);实验组细胞呈梭形,核质比增大;与实验相比,两对照组E-cadhein蛋白表达水平分别增加52.76%和46.93%(P<0.001),而Twist、Slug和Vimentin蛋白分别降低了50.49%和54.63%、43.74%和51.55%、60.35%和53.77%(P<0.001);两对照组的瘤重明显低于实验组,分别低84.91%和80.89%(P<0.01),其RI和E-cadherin蛋白表达水平降低,而Vimentin蛋白表达水平则增加。结论沉默RI基因能明显增加T24细胞的转移、侵袭及EMT的能力。     

吴茱萸碱对人结肠癌lovo细胞生长的抑制作用

目的探讨吴茱萸碱(Evodiamine,Evo)在体内外对人结肠癌lovo细胞生长的抑制作用及其机制。方法采用MTT法检测Evo对人结肠癌lovo细胞、人乳腺癌MDA-MB-231细胞以及人肺癌A549细胞生长的影响;流式细胞术分析Evo对lovo细胞细胞周期和细胞凋亡的影响;采用Evo治疗lovo细胞荷瘤裸鼠,并绘制肿瘤生长曲线;Westernblot法检测Evo对lovo细胞和肿瘤组织中Bcl-2及procaspase-3表达的影响。结果 Evo能抑制lovo细胞生长(P<0.01),促进MDA-MB-231细胞增殖(P<0.01),对A549细胞无明显作用(P>0.05);可将lovo细胞阻滞于S期(P<0.01),并呈时间依赖性诱导其凋亡(P<0.01);能抑制lovo细胞荷瘤裸鼠肿瘤生长(P<0.05);Westernblot检测结果显示,Evo在体内外均可降低lovo细胞Bcl-2及procas-pase-3的表达。结论 Evo可通过抑制Bcl-2表达,激活caspase-3,诱导凋亡,从而抑制lovo细胞的生长。     

骨髓间充质干细胞对海藻酸钙胶珠内神经干细胞增殖与分化的作用

神经干细胞(neural stem cells,NSCs)移植治疗神经损伤被认为是具有潜在应用价值的手段,但其来源困难;骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)以其所具有的诸多优点,为神经损伤的治疗提供了一个新的思路。而BMSCs是否是通过作用于内源性的NSCs来促进神经修复,仍存在着争议。今采用海藻酸钙胶珠将NSCs包囊培养至一定大小的神经球后,再与BMSCs进行共培养,考察BMSCs对生长在海藻酸钙胶珠内的NSCs增殖与分化的作用,探讨BMSCs移植治疗神经疾病与损伤的作用机理。共培养过程中观察神经球结构的变化;共培养结束后计算NSCs的增殖倍数,对增殖条件下共培养的NSCs表型和多向分化潜能进行免疫荧光染色鉴定;对分化条件下共培养的NSCs向不同神经细胞分化的能力进行流式细胞仪检测。结果表明,BMSCs可使生长于支架内的NSCs迁出细胞球,对NSCs的增殖没有明显影响;但能够明显影响NSCs的分化,使其向少突胶质细胞分化的能力增加3倍,向星形胶质细胞分化的能力减弱1倍,而向神经元细胞分化的能力没有明显变化。BMSCs有可能是通过分泌某些因子增加了NSCs迁移及向少突胶质细胞分化的能力,从而促进神经损伤的修复。     

Hyperactive RAS/PI3-K/MAPK Signaling Cascade in Migration and Adhesion of Nf1 Haploinsufficient Mesenchymal Stem/Progenitor Cells

