Differential Expression of O-glycoprotein Glycans in Cholangiocarcinoma Cell Lines

Protein glycosylation is the most common posttranslational modification in mammalian cells. Aberrant protein glycosylation has been reported in various diseases, including cancer. We identified and quantified the glycan structures of O-linked glycoprotein from cholangiocarcinoma (CCA) cell lines from different histological types and compared their profiles by nanospray ionization-linear ion trap mass spectrometry (NSI-MSn). Five human CCA cell lines, K100, M055, M139, M213 and M214 were characterized. The results showed that the O-linked glycans of the CCA cell lines comprised tri- to hexa-saccharides with terminal galactose and sialic acids: NeuAc1Gal1GalNAc1, Gal2GlcNAc1GalNAc1, NeuAc2Gal1GalNAc1 NeuAc1Gal2GlcNAc1GalNAc1 and NeuAc2Gal2GlcNAc1GalNAc1 All five CCA cell lines showed a similar glycan pattern, but with differences in their quantities. NeuAc1Gal1GalNAc1 proved to be the most abundant structure in poorly differentiated adenocarcinoma (K100; 57.1%), moderately differentiated adenocarcinoma (M055; 42.6%) and squamous cell carcinoma (M139; 43.0%), while moderately to poorly differentiated adenocarcinoma (M214; 40.1%) and adenosquamous cell carcinoma (M213; 34.7%) appeared dominated by NeuAc2Gal1GalNAc1. These results demonstrate differential expression of the O-linked glycans in the different histological types of CCA. All five CCA cell lines have abundant terminal sialic acid (NeuAc) O-linked glycans, suggesting an important role for sialic acid in cancer cells. Our structural analyses of glycans may provide important information regarding physiology of disease-related glycoproteins in CCA.     

CircHIPK3通过调控p53-Akt-Mdm2通路影响食管鳞癌的恶性表型

目的 研究食管鳞癌组织中CircHIPK3的表达差异,以寻找食管鳞癌诊断和靶向治疗的新思路。方法 qRT-PCR、CCK-8法、Transwell法和流式细胞术检测CircHIPK3在食管鳞癌和癌旁组织中的表达差异以及其对细胞增殖、侵袭、迁移和凋亡的影响;生物信息数据分析确定CircHIPK3的可能作用通路,Western blot对其通路相关功能蛋白加以验证。结果 11例患者的癌组织和对应的7例癌旁组织中CircHIPK3表达异常（P=0.027）;CircHIPK3高表达的细胞增殖（P<0.001）、迁移（P<0.001）、侵袭（P<0.001）能力增强;CircHIPK3过表达的EC9706细胞凋亡受到抑制,抑癌基因p53被明显抑制,而Akt-Mdm2信号通路处于激活状态,p53-Akt-Mdm2通路蛋白的表达量随之增加,最终导致癌细胞增殖、迁移、侵袭以及相关癌症蛋白的表达增加。结论 CircHIPK3可能通过调节p53-Akt-Mdm2信号通路促进人食管鳞癌细胞的增殖、迁移和侵袭,并抑制细胞凋亡,其可能是食管鳞癌诊断和治疗的潜在靶点。     

