H—ras癌基因与肝癌转移行为的关系探讨

我们应用基因转移技术,研究了Ｈｒａｓ癌基因对肝癌细胞株ＳＭＭＣ７７２１细胞转移的影响,以探讨Ｈｒａｓ基因与肝癌转移行为的关系。１　材料与方法１．１　材料载体质粒ＰＳＶ２ｎｅｏ及载有活化的Ｈｒａｓ基因的重组质粒ＰＳＶ２ｎｅｏｒａｓ由上海复旦大学生物物理系惠赠,人肝癌细胞株ＳＭＭＣ７７２１由本所细胞室提供。磷酸钙转染试剂盒购自Ｐｒｏｍｅｇａ公司。ＤＡＣＤｐ２１ｒａｓ抗体购自Ｓｉｇｍａ公司,它可特异性识别ｐ２１ＨｒａｓＣ末端１２６～１４０氨基酸区域ｃⅣａｓｅ抗体,ＥＧＦＲ抗体购自Ｏｎｃ…     

克隆化人肝癌裸鼠移植瘤株的建立及其转移性状观察

应用单个细胞克隆化技术，从已建系的人肝癌细胞（ＳＭＭＣ－７７２１）中选育出３个亚系７７２１－ＣＡ，７７２１－ＣＢ，７７２１－ＣＳ（ＣＡ、ＣＢ、ＣＳ），并进行扩大培养，将上述各亚系肝癌细胞接种到裸鼠体内，成瘤率与自发转移率以ＣＳ组最高，ＣＢ组最低。主要的转移器官是肺。原发灶和转移灶肿瘤形态结构一致。ＣＳ细胞形态及其接种的裸鼠移植瘤形态结构和ＳＭＭＣ－７７２１母系细胞基本相似。用ＡＢＣ免疫酶标法检测ＨＢｓＡｇ、ｋｅｒａｔｉｎ、ＡＦＰ、Ｈ－ｒａｓ、Ｃ－ｅｒｂ－２、Ｐ５３癌基因蛋白等肿瘤标记物，ＣＳ组裸鼠移植瘤对Ｈ－ｒａｓ、Ｃ－ｅｒｂ－２癌基因蛋白呈阳性反应，对ＡＦＰ、Ｐ５３呈弱阳性反应，而ＣＢ组裸鼠移植瘤仅对Ｋｅｒａｔｉｎ、Ｃ－ｅｒｂ－２呈弱阳性反应，对其它肿瘤标记物均呈阴性反应。     

H—ras癌基因与肝癌浸润转移相关性的初步探讨

Ｈ－ｒａｓ癌基因与肝癌浸润转移相关性的初步探讨＊翁毅林芷英汪青作者单位：上海医科大学肝癌研究所（２０００３２）＊本研究系美国中华医学基金（ＣＭＢ）资助项目关键词肝肿瘤基因，ｒａｓ浸润肿瘤转移胶原蛋白免疫组织化学Ｃ－Ｈ－ｒａｓ癌基因及其蛋白产物ｐ２１广...     

nm23H1对肝癌细胞增殖及体内肿瘤形成能力的影响

目的 研究ｎｍ２３Ｈ１对肝癌细胞增殖、体内肿瘤形成和转移的影响。方法 构建正反义ｎｍ２３Ｈ１ ｃＤＮＡ表达载体并转染肝癌细胞ＳＭＭＣ－７７２１,得到ｎｍ２３Ｈ１稳定最高和最低表达的两种细胞克隆,并进行细胞生长曲线测定、裸鼠皮下及脾包膜下移植试验。结果 转染反义表达载体后,肝癌细胞ｍＲＮＡ和蛋白表达下降,在体内、体外增殖加速;转染正义表达载体后,出现与之相反的结果。正义表达细胞接种组裸鼠肿瘤结节形成     

肺癌p14ARF和p16INK4a基因协同表达缺失及其意义

目的：研究抑癌基因位点ＩＮＫ４ａ－ＡＲＦ在肺肿瘤细胞中的表达状况,揭示ｐ１４ＡＲＦ和ｐ１６ＩＮＫ４ａ协同表达缺失与肺癌发生发展的相关性。方法：用ＲＴ－ＰＣＲ和Ｗｅｓｔｅｒｎ ｂｌｏｔ对６株肺癌细胞（ＳＰＣ－Ａ－１,Ｃａｌｕ－１,Ｈ４４６,ＳＨ７７,Ａ５４９,Ｈ４６０）的ＩＮＫ－４ａ－ＡＲＦ基因位点在ｍＲＮＡ、蛋白水平上进行检测,对ＰＣＲ产物进行纯化和测序分析。结果：６株肺癌细胞中,有３株细胞（Ｈ４     