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in the NF1 tumor suppressor gene, which affect approximately 1 out of 3000 individuals. Patients with NF1 suffer from a range of malignant and nonmalignant manifestations such as plexiform neurofibromas and skeletal abnormalities. We previously demonstrated that Nf1 haploinsufficiency in mesenchymal stem/progenitor cells (MSPCs) results in impaired osteoblastic differentiation, which may be associated with the skeletal manifestations in NF1 patients. Here we sought to further ascertain the role of Nf1 in modulating the migration and adhesion of MSPCs of the Nf1 haploinsufficient (Nf1^+/−) mice. Nf1^+/− MSPCs demonstrated increased nuclear-cytoplasmic ratio, increased migration, and increased actin polymerization as compared to wild-type (WT) MSPCs. Additionally, Nf1^+/− MSPCs were noted to have significantly enhanced cell adhesion to fibronectin with selective affinity for CH271 with an overexpression of its complimentary receptor, CD49e. Nf1^+/− MSPCs also showed hyperactivation of phosphoinositide 3-kinase (PI3-K) and mitogen activated protein kinase (MAPK) signaling pathways when compared to WT MSPCs, which were both significantly reduced in the presence of their pharmacologic inhibitors, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and PD0325901, respectively. Collectively, our study suggests that both PI3-K and MAPK signaling pathways play a significant role in enhanced migration and adhesion of Nf1 haploinsufficient MSPCs.     

Ipr1和GFP基因真核共表达穿梭质粒的构建及其在人肺癌细胞中的表达

目的构建胞内病原体抗性基因1(Ipr1)和绿色荧光蛋白(GFP)基因真核共表达穿梭质粒,并在人肺腺癌细胞A549中表达。方法采用PCR方法,分别从质粒pEGFP-C1-Ipr1和pEGFP-C1中扩增Ipr1和GFP基因,将GFP基因、分枝杆菌复制子OriM和Ipr1基因同时克隆入多启动子真核共表达载体pBudCE4.1中,构建pBud-GFP-OriM-Ipr1穿梭质粒,脂质体法转染A549细胞,荧光显微镜观察GFP的表达,免疫组化方法检测Ipr1蛋白的表达。结果酶切和测序分析表明,pBud-GFP-OriM-Ipr1真核共表达穿梭质粒构建正确。转染A549细胞后,荧光显微镜下可观察到转染细胞中有GFP表达,免疫组化法可检测到Ipr1蛋白的表达,且定位于细胞核内。结论已成功构建Ipr1和GFP基因真核共表达穿梭质粒,为进一步研究Ipr1抗结核的功能奠定了基础。     

Over-Expression of Platelet-Derived Growth Factor-D Promotes Tumor Growth and Invasion in Endometrial Cancer

The platelet-derived growth factor-D (PDGF-D) was demonstrated to be able to promote tumor growth and invasion in human malignancies. However, little is known about its roles in endometrial cancer. In the present study, we investigated the expression and functions of PDGF-D in human endometrial cancer. Alterations of PDGF-D mRNA and protein were determined by real time PCR, western blot and immunohistochemical staining. Up-regulation of PDGF-D was achieved by stably transfecting the pcDNA3-PDGF-D plasmids into ECC-1 cells; and knockdown of PDGF-D was achieved by transient transfection with siRNA-PDGF-D into Ishikawa cells. The MTT assay, colony formation assay and Transwell assay were used to detect the effects of PDGF-D on cellular proliferation and invasion. The xenograft assay was used to investigate the functions of PDGF-D in vivo. Compared to normal endometrium, more than 50% cancer samples showed over-expression of PDGF-D (p < 0.001), and high level of PDGF-D was correlated with late stage (p = 0.003), deep myometrium invasion (p < 0.001) and lympha vascular space invasion (p = 0.006). In vitro, over-expressing PDGF-D in ECC-1 cells significantly accelerated tumor growth and promoted cellular invasion by increasing the level of MMP2 and MMP9; while silencing PDGF-D in Ishikawa cells impaired cell proliferation and inhibited the invasion, through suppressing the expression of MMP2 and MMP9. Moreover, we also demonstrated that over-expressed PDGF-D could induce EMT and knockdown of PDGF-D blocked the EMT transition. Consistently, in xenografts assay, PDGF-D over-expression significantly promoted tumor growth and tumor weights. We demonstrated that PDGF-D was commonly over-expressed in endometrial cancer, which was associated with late stage deep myometrium invasion and lympha vascular space invasion. Both in vitro and in vivo experiments showed PDGF-D could promote tumor growth and invasion through up-regulating MMP2/9 and inducing EMT. Thus, we propose targeting PDGF-D to be a potent strategy for endometrial cancer treatment.     