两种巨噬细胞系中PKC亚型表达的差别与其抗瘤活性的比较:依赖一氧化氮的杀瘤机制

目的探讨 LPS 诱导的巨噬细胞杀瘤效应以及与 PKC 相关的分子机制。方法两种巨噬细胞系 P388D1和 RAW264.7用 LPS 刺激、或先用 PMA 长期处理下调 PKC 表达、或用 L-NAME 阻断 iNOS 后再以LPS 刺激,检测其杀伤肿瘤细胞系 P815(MTT 法)及分泌 IL-1,TNF-α(ELISA 法)和 NO(Griess 试剂)的作用;并用 Western blot 法检测 PMA 长期作用后两株细胞系中 PKC 亚型的表达。结果 LPS 刺激的 RAW264.7细胞有杀伤靶细胞的功能,而 P388D1几乎没有杀伤作用。PMA 预处理(1μg/mL,24h)后可明显抑制 LPS 诱导的杀瘤作用。因上述结果提示 PKC 在巨噬细胞杀瘤活性中可能的重要作用,比较了 PMA 处理后两株细胞 PKC 亚型的表达:Western blot 结果显示,在所检测的 PKCα,β1,β2,δ及ε5种亚型中,在 P388D1细胞均有表达,而在 RAW264.7细胞仅有 PKCα,PKCβ1,PKCδ表达;1μg/mL PMA 作用24h 后明显下调了 RAW264.7细胞 PKCα,PKCβ1和PKCδ的表达,在 P388D1则有 PKCα、PKCδ和 PKCε下调,而 PKCβ1和 PKCβ2不被下调。结合 LPS 诱导的与 PKC 活化有关的 IL-1、TNF-α及 NO 的产生,发现在 P388D1细胞几乎不产生 NO,而在 RAW264.7细胞,NO 合成酶抑制剂 L-NAME 不仅能阻断 LPS 诱导的 NO 的产生,而且明显抑制 LPS 诱导的杀瘤活性。结论 LPS 诱导的巨噬细胞杀瘤作用主要是 NO 的杀伤作用来完成而非巨噬细胞直接与瘤细胞作用所致;而该作用与 PKCβ活性密切相关。     

两种巨噬细胞系中PKC亚型表达的差别与其抗瘤活性的比较:依赖一氧化氮的杀瘤机制

目的 探讨LPS诱导的巨噬细胞杀瘤效应以及与PKC相关的分子机制。方法 两种巨噬细胞系P388D1和RAW264．7用LPS刺激、或先用PMA长期处理下调PKC表达、或用L-NAME阻断iNOS后再以LPS刺激,检测其杀伤肿瘤细胞系P815(MTT法)及分泌IL-1,TNF-α(ELISA法)和NO(Griess试剂)的作用;并用Western blot法检测PMA长期作用后两株细胞系中PKC亚型的表达。结果 LPS刺激的RAW264．7细胞有杀伤靶细胞的功能,而P388D1几乎没有杀伤作用。PMA预处理(1μg／mL,24h)后可明显抑制LPS诱导的杀瘤作用。因上述结果提示PKC在巨噬细胞杀瘤活性中可能的重要作用,比较了PMA处理后两株细胞PKC亚型的表达：Western blot结果显示,在所检测的PKCα,β1,β2,δ及ε5种亚型中,在P388D1细胞均有表达,而在RAW264．7细胞仅有PKcd,PKCα,PKCβ1,PKCδ表达;1μg/mL PMA作用24h后明显下调了RAW264．7细胞PKCα,PKCβ1和PKCδ的表达,在P388D1则有PKCα、PKCδ和PKCε下调,而PKCβ1和PKCβ2不被下调。结合LPS诱导的与PKC活化有关的IL-1、TNF-α及NO的产生,发现在P388D1细胞几乎不产生NO,而在RAW264．7细胞,NO合成酶抑制剂L-NAME不仅能阻断LPS诱导的NO的产生,而且明显抑制LPS诱导的杀瘤活性。结论 LPS诱导的巨噬细胞杀瘤作用主要是NO的杀伤作用来完成而非巨噬细胞直接与瘤细胞作用所致;而该作用与PKCβ活性密切相关。     

Deletion of Atbf1/Zfhx3 In Mouse Prostate Causes Neoplastic Lesions,Likely by Attenuation of Membrane and Secretory Proteins and Multiple Signaling Pathways

