A new human ovarian carcinoma cell line: establishment and analysis of tumor-associated markers

In the present study we describe the establishment and characteristics of a new human tumor cell line (OV-1063) positive for carcinoembryonic antigen (CEA) originating from ovarian metastatic tumor cells. Analysis of the cultured cells during their in vitro adaptation period revealed while the primary culture exhibited a low proportion of CEA-positive cells, this proportion increased with culture passages and eventually more than 90% of the cells in the established line were CEA-positive. Thus, during the period of adaptation to in vitro growth, a selection for CEA-positive cells took place but the amount of CEA secreted per each positive cell seemed to be constant. Several tumor-associated characteristics were found positive on the established OV-1063 cell line. The in vitro growing cell line exhibited an abnormal chromosome pattern with a near-trisomy karyotype for some chromosomes, colony formation in soft agar as well as positive staining with a monoclonal antibody B38.1. Culture supernatants of the OV-1063 cells contained significant amounts of CEA as well as CA-125 antigen which is an ovarian-carcinoma-associated antigen.     

Overexpression of SPAG9 correlates with poor prognosis and tumor progression in hepatocellular carcinoma

Sperm-associated antigen 9 (SPAG9) was reported as a novel biomarker for several cancers and associated with the malignant behavior of cancer cells. However, its expression pattern and biological role in human hepatocellular carcinoma (HCC) have not been reported. In the present study, we analyzed SPAG9 expression in human HCC tissues by immunohistochemistry and found that SPAG9 overexpression is correlated with tumor stage (p?p?=?0.019), tumor size (p?=?0.034), AFP levels (p?=?0.006), and tumor relapse (p?=?0.0017). Furthermore, SPAG9 overexpression is correlated with poor overall survival (p?p?=?0.002). Transfection of SPAG9 small interfering RNA (siRNA) was performed in Bel-7402 cell line. Colony formation and MTT showed that SPAG9 siRNA knockdown inhibited HCC cell proliferation. We also found that SPAG9 depletion could increase cell apoptosis. In addition, the level of cyclin D1 and cyclin E protein expression was downregulated after siRNA treatment. In conclusion, SPAG9 is overexpressed in human HCC and serves as a prognostic marker. SPAG9 contributes to cancer cell growth through regulation of cyclin proteins.     

RNAi沉默MCM7基因对人肝癌细胞SMMC-7721裸鼠移植瘤影响研究

目的探讨微小染色体维持蛋白7(miniehromosome maintenanceprotein7,MCM7)基因RNAi(RNA interference)的重组慢病毒载体,对人肝癌细胞SMMC-7721 MCM7基因表达和裸鼠移植瘤生长的影响。方法构建重组逆转录慢病毒载体MCM7-shRNA,以MCM7基因沉默重组慢病毒颗粒(LV-shRNA-MCM7)感染SMMC-7721细胞,作为实验组;以对照慢病毒颗粒(LV-shRNA-NC)感染SMMC-7721细胞,作为阴性对照组;空白对照组常规培养,不做任何处理。通过嘌呤霉素筛选出稳定转染细胞株。3组细胞分别接种至裸鼠皮下,建立人肝癌裸鼠移植瘤模型。观察裸鼠成瘤情况、移植瘤生长情况并绘制肿瘤生长曲线;4周后测定肿瘤体积和质量,并用RT-PCR、实时荧光定量PCR、蛋白质印迹法及免疫组织化学法检测移植瘤中MCM7的表达情况。结果 MCM7-shRNA慢病毒载体构建成功。裸鼠接种癌细胞后第6天均有肿瘤形成,与空白对照组和阴性对照组相比,实验组的肿瘤生长速度明显减慢,实验组、阴性对照组和空白对照组的瘤体平均体积分别为(27.72±7.80)、(81.86±10.91)和(79.75±16.61)mm3,差异有统计学意义,F=61.949,P<0.05;实验组、阴性对照组和空白对照组的瘤体平均质量分别为(0.19±0.06)、(0.501±0.14)和(0.509±0.18)g,差异有统计学意义,F=18.41,P<0.05。实验组MCM7mRNA的相对表达量为0.253±0.198,阴性对照组1.213±0.548,空白对照组1.201±0.744,实验组相比阴性对照组和空白对照组明显下降,差异有统计学意义,F=4.091,P<0.05;实验组MCM7蛋白相对表达量为0.207±0.015,阴性对照组1.116±0.062,空白对照组1.088±0.040,实验组相比阴性对照组和空白对照组明显下降,差异有统计学意义,F=292.778,P<0.05。MCM7蛋白在阴性对照组和空白对照组中阳性表达率均为100%(10/10),在实验组中为30%(3/10),实验组明显低于阴性对照组和空白对照组,差异有统计学意义,χ2=18.261,P<0.001。结论慢病毒沉默SMMC-7721细胞MCM7基因表达能有效抑制人肝癌裸鼠移植瘤的生长,MCM7基因可能成为肝癌基因治疗的有效靶点。     