H-ras和ICAM-1基因在人肝癌细胞中的表达及分布

当激活的ｒａｓ基因被转染进入ＮＩＨ３Ｔ３细胞时，可产生许多转移现象。细胞间粘附分子（ＩＣＡＭ－１）能介导癌细胞与内皮细胞、淋巴细胞结合，在肿瘤转移中有肯定的作用。为此，我们用原位核酸杂交技术观察Ｈ－ｒａｓ和ＩＣＡＭ－１ｍＲ－ＮＡ在人肝癌细胞中的原位表...     

c－ets－2、c－myc、N－ras三个癌基因联合反义RNA对肝癌细胞恶性表型的逆转

作者构建了一个能表达ｃ－ｅｔｓ－２、ｃ－ｍｙｃ及Ｎ－ｒａｓ三个癌基因联合反义ＲＮＡ的重组逆转录病毒载体，经病毒包装细胞ＰＡ３１７包装成假型逆转录病毒，利用此病毒成功地感染了人肝癌细胞株ＳＭＭＣ－７７２１，经Ｇ４１８筛选得到Ｇ４１８抗性细胞，基因组ＤＮＡ杂交结果表明重组病毒稳定地整合入７７２１细胞基因组中。ＲＮＡ杂交结果表明转化细胞中有较高水平的联合反义ＲＮＡ表达。初步结果表明，反义ＲＮＡ使７７２１细胞生长速率下降约７０％，软琼脂集落形成能力及裸鼠致瘤能力显著下降。这一结果表明，针对多个癌基因的联合反义ＲＮＡ可能给肿瘤基因治疗提供新的途径，有进一步探索的价值。     

阴阳莲提取物3,4‘,5—三羟基芪—3—β—单—D—葡萄糖苷对人大肠癌细胞？…

目的：研究蓼科植物阴阳莲提取物３,４’,５－三羟基芪－３－β－单－Ｄ－葡萄糖苷（３,４’,５,ｔｒｉｈｙｄｒｏｘｙｓｔｉｂｅｎｅ－３－β－ｍｏｎｏ－Ｄ－ｇｌｕｃｃｏｓｉｄｅ,ＴＨＭＧ）对大肠癌细胞的体外生长特性、粘附性及浸润力的影响;从细胞层次探讨ＴＨＭＧ抗转移作用的基本环节。方法：检测４种大肠癌细胞（ＨＲ８３４８,Ｈｃｅ８６９３,ＨＴ２９,ＬｏＶｏ）在ＴＨＭＧ０．４ｍｍｏｌ／Ｌ,０．８ｍｍｏｌ／     

滤泡性淋巴瘤与反应性淋巴滤泡增生的免疫组化及DNA图像分析研究

目的：观察滤泡性淋巴瘤（ＦＬ）与反应性淋巴滤泡增生（ＲＦＨ）中滤泡生发中心细胞（ＧＣＣｓ）核ＤＮＡ含量、倍体情况以及ｂｃｌ－２癌基因蛋白、免球蛋白轻链（ＩｇＬ）表达情况，探讨它们对于二都鉴别诊断的意义。方法：对２１例ＦＬ及２１例ＲＦＨ进行ｂｃｌ－２蛋白、Ｋｆｐｐａ、Ｌａｍｂｄａ轻链蛋白免疫组化检测及ＤＮＡ图像细胞分析。结果：６１．９％ＦＬ中ＧＣＣｓ有ｂｃｌ－２表达而ＲＦＨ的ＧＣＣｓ均为ｂｃｌ－２阴     

ＨＭＧＢ１表达载体转染结肠癌细胞对ＶＥＧＦ-Ｄ表达的影响

目的：构建ＨＭＧＢ１表达载体,转染结肠癌细胞,研究其对结肠癌细胞中血管内皮生长因子Ｄ（ＶＥＧＦ-Ｄ）表达的影响。方法：将分离得到的淋巴细胞总ＲＮＡ反转录合成ｃＤＮＡ,以此为模板进行ＰＣＲ扩增得到ＨＭＧＢ１基因;随后酶切转入载体ｐＭＤ１８Ｔ,通过亚克隆转入载体ｐＬｘｓｎ,得到重组质粒。重组体质粒经酶切鉴定后,并对插入的ＨＭＧＢ１基因片段进行测序,将已鉴定的阳性重组质粒用脂质体介导转染ＨＣＴ１１６细胞。通过ＲＴ-ＰＣＲ和Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ检测ＨＭＧＢ１和ＶＥＧＦ-Ｄ的表达情况。结果：成功构建含ＨＭＧＢ１的表达载体。ＲＴ-ＰＣＲ和Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ检测发现转染表达ＨＭＧＢ１载体的ＨＣＴ１１６细胞中ＨＭＧＢ１和ＶＥＧＦ-Ｄ的表达均增高。结论：ＨＭＧＢ１可以通过促进结肠癌细胞中ＶＥＧＦ-Ｄ的表达,诱导淋巴管生成,从而促进其淋巴结转移。     