The ATBF1/ZFHX3 gene at 16q22 is the second most frequently mutated gene in human prostate cancer and has reduced expression or mislocalization in several types of human tumors. Nonetheless, the hypothesis that ATBF1 has a tumor suppressor function in prostate cancer has not been tested. In this study, we examined the role of ATBF1 in prostatic carcinogenesis by specifically deleting Atbf1 in mouse prostatic epithelial cells. We also examined the effect of Atbf1 deletion on gene expression and signaling pathways in mouse prostates. Histopathologic analyses showed that Atbf1 deficiency caused hyperplasia and mouse prostatic intraepithelial neoplasia (mPIN) primarily in the dorsal prostate but also in other lobes. Hemizygous deletion of Atbf1 also increased the development of hyperplasia and mPIN, indicating a haploinsufficiency of Atbf1. The mPIN lesions expressed luminal cell markers and harbored molecular changes similar to those in human PIN and prostate cancer, including weaker expression of basal cell marker cytokeratin 5 (Ck5), cell adhesion protein E-cadherin, and the smooth muscle layer marker Sma; elevated expression of the oncoproteins phospho-Erk1/2, phospho-Akt and Muc1; and aberrant protein glycosylation. Gene expression profiling revealed a large number of genes that were dysregulated by Atbf1 deletion, particularly those that encode for secretory and cell membrane proteins. The four signaling networks that were most affected by Atbf1 deletion included those centered on Erk1/2 and IGF1, Akt and FSH, NF-κB and progesterone and β-estradiol. These findings provide in vivo evidence that ATBF1 is a tumor suppressor in the prostate, suggest that loss of Atbf1 contributes to tumorigenesis by dysregulating membrane and secretory proteins and multiple signaling pathways, and provide a new animal model for prostate cancer.     

Induction of Apoptosis in Human Leukemic Cell Lines by Diallyl Disulfide via Modulation of EGFR/ERK/PKM2 Signaling Pathways

Background: Diallyl disulfide (DADS) may exert potent anticancer action both in vitro and in vivo. Althoughits effects on cancer are significant, the underlying mechanisms remain unknown. In this study, we sought toelucidate possible links between DADS and pyruvate kinase (PKM2). Materials and Methods: KG1α, a leukemiacell line highly expressing PKM2 was used with a cell counting kit (CCK)-8 and flow cytometry (FCM) toinvestigate the effects of DADS. Relationships between PKM2 and DADS associated with phosphorylation ofEGFR, ERK1/2 and MEK, were assessed by western blot analysis. Results: In KG1α cells highly expressingPKM2, we found that DADS could affect proliferation, apoptosis and EGFR/ERK/PKM2 signaling pathways,abrogating EGF-induced nuclear accumulation of PKM2. Conclusions: These results suggested that DADSsuppressed the proliferation of KG1α cells, providing evidence that its proapoptotic effects are mediated throughthe inhibition of EGFR/ERK/PKM2 signaling pathways.     

塞来昔布对前列腺癌细胞株PC-3中VEGF-C及bcl-2 mRNA表达的影响

 目的探讨环氧化酶-2(COX-2)选择性抑制剂塞来昔布(celecoxib)对前列腺癌细胞株PC-3中VEGF-CmRNA及bcl-2mRNA表达的影响,研究COX-2抑制剂抗肿瘤的作用机制。方法将培养的PC-3细胞分为四组:试验组(分别以10μmol/L、20μmol/L、40μmol/L不同浓度塞来昔布处理)和对照组,采用RT-PCR的方法检测四组中VEGF-C及bcl-2mRNA的表达。结果10μmol/L塞来昔布组VEGF-C/GAPDH值及bcl-2/GAPDH值与对照组相比差别均无统计学意义(P>0.05);20μmol/L及40μmol/L塞来昔布组VEGF-C/GAPDH值分别为0.370±0.063、0.263±0.062,bcl-2/GAPDH值分别为0.339±0.047、0.272±0.042,两组中上述两项指标均较对照组明显下降(P均<0.01)。结论塞来昔布可能通过对前列腺癌细胞株PC-3中VEGF-C及bcl-2mRNA表达的抑制发挥其抗肿瘤作用。     