Proteome analysis of the effects of sorafenib on human hepatocellular carcinoma cell line HepG2

Sorafenib is a multi-target oral anticancer drug used as first-line treatment for patients with advanced human hepatocellular carcinoma (HCC). But the exact mechanism of sorafenib involved in HCC treatment is not clear yet. In this study, a comparative proteomic approach was performed to identify novel sorafenib-related proteins in HCC. Proteomes of HepG2 cells treated with sorafenib and the control (without sorafenib) were obtained by two-dimensional differential gel electrophoresis. Comprehensive analysis of proteins was focused on total protein spots to filtrate the different protein spots between the two groups. The differentially expressed proteins were identified by peptide mass fingerprinting with high-performance liquid chromatography-tandem mass spectrometry. Then, Western blot and immunohistochemistry were used to verify the expression of some candidate proteins. Results indicated that 19 protein spots were differentially expressed with significant changes, including 6 up-regulated proteins and 13 down-regulated proteins. It was confirmed by Western blot that expressions of Annexin A1 and cyclophilin A were down-regulated in sorafenib-treated HCC cell lines. Immunohistochemical study revealed their oncogenic role in HCC tissues. These observations might be novel findings leading to bring new insights into the exact mechanism of sorafenib and identify possible therapeutic targets.     

沉默SIAH2基因抑制人肝癌细胞HepG2裸鼠移植瘤生长实验研究

目的 本研究前期实验发现,靶向SIAH2的shRNA载体能显著抑制HepG2细胞SIAH2 mRNA和蛋白的表达,并抑制细胞体外增殖.本研究从体内水平验证靶向SIAH2的干扰载体对人肝癌细胞HepG2细胞裸鼠移植瘤生长的抑制作用.方法 转染pGenesil-SIAH2的HepG2细胞作为实验组,命名为HepG2-SIAH2;转染空载体pGenesil-1的HepG2细胞作为阴性对照组,命名为HepG2-neo;未转染任何质粒的HepG2细胞作为空白对照组,通过G418筛选出稳定转染细胞株.将15只裸鼠随机分为3组,接种肿瘤细胞后观察裸鼠成瘤情况.4周后测量肿瘤的体积和瘤体质量,绘制移植瘤生长曲线,并用实时荧光定量PCR和蛋白质印迹法检测移植瘤中SIAH2的表达情况.结果 稳定转染pGenesil-SIAH2的细胞株构建成功,3组裸鼠接种癌细胞后均有肿瘤形成.与空白对照组和阴性对照组相比,实验组肿瘤生长速度明显减慢,实验组平均瘤体积(261.57±41.141)mm3,明显低于阴性对照组(494.35±93.236) mm3(P=0.015)和空白对照组(418.3±28.576 5)mm3(P=0.012),差异有统计学意义;实验组平均瘤体质量为(0.162±0.02)g,明显低于阴性对照组(0.358±0.12)g(P=0.032)和空白对照组(0.322±0.24)g(P=0.028).实验组瘤体内SIAH2 mRNA相对表达量为0.83±0.35,明显低于阴性对照组2.35±0.96(P=0.003)和空白对照组2.57±0.41(P=0.006),差异有统计学意义.实验组瘤体内SIAH2蛋白表达量为0.72±0.02,明显低于阴性对照组2.61±0.67(P=0.004)和空白对照组2.49±0.91(P=0.007),差异有统计学意义.结论 靶向SIAH2的shRNA干扰载体能有效抑制人肝癌裸鼠移植瘤的生长,SIAH2有可能成为肝癌基因治疗新的分子靶.     