高转移潜能人肝癌细胞特异性结合肽对肝癌侵袭和转移的影响

目的 研究高转移潜能人肝癌细胞特异性结合肽(AWYPLPP肽)对肝癌侵袭和转移的影响.方法 采用Matrigel侵袭实验、迁移实验、二苯基溴化四氮唑蓝(MTT)实验以及黏附实验,探讨AWYPLPP肽对高转移潜能人肝癌细胞HCCLM3侵袭表型的影响.裸鼠皮下接种HCCLM3细胞,建立肿瘤肺转移模型,观察AWYPLPP肽对HCCLM3肺转移的影响.结果 侵袭实验结果显示,在AWYPLPP肽浓度为0.1～100 μmol/L时,能显著促进HCCLM3细胞的侵袭能力,呈剂量效应关系.迁移实验、MTT实验和黏附实验表明,AWYPLPP肽对HCCLM3细胞的迁移、增殖和黏附能力无影响.HCCLM3细胞皮下接种30 d后处死裸鼠,AWYPLPP肽组出现明显的肺转移,肺转移率为88.9%(8/9),与PBS组比较,差异有统计学意义(P<0.05),但对皮下肿瘤的生长无明显影响.结论 AWYPLPP肽能促进高转移潜能人肝癌细胞体外侵袭能力以及肿瘤肺转移;进一步寻找肿瘤细胞表面与AWYPLPP肽结合的受体,可能为深入研究肝癌侵袭转移的机制和设计干预治疗的靶点提供新思路.     

薏苡仁油注射液对人体肝癌SMMC-7721细胞株体外抗肿瘤作用及机制研究

目的：研究薏苡仁油注射液（KLT）对人体肝癌SMMC-7721的体外抗肿瘤作用及机制。方法：在人肝癌SMMC-7721细胞模型上采用CCK-8细胞增殖试验、划痕试验、Transwell小室穿膜试验、Matrigel克隆形成试验观察KLT对细胞增殖、迁移及侵袭的影响;应用流式细胞术检测KLT对肿瘤细胞周期及细胞凋亡的影响;Western blot检测KLT对肿瘤细胞中目的基因的表达情况。结果：经KLT作用后的人肝癌细胞生长、迁移及侵袭功能被抑制;流式细胞术检测发现KLT处理的肝癌细胞阻滞于G2/M期,细胞晚期凋亡较明显;KLT上调了cyclin B1的表达,下调了cyclin D1、cyclin E的表达。结论：KLT在体外对肝癌细胞具有良好的抗肿瘤活性,其作用机制可能与其诱导的细胞周期阻滞、细胞凋亡、抑癌基因的上调、癌基因的下调有关。     

Involvement of epidermal growth factor receptor overexpression in the promotion of breast cancer brain metastasis

BACKGROUND:

Brain‐metastatic breast cancer (BMBC) is increasing and poses a severe clinical problem because of the lack of effective treatments and because the underlying molecular mechanisms are largely unknown. Recent work has demonstrated that deregulation of epidermal growth factor receptor (EGFR) may correlate with BMBC progression. However, the exact contribution that EGFR makes to BMBC remains unclear.

METHODS:

The role of EGFR in BMBC was explored by serial analyses in a brain‐trophic clone of human MDA‐MB‐231 breast carcinoma cells (231‐BR cells). EGFR expression was inhibited by stable short‐hairpin RNA transfection or by the kinase inhibitor erlotinib, and it was activated by heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF). Cell growth and invasion activities also were analyzed in vitro and in vivo.

RESULTS:

EGFR inhibition or activation strongly affected 231‐BR cell migration/invasion activities as assessed by an adhesion assay, a wound‐healing assay, a Boyden chamber invasion assay, and cytoskeleton staining. Also, EGFR inhibition significantly decreased brain metastases of 231‐BR cells in vivo. Surprisingly, changes to EGFR expression affected cell proliferation activities less significantly as determined by a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, an anchorage‐independent growth assay, and cell cycle analysis. Immunoblot analysis suggested that EGFR drives cells' invasiveness capability mainly through phosphoinositide 3‐kinase/protein kinase B and phospholipase C γ downstream pathways. In addition, EGFR was involved less in proliferation because of the insensitivity of the downstream mitogen‐activated protein kinase pathway.