Differential Expression of Progesterone Receptor Isoforms A and B in the Normal Ovary, and in Benign, Borderline, and Malignant Ovarian Tumors

Human epithelial ovarian neoplasm is well-known to be sex steroid-related, but the possible biological significance of progesterone actions in these tumors remains controversial. In this study, we examined the differential expression patterns of the two progesterone receptor (PR) isoforms, PRA and PRB, using immunohistochemistry and real-tune quantitative RT-PCR in normal and neoplastic ovarian tissues, and in cell lines derived from a normal ovarian surface epithelium and an ovarian epithelial carcinoma in order to further elucidate the possible involvement of progesterone in the development of ovarian neoplasms. The median H scores for PR isoforms in normal ( n =8), benign ( n =10), borderline ( n =8) and malignant ( n =24) ovarian tissues were as follows; PRA: 194.0, 171.0, 49.5, 0 ( P <0.05), and PRB: 175.0, 180.5, 251.5, 168.5, respectively. In ovarian cancer cell lines (OVCAR–3 and Caov–3), the PRB/PRAB mRNA ratio was increased by 17β-estradiol, both tune-and dose-dependently. However, this ratio was unaltered following the addition of 17β-estradiol in a normal ovarian epithelial cell line (NOV–31). Immunoblotting analysis demonstrated that PRB protein expression was markedly up-regulated in OVCAR–3, whereas the PRA and PRB isoforms both appeared to be increased in NOV–31. These results suggest that down-regulation of PRA is associated with the development of ovarian epithelial carcinoma.     

非小细胞肺癌细胞株TGFBR3基因缺陷表达及其分子机制研究

背景与目的国外有研究表明,非小细胞肺癌(non-small cell lung cancer,NSCLC)中转化生长因子受体III(TGFBR3)存在缺陷表达,但是分子机制尚未明确。本研究以正常支气管上皮细胞(human bronchial epithelial cell,HBEpiC)为对照,分析NSCLC细胞株中TGFBR3基因的表达情况,并探讨TGFBR3基因表达失活的分子机制。方法采用Western blot检测HBEpiC和NSCLC细胞株中TGFBR3的表达情况并做相对定量分析;采用DNA直接测序检测TGFBR3基因启动子基本转录元件区的突变情况;应用亚硫酸氢钠处理后测序法(bisulfite sequence-PCR,BSP)检测TGFBR3基因启动子区甲基化状态。结果NSCLC细胞株中TGFBR3表达水平明显低于HBEpiC;高转移细胞株95D明显低于非转移细胞株LTEP-α-2、A549、NCI-H460;HBEpiC与NSCLC细胞株中TGFBR3基因近端启动子区-165到-75区域无遗传突变,且未见甲基化,远端启动子区-314到-199区域均为高甲基化。结论TGFBR3基因在NSCLC细胞株中表达下调,在高转移细胞株95D中尤其明显,提示该基因的表达缺陷对NSCLC发生发展起重要作用,可能与NSCLC的侵袭和转移相关;然而,TGFBR3基因启动子区重要转录元件区域的甲基化状态并不是导致TGFBR3基因表达下调的主要原因。     