尼美舒利对肝癌细胞生长的影响

目的:探讨选择性环氧化酶-2(COX-2)抑制剂尼美舒利对肝癌细胞HepG2生长的影响.方法: MTs法检测对照组和实验组(尼美舒利25、50、100、200、400μmol/L)作用后HepG2细胞增殖变化;细胞侵袭小室法检测尼美舒利对HepG2迁移作用的影响.结果: 对肝癌细胞HepG2的增殖有抑制作用,且呈剂量依赖性,尼美舒利对HepG2细胞迁移有明显抑制作用.结论: 选择性环氧化酶2抑制剂尼美舒利可以抑制肝癌细胞的增殖和迁移.     

Orthotopic implantation is essential for the selection,growth and metastasis of human real cell cancer in nude mice

Human neoplasms are heterogeneous for a variety of biological properties that include invasion and metastasis. The presence of a small subpopulation of cells with a highly metastatic phenotype has important clinical implications for diagnosis and therapy of cancer. For this reason, it is important to develop an animal model for the selection and isolation of metastatic variants from human neoplasms and for testing the metastatic potential of human tumor cells.We have implanted human renal cell carcinoma (HRCC) cells (obtained from a surgical specimen) into different organs of nude mice and then recovered the tumors and established each in culture. The 5 established lines differed in their biological-metastatic properties and had a unique karyotype, indicating that growth at different organs selects for different subpopulations of HRCC. Moreover, the HRCC did not metastasize unless they were implanted orthotopically. These findings indicate that the appropriate nude mouse model for studying the biology and therapy of HRCC must be based on the orthotopic implantation of tumor cells.     

高表达CypA肝癌细胞系的建立及其功能初步研究

目的：构建含人全长CypA cDNA的真核表达质粒,建立高表达CypA蛋白的肝癌细胞系,为进一步研究CypA在肝癌中的功能和作用机制提供细胞模型。方法：将人全长CypA cDNA序列克隆后,连接至真核表达载体pcDNA3.1中,再将构建的真核表达载体转染至FHCC98肝癌细胞中,经G418加压筛选后获得稳定表达CypA的肝癌细胞株;免疫印迹法检测目的蛋白的表达情况;MTT法测定细胞的增殖速率。结果：真核表达载体pcDNA3.1-CypA经酶切、测序鉴定,结果正确;免疫印迹结果显示,稳定转染后细胞的CypA表达水平显著高于空载体转染和未转染的细胞;与对照相比,稳定高表达CypA的细胞增殖速度加快。结论：成功构建了含有人全长CypA cDNA序列的真核表达载体pcDNA3.1-CypA;建立了稳定高表达CypA的FHCC-98肝癌细胞系;CypA的高表达可以促进FHCC98细胞的增殖。     

Adrenal carcinoma tumor progression and penultimate cell surface oligosaccharides.

Many previous studies have implicated cell surface saccharides, and sialylglycoconjugates in particular, as important mediators of tumor cell metastasis. In this report, we have used three different specific sialidases and a highly sensitive high-performance liquid chromatographic sialic acid assay to probe the cell surfaces of several murine adrenal carcinoma variants. In contrast to several earlier studies on other metastatic variants, we find no significant differences in the overall levels of cell surface or total cellular sialic acid among three Y1 murine adrenal carcinoma variants with widely different metastatic phenotypes. However, using highly purified, linkage-specific sialyltransferases, in conjunction with V. cholerae sialidase, to probe the cell surface saccharide topography of specific penultimate oligosaccharides, we do find striking differences in oligosaccharide structures underlying the sialic acid moieties. Two tumorigenic and metastatic variants (F2 and F4) contain about 6-fold more penultimate Gal beta 1----4GlcNAc sialylation sites than a related tumorigenic but nonmetastatic variant (HSR) when CMP-[3H]-N-acetylneuraminic acid and the Gal beta 1----4GlcNAc alpha 2,6 sialyltransferase are used to probe the adrenal carcinoma cell surfaces. The metastatic variants also are found to contain 4- to 4.5-fold more Gal beta 1----3GalNAc sialylation sites than the nonmetastatic variant when the Gal beta 1----3GalNAc alpha 2,3 sialyltransferase is used as a cell surface probe. Earlier work, which used the same sialyltransferase probes on sialidase-treated murine melanoma variants (A. Passaniti and G. W. Hart, J. Biol. Chem., 263: 7591-7603, 1988), also showed similar quantitative differences in penultimate structures between metastatic variants. However, in contrast to the adrenal carcinoma cells, the highly metastatic melanoma cells have severalfold lower levels of sialylatable penultimate Gal beta 1----4GlcNAc and Gal beta 1----3GalNAc saccharides compared to their nonmetastatic counterparts. Thus, while the precise structural alterations or surface accessibilities of penultimate saccharides appear to be cell type dependent, these results suggest that pronounced changes in penultimate cell surface sialo-oligosaccharide moieties occur during progression to a malignant phenotype in two widely different tumor systems. These types of alterations in the underlying penultimate oligosaccharide structures of cell surface sialoglycoconjugates may be a common feature of highly metastatic cells arising from very different tumor cell types.     