CONCLUSIONS:

The current results indicated that EGFR plays more important roles in cell migration and invasion to the brain than in cell proliferation progression on 231‐BR cells, providing new evidence of the potential value of EGFR inhibition in treating BMBC. Cancer 2012. © 2012 American Cancer Society.     

Low Dose Berberine Suppresses Cholangiocarcinoma Cell Progression as a Multi-Kinase Inhibitor

Background: Berberine (BBR), a natural isoquinoline alkaloid, possesses diverse pharmacological properties and anti-cancer effects that have been demonstrated in many in vitro and in vivo studies. In this study, the inhibitory effects and molecular mechanism of low dose BBR on EMT-induced cell migration, and invasion capability of cholangiocarcinoma (CCA) cell lines were demonstrated. Methods: The commercially available BBR chloride powder with purity ≥ 95% was used in this study. Effects of BBR on cell growth of two human CCA cell lines, KKU-213A and KKU-213B were measured using MTT assay. The progressive phenotypes-cell adhesion, migration, and invasion were evaluated using cell adhesion, wound healing, and Boyden chamber assays. Molecular docking analysis was performed to assess the possible binding mode of BBR against EGFR, Erk, STAT3 and Akt. The effects of BBR on the activations of EGF/EGFR and its downstream effectors were demonstrated using Western blotting. Results: BBR inhibited growth of CCA cells in a dose dependent manner. At sub-cytotoxic dose, BBR significantly inhibited cell adhesion, migration, invasion and decreased expression of vimentin, slug, and VEGFA of both CCA cell lines. Molecular docking suggested the simultaneous inhibitory activity of BBR on EGFR, Erk, STAT3 and Akt. The Western blot analyses revealed that upon the EGF/EGFR activation, BBR considerably attenuated the activations of EGFR, Erk, STAT3 and Akt. Conclusion: Low dose of BBR suppresses EMT and thus aggressiveness of CCA cells, in part by its multi-kinase inhibitor property on EGFR and its downstream pathways.  BBR might be beneficial for therapy of human CCA.     

Circumvention of Cisplatin resistance in h-ras transformed-nih/3t3 cells by suppressor mutant of h-ras

The expression of H-ras oncogene, it has been shown, induces cisplatin resistance in vitro. Using two types of flat revertants (R1, F32/F33) which lost the transformed phenotypes, we studied the mechanism of the cisplatin resistance. R1 cells, which expressed an activated c-H-ras oncogene, exhibited increased cisplatin resistance. Further, F32/F33 cell lines, which were suppressed the H-ras function by a suppressor mutant of H-ras, restored the cisplatin sensitivity. These results implicate that the cisplatin resistance was directly related to the expression of H-ras and can be circumvented by suppression of the H-ras functions.     

Growth properties, differentiation capacity and oncogene expression in metastatic and nonmetastatic Friend leukemia cell variants.

The aims of this study were: (1) to characterize the biologic properties of the WGA-resistant (WR) Friend leukemia cells (FLC) as compared to the original nonmetastatic or highly metastatic FLC; (2) to investigate the possible correlations between the expression of some oncogenes (i.e., c-myc, H-ras and K-ras) and the in vitro and in vivo behavior of FLC. The tumorigenic behavior of the different FLC types strongly depended on the site of tumor injection. Both WR FLC and in vitro passaged FLC did not grow as ascites (when injected intraperitoneally) and developed large solid tumors (when injected subcutaneously), without forming any spleen or liver metastasis. In contrast, in vivo passaged FLC rapidly formed hemorrhagic ascites when injected intraperitoneally; the subcutaneous injection of these cells resulted in the development of solid tumors, which were smaller than the other FLC tumors, but capable of metastasizing to the liver and to the spleen. No significant differences were observed in the in vitro growth characteristics and cell cycle parameters among the different FLC types under various experimental conditions (i.e., FCS concentration or cell seeding densities). Similarly to the metastatic in vivo passaged parental cells, WR FLC exhibited a much lower erythroid differentiation after in vitro addition of either dimethyl sulfoxide or hexamethylene bisacetamide than the in vitro passaged FLC. High levels of c-myc oncogene mRNA were expressed in all FLC variants; no major variations in the c-myc expression were observed in FLC cultivated in medium supplemented with different FCS concentrations and/or seeded at various cell densities. In addition, no changes in the expression of H-ras or K-ras were observed between the different FLC types.     