Regulation of Human B Cell Lymphopoiesis by Adhesion Molecules and Cytokines

Recent advances in the ability to culture normal human B cell precursors have emphasized the supportive relationship between these cells and stromal cells in the bone marrow microenvironment. It is now possible to examine the role of adhesion molecules and cytokines in the regulation of different stages of human lymphopoiesis using these culture systems. Direct cell-cell adhesion mediated by the integrin adhesion molecule VLA-4 plays a critical role in supporting stromal dependent proliferation of human B cell precursors. In addition, human B precursor cell lines migrate underneath the stromal layer. This transmigration is VLA-4 dependent but not inhibitable by antibody to known VLA-4 ligands. IL-7 is secreted by the stromal layer, and is necessary for stromal-dependent proliferation of early human B cell precursors. Proliferation of early human B cell precursors is inhibited by multiple cytokines, some of which stimulate growth of late B cell precursors or mature B cells. Since the bone marrow stroma is a source of cytokines with B cell precursor growth stimulatory activity, it is possible that adhesion interactions may play a co-stimulatory role with respect to cytokine secretion or response. As the cytokine requirements for human B cell lymphopoiesis become more completely defined, it will be important to uncover the cell-cell signals that regulate lymphophoiesis either directly or through modulation of cytokine secretion by supporting cells in the bone marrow microenvironment. The dependent relationship between human B cell precursors and the bone marrow microenvironment provides a model system for these cell-cell interactions which may be applicable to progenitor development in other lineages.     

Differential Regulation of MMP-9 and TIMP-2 Expression in Malignant Melanoma Developed in Metallothionein/RET Transgenic Mice

We recently established a metallothionein-I(MT)/RET transgenic mouse line in which skin melanosis, benign melanocytic tumor and malignant melanoma develop stepwise. Malignant melanoma cells but not benign melanocytic tumor cells had metastatic ability in transgenic mice. In the present study, we investigated the expression of several matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) , including MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MT1– MMP, TIMP-1 and TIMP-2 , in these tumors. Western and northern blot analyses revealed that malignant transformation of melanocytic tumors developed in MT/RET transgenic mice accompanied with upregulation of MMP-9 and downregulation of TIMP-2. Expression of other MMP and TIMP genes examined was very low or undetectable in both benign and malignant tumors. Since activation of MMP-9 in malignant tumors was detected by gelatin zymography, these results suggest that imbalance of expression of the MMP-9 and TIMP-2 genes might be associated with metastatic ability of melanoma cells developed in MT/RET transgenic mice.     

转移相关基因CD44V6在不同恶性肿瘤细胞系中的表达及意义

目的 寻找具有高转移潜能的肿瘤细胞系。方法 采用免疫组织化学染色ＳＰ法,检测ＣＤ４４Ｖ６在不同恶性肿瘤细胞系中表达。结果 仅有Ｃｏｌｏ－２０５细胞表达ＣＤ４４Ｖ６,其表现为细胞膜表面有呈连续线状分布的棕色颗粒。结论 大肠癌Ｃｏｌｏ－２０５细胞可能具有较高的转移能力。     

Differential Expression of HER2 and SKP2 in Benign and Malignant Colorectal Lesions

Background: Colorectal cancer (CRC) is the fourth most common cancer worldwide. Both HER2 and SKP2 have a carcinogenic role in CRC making them attractive targets for tailored treatment. This work aims to correlate HER2 and SKP2 protein expression as well as HER2 gene amplification with clinicopathological parameters aiming at identifying potential candidates for targeted therapy. Methods: This Study was conducted on 127 paraffin-embedded tissue samples of different colorectal lesions [controls, chronic colitis, ulcerative colitis (UC), hyperplastic polyps (HPs), adenomas and CRCs] to investigate HER2 and SKP2 expression by immunohistochemistry (IHC), Selected CRC cases [equivocal (2+) and positive (3+) by IHC] were further evaluated by ISH (CISH and SISH ) to assess HER2 gene amplification. Results: Chronic colitis, UC, HPs and adenomas were HER2-negative. HER2 positivity (scores 2+ and 3+) was found only in15% of CRCs. Both SISH and CISH showed the same results with high concordance as 66.7% of equivocal and 100% of positive cases showed amplification of HER2 gene. SKP2 positivity was detected in 26.7% and 45% of adenomas and CRCs respectively, while other studied groups were negative. A significant correlation was noted between HER2 and SKP2 expression. Conclusion: A small percent of CRCs exhibited HER2 gene amplification, which would be potential candidates for anti HER2 therapy whereas IHC could be a primary screening test for patient selection. A potential carcinogenic role of SKP2 was suggested by the findings that SKP2 expression was undetectable in normal colonic mucosa but significantly increases from adenoma to carcinoma, hoping adenoma patients to get benefit from targeted therapy.     