A human squamous cell carcinoma cell line.

An epithelial cell line COLO 16 has been established from a human squamous carcinoma, characterized and maintained for over two years. The cells produce a parathyroid-like hormone and carcinoembryonic antigen. The line is definitely not a "HeLa contaminant." The cell line is available to other investigators.     

Orthotopic reconstitution of human small-cell lung carcinoma after intravenous transplantation in SCID mice.

We have constructed an orthotopically reconstituted model of human small-cell lung carcinoma (SCLC) by intravenous transplantation in severe combined immunodeficient (SCID) mice. Two human SCLC xenografts, H-69 and Lu-130, were disaggregated and injected through the tail vein of SCID mice. Human SCLCs were orthotopically reconstituted with multi-focal lung tumor growth in all SCID mice after intravenous injection of 5 x 10(6) tumor cells per mouse. The heart and liver were also seeded with actively growing SCLC. This orthotopic reconstitution model of human SCLC in SCID mice should be useful for further studies on the biological behavior and treatment of human SCLC.     

Overexpression of mitochondrial manganese superoxide dismutase protects against radiation-induced cell death in the human hepatocellular carcinoma cell line HLE.

We investigated the potential role of mitochondrial manganese superoxide dismutase (Mn-SOD) in protective activity against irradiation by analyzing cell viability by a colony formation assay and by detecting apoptosis in stably human Mn-SOD gene-transfected HLE, a hepatocellular carcinoma cell line. We found that overexpression of Mn-SOD reduced the levels of reactive oxygen species in the mitochondria and intracellular phospholipid peroxidation product (4-hydroxy-2-nonenal) and prevented cell death. The production of intracellular nitric oxide after irradiation was not changed by Mn-SOD overexpression. The results suggested that Mn-SOD might play an important role in protecting cells against radiation-induced cell death by controlling the generation of mitochondrial reactive oxygen species and intracellular lipid peroxidation.     

番荔枝内酯Bullatacin对人肝癌细胞株HpG2凋亡影响的探讨

目的:研究番荔枝内酯 Bullatacin在诱导人肝癌细胞株HpG2凋亡中的作用.方法:MTT法测定Bullatacin对HpG2细胞增殖的影响;采用碘化丙锭染色(PI)及荧光标记的单克隆抗体(mAb)结合流式细胞仪(FCM)分析Bullatacin对HpG2细胞周期和凋亡及Fas蛋白表达的作用;RT-PCR检测Bullatacin对Fas基因表达的影响.结果:Bullatacin (100 gg/mL)能够明显抑制人肝癌细胞株HpG2的增殖,作用96 h其生长抑制率达66.3%;Bullataein将HpG2细胞周期抑制在G0/G1期,阻止其进入G2/M期,同时能诱导细胞凋亡;Bullatacin能够明显上调HpG2细胞表面Fas蛋白的表达(P=0.006),且作用优于顺铂(P=0.03);进一步研究发现,Bullatacin能够诱导Fas基因的表达.结论:Bullatacin能够诱导HpG2细胞凋亡,其可能机制是诱导Fas基因表达从而上调Fas蛋白.本研究以Bullatacin作为一种抗肿瘤药物的研发提供理论和实验依据.     