MMP-2反义寡核苷酸对人胃癌SGC7901细胞增殖和侵袭能力的影响

目的研究基质金属蛋白酶-2(MMP-2)反义寡核苷酸(ASODN)对人胃癌SGC7901细胞增殖及侵袭能力的影响。方法将不同浓度的硫代磷酸化修饰的MMP-2ASODN转染入人中低分化胃腺癌SGC7901细胞株,RT-PCR法检测细胞MMP-2基因mRNA的转录水平;Transwell试验检测细胞体外迁移和侵袭能力变化;MTT法检测细胞的增殖活性。结果转染MMP-2ASODN后,SGC7901细胞中MMP-2基因mRNA的转录水平显著降低,细胞的体外迁移和侵袭能力受到抑制,且抑制作用随ASODN浓度的增加而增强;而细胞的增殖活性无明显变化。结论MMP-2ASODN可抑制胃腺癌SGC7901细胞MMP-2基因mRNA的转录水平及细胞的体外迁移和侵袭能力,但与胃癌细胞的增殖活性无明显相关性。     

人肝细胞生长因子基因真核表达质粒的构建及其在人骨髓间充质干细胞中的表达

目的构建人肝细胞生长因子(Human hepatocyte growth factor,hHGF)真核表达质粒,并检测其在人骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)中的表达及其对细胞生长的影响。方法采用密度梯度法从人骨髓中分离BMSCs,流式细胞术检测细胞表型,成脂及成骨诱导其分化。PCR扩增hHGF基因,定向克隆至pEGFP-N1载体中,构建重组表达质粒pEGFP-N1-hHGF,通过电穿孔法转染BMSCs,荧光显微镜下观察增强型绿色荧光蛋白的表达,RT-PCR及Western blot法检测hHGF基因mRNA的转录及蛋白的表达,MTT法检测hHGF对BMSCs增殖活力的影响。结果 BMSCs高表达CD29和CD44,不表达CD34和CD45;BMSCs在体外有向成脂及成骨细胞诱导分化的能力。酶切及基因测序证实,重组表达质粒pEGFP-N1-hHGF构建正确;转染后48 h观察到转染细胞中有绿色荧光蛋白表达;RT-PCR法和Western blot检测到hHGF基因在BMSCs中表达;转染hHGF基因的BMSCs增殖活力明显高于空白对照组和空载体转染组(P<0.05)。结论成功构建了hHGF基因真核表达质粒,转染人BMSCs后获得表达,表达的hHGF可促进BMSCs增殖。     

Effect of NK4 Transduction in Bone Marrow-Derived Mesenchymal Stem Cells on Biological Characteristics of Pancreatic Cancer Cells

Pancreatic cancer usually has a poor prognosis, and no gene therapy has yet been developed that is effective to treat it. Since a unique characteristic of bone marrow-derived mesenchymal stem cells (MSCs) is that they migrate to tumor tissues, we wanted to determine whether MSCs could serve as a vehicle of gene therapy for targeting pancreatic cancer. First, we successfully extracted MSCs from SD rats. Next, MSCs were efficiently transduced with NK4, an antagonist of hepatocyte growth factor (HGF) which comprising the N-terminal and the subsequent four kringle domains of HGF, by an adenoviral vector. Then, we confirmed that rat MSCs preferentially migrate to pancreatic cancer cells. Last, MSCs expressing NK4 (NK4-MSCs) strongly inhibited proliferation and migration of the pancreatic cancer cell line SW1990 after co-culture. These results indicate that MSCs can serve as a vehicle of gene therapy for targeting pancreatic cancer.