卵巢癌细胞系BRCA1蛋白表达的雌激素调控

目的：观察不同浓度雌激素对卵巢癌细胞系乳腺癌／卵巢癌易感基因（BRCAI）蛋白表达的影响。方法：采用免疫组化及计算机图象分析方法，检测体外不同浓度（10－9mol／L~10－6mol／L．）的17－β雌二醇（F2）对卵巢癌OVCAR3和SKOV3细胞系BRCA1蛋白表达的影响。结果：OVCAB3和SKOV3细胞在E2的作用下，BRCA1的表达明显增加，且呈现正性剂量效应关系。结论：雌激素能够诱导卵巢癌OVCAB3和SKOV3细胞系BRCA1蛋白的表达，可通过该方法探索治疗卵巢癌的新途径。     

细胞间隙连接基因connexin32、connexin43及其蛋白在肝癌细胞系中的表达

为了探讨细胞间隙连接ｃｏｎｎｅｘｉｎ（ｃｘ）基因及其蛋白产物在肝癌细胞系的表达及意义,应用核酸分子原位杂交和流式细胞仪分析技术,研究肝癌细胞系ＨＨＣＣ、ＳＭＭＣ７７２１和正常肝细胞系ＱＺＧ中,间隙连接基因ｃｘ３２、ｃｘ４３ｍＲＮＡ及其蛋白产物的表达规律。原位杂交显示,ｃｘ３２、ｃｘ４３ｍＲＮＡ在ＨＨＣＣ、ＳＭＭＣ７７２１和ＱＺＧ中均呈强阳性。流式细胞仪分析,Ｃｘ３２蛋白在ＨＨＣＣ、ＳＭＭＣ７７２１和ＱＺＧ中阳性细胞计数率分别为１．９％、１．７％和９９．０％,Ｃｘ４３蛋白阳性细胞计数率分别为７．３％、２．３％和９９．１％。Ｃｘ３２、Ｃｘ４３蛋白在ＨＨＣＣ、ＳＭＭＣ７７２１中阳性细胞计数率与ＱＺＧ相比,有非常显著性差异（Ｐ＜０．０１）。ｃｘ３２、ｃｘ４３基因在转录后水平的调控异常所导致的蛋白表达降低与ＨＣＣ的发生密切相关。     

Alkylglyceronephosphate Synthase (AGPS) Alters Lipid Signaling Pathways and Supports Chemotherapy Resistance of Glioma and Hepatic Carcinoma Cell Lines

Chemotherapy continues to be a mainstay of cancer treatment, although drug resistance is a majorobstacle. Lipid metabolism plays a critical role in cancer pathology, with elevated ether lipid levels. Recently,alkylglyceronephosphate synthase (AGPS), an enzyme that catalyzes the critical step in ether lipid synthesis,was shown to be up-regulated in multiple types of cancer cells and primary tumors. Here, we demonstrated thatsilencing of AGPS in chemotherapy resistance glioma U87MG/DDP and hepatic carcinoma HepG2/ADM celllines resulted in reduced cell proliferation, increased drug sensitivity, cell cycle arrest and cell apoptosis throughreducing the intracellular concentration of lysophosphatidic acid (LPA), lysophosphatidic acid-ether (LPAe)and prostaglandin E2 (PGE2), resulting in reduction of LPA receptor and EP receptors mediated PI3K/AKTsignaling pathways and the expression of several multi-drug resistance genes, like MDR1, MRP1 and ABCG2.β-catenin, caspase-3/8, Bcl-2 and survivin were also found to be involved. In summary, our studies indicate thatAGPS plays a role in cancer chemotherapy resistance by mediating signaling lipid metabolism in cancer cells.