Clonal heterogeneity, experimental metastatic ability, and p21 expression in H-ras-transformed NIH 3T3 cells

We examined individual clones of murine NIH 3T3 cells, transformed with the human bladder cancer (T24) H-ras oncogene, for p21 expression and for experimental metastatic ability in the immunodeficient chick embryo. We found that the clones were heterogeneous for both of these properties. In general, p21 expression was a good predictor of metastatic ability of the clones. Cells from poorly metastatic clones were passaged in the chick embryo metastasis assay to determine whether cells with increased metastatic ability could be selected. We found that the selected cells were more metastatic and that substantial increases in expression of p21 also accompanied this increase in metastatic ability. The relationship between p21 expression and metastatic ability appeared linear, with a high correlation coefficient (r = .85), suggesting that in this model system quantitative increases in metastatic properties can result from increased expression of the ras oncogene protein product p21.     

Biological characteristics of a specific brain metastatic cell line derived from human lung adenocarcinoma

To study the expression of VEGF, MMP-9, EGFR, and S100B in a highly brain metastases sub-clone cell line, PC14/B. The in vitro metastases-related behaviors of PC14 /B cells, such as adhesion to extracellular matrix (ECM), migration, and invasion were determined and compared with primary PC14 cells and A549 cells that do not metastasize to brain. The expression of vascular epithelial growth factor (VEGF), matrix metalloproteinase 9 (MMP-9), S100B, and epidermal growth factor receptor (EGFR) in the above three cell lines were measured by immunohistochemical staining and Western blot assay.The PC14/B cells have enhanced abilities of adhesion, migration, and invasion than PC14 cells and A549 cells. The expression levels of VEGF and MMP-9 in PC14/B cells are much higher than in PC14 and A549 cells. Two protein polymers of S100B are expressed specially in PC14/B cells. The expression of EGFR has a significant lower level in PC14 cells than in the other two cell lines. The increased expression of VEGF and MMP-9 may lead to the enhancement of adhesion, migration, and invasion of PC14/B cells. The expression of EGFR in PC14/B cells may have negative correlation with their capacities of metastasizing to brain. The specific expression of S100B in PC14/B cells strongly suggest that S100B might be a potential target for developing new therapy to brain metastases of lung cancer.     

BMP4 通过诱导EMT 促进肝癌迁移侵袭*

目的:检测BMP 4 在肝癌中的表达并探讨BMP 4 在诱导肝癌EMT 中的作用,进而研究其对肝癌细胞迁移侵袭能力的影响。方法:采用免疫组织化学方法检测肝癌组织中BMP 4 的表达,分析其与肝癌临床病理资料之间的关系。将BMP 4 表达质粒转染至肝癌细胞系HepG2 中,诱导BMP 4 外源性过表达。观察BMP 4 转染前、后HepG2 的细胞形态学改变;Westernblot检测转染前、后HepG2 中BMP 4、EMT 相关蛋白（E-cadherin、Vimentin）表达变化情况;划痕和侵袭实验检测BMP 4 对细胞迁移侵袭能力的影响。结果:BMP 4 与患者的年龄、病理分级、临床分期、不良预后密切相关。BMP 4 过表达后HepG2 呈现典型的EMT 形态学改变,E-cadherin 表达下调、Vimentin 表达上调、细胞的迁移侵袭能力显著增强。结论:BMP 4 与肝癌临床病理资料密切相关,并可能通过诱导EMT 促进肝癌细胞的迁移侵袭能力。      

Flow cytometric analysis of oncogene products

Flow cytometry (FCM) of oncogene products which opens new avenues of cell biological investigation of human neoplasia is being reviewed. Using H-ras p21/DNA dual FCM, patients with DNA-aneuploid multiple myeloma (MM) were examined. The patients whose MM cells expressed high level of H-ras p21 had poor prognosis. Specificity of this assay was appraised extensively. It is not likely that H-ras p21 expressed in MM is of oncogenic form since point mutation of H-ras gene was not reported in B cell chronic lymphocytic leukemia which is closely located to MM in B lymphocyte differentiation lineage. High expression of H-ras p21 in MM seems to be related to cell proliferation and/or differentiation. H-ras p21/DNA dual FCM is applicable to analyse the pathophysiology of tumor cells. FCM analyses of other oncogene products and proteins related to cell proliferation, c-myc, p53 and Ki-67, were also described. Multiparameter FCM analysis is quite suited to examine expression of these proteins in situ.