Establishment of experimental implantation tumor models of hepatocellular carcinoma in Wistar rats

Our aims were to investigate and establish simple and reliable implanted hepatocellular carcinoma (HCC) models in Wistar rats. Concentrated suspensions of CBRH-7919 cancer cell lines were injected subcutaneously into the scapular regions of nude mice. The developing tumor tissues were then implanted into the livers of 45 adult Wistar rats. Dexamethasone (2.5 mg/day) was injected intramuscularly daily for 1 week preoperatively and 2 weeks postoperatively. After 4 weeks of implantation, ultrasonography and nuclear magnetic resonance imaging (MRI) were performed to identify model rats with liver tumor growth and to analyze the growth and characteristics of the tumors. Five of these model rats were then sacrificed, and the tumors were removed from the liver for pathological examination. Three rats died during the operation; among the remaining 42 rats, 36 possessed a total of 43 liver tumors. The success rate of tumor implantation was 85.7 % (36/42), and the diameters of the tumors ranged from 5 to 10 mm. All tumor specimens were confirmed to be HCC by pathological examination. This study provides a new approach for establishing implanted HCC models in Wistar rats, which can be used for studying numerous biological features of HCC.     

Role of the pituitary tumor transforming gene 1 in the progression of hepatocellular carcinoma

In order to demonstrate the role of the pituitary tumor transforming gene 1 (PTTG1) in the development of hepatocellular carcinoma (HCC) and its value as a molecular target for cancer therapy, we analyzed the expression of PTTG1 mRNA and protein, and their relation to clinicopathological characteristics and basic fibroblast growth factor (bFGF) expression in HCC. It was observed that the level of PTTG1 mRNA and the positive rate of PTTG1 protein in cancerous tissues were significantly higher than that in adjacent non-cancerous tissues (both P< 0.001). The PTTG1 protein levels were correlated with several clinicopathological parameters, including alpha-fetoprotein level, portal vein tumor thrombosis, tumor stage, and bFGF protein level (P< 0.05). The proliferation indices were significantly less and the apoptotic rates were significantly higher in the HepG2 and SMMC-7721 cells treated with PTTG1 siRNA transfection than their untransfected counterparts. The expressions of Caspase-3, Bax, p21 and p53 in HepG2 and SMMC-7721 cells were significantly increased after siRNA knockdown of PTTG1 expression. In conclusion, the PTTG1 gene is up-regulated in the cancerous tissue from patients with HCC and involved in the progression of HCC. Inhibiting PTTG1 expression decreases cell proliferation and induces apoptosis in hepatic cancer cell lines, indicating that PTTG1 may be a new therapeutic target for HCC treatment.     

Role of epigenetic aberrations in the development and progression of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers in humans. The molecular mechanisms leading to the development of HCC are extremely complicated and consist of prominent genetic, genomic, and epigenetic alterations. This review summarizes the current knowledge of the role of epigenetic aberrations, including changes in DNA methylation, histone modifications, and expression of microRNAs in the pathogenesis of HCC. It also emphasizes that identification of the underlying epigenetic alterations that drive cell transformation and promote development and progression of HCC is crucially important for understanding mechanisms of hepatocarcinogenesis, its detection, therapeutic intervention, and prevention.     

人肝癌顺铂耐药细胞系的建立及其生物学特征

背景与目的：药物耐受性是当前肿瘤研究的热点,国内外普遍应用体外建立的耐药细胞系作为研究模型。为探讨肝癌对顺铂耐药的机理。本研究首次建立了人肝癌顺铂耐药细胞系并研究其生物学特性。方法：采用逐步递增顺铂浓度,间歇作用体外诱导法建立人肝癌顺铂耐药细胞系QGY/cDDP;MTT法测定药物敏感性,光镜,电镜,活细胞计数法,流式细胞仪及染色体分析等方法观察其生物学特征的改变。结果：历时3个月建成人肝癌顺铂耐药细胞系QGY/cDDP,其对顺铂的耐药指数为10.81,并且与5-氟尿嘧啶,表阿霉素等多种抗癌药有不同程度的交叉耐药性;QGY/cDDP的细胞形态及染色体数目发生改变;体外群体倍增时间较亲代细胞延长;细胞周期分析发现其S期与G2/M期细胞减少,G0/G1期细胞增多。结论：QGY/cDDP细胞具有耐药表型,且耐药性能稳定。