HER-2 Expression in Brain Metastases from Colorectal Cancer and Corresponding Primary Tumors: A Case Cohort Series

Brain metastases (BM) from colorectal cancer (CRC) are a rare but increasing event. Surgical resection of oligometastatic disease, including BM, may produce a survival benefit in selected patients. Previous studies described the HER-2 expression patterns in CRC patients, but its prognostic role still remains controversial. Information on the HER-2 expression in BM from CRC is currently lacking. Among the over 500 patients treated at our Department of Neurosurgery in the last 13 years (1999–2012), we identified a cohort of 50 consecutive CRC patients resected for BM. Clinical data were retrospectively reviewed using electronic hospital charts and surgical notes. Formalin-fixed, paraffin-embedded tissue samples were retrieved and histologically reviewed. HER-2 status was assessed on 4-μm sections by HerceptTest™, and scored by two pathologists according to gastric cancer HER-2 status guidelines. In score 2+ cases HER-2 gene copy number was analyzed by FISH, performed using the PathVysion HER-2 DNA Probe Kit. Median age at time of BM resection was 65 years (35–82); most patients were males (60%) with a good performance status. The majority of the BM were single (74%) and sited in the supratentorial area (64%); 2–4 lesions were diagnosed in 9 patients (18%), and >4 in 3 patients (6%). The rate of HER-2 positivity (defined as IHC score 3+ or IHC score 2+ and FISH gene amplification) was 8.1% for the primary CRC tumors and 12% for their corresponding BM. The concordance rate between primary tumors and matched BM was 89%. Median overall survival after neurosurgery was 6.5 months for HER-2 IHC score 0 vs. 4.6 months for HER-2 IHC score 1+/2+/3+; the difference was statistically significant (p = 0.01, Log-rank test). HER-2 positivity of our case cohort was low but comparable to literature. Concordance rate of HER-2 expression between BM and corresponding primary tumors is high and similar to those reported for breast and gastric cancers. Our data suggest a potential negative prognostic value of HER-2 expression in brain lesions from CRC.     

An Institutional Retrospective Analysis of 93 Patients with Brain Metastases from Breast Cancer: Treatment Outcomes,Diagnosis-Specific Prognostic Factors

To evaluate the prognostic factors and indexes of a series of 93 patients with breast cancer and brain metastases (BM) in a single institution. Treatment outcomes were evaluated according to the major prognostic indexes (RPA, BSBM, GPA scores) and breast cancer subtypes. Independent prognostic factors for overall survival (OS) were identified. The median OS values according to GPA 0–1, 1.5–2, 2.5–3 and 3.5–4, were 4.5, 9.5, 14.2 and 19.1 months, respectively (p < 0.0001) and according to genetic subtypes, they were 5, 14.2, 16.5 and 17.1 months for basal-like, luminal A and B and HER, respectively (p = 0.04). Using multivariate analysis, we established a new grading system using the six factors that were identified as indicators of longer survival: age under 60 (p = 0.001), high KPS (p = 0.007), primary tumor control (p = 0.05), low number of extracranial metastases and BM (p = 0.01 and 0.0002, respectively) and triple negative subtype (p = 0.002). Three groups with significantly different median survival times were identified: 4.1, 9.5 and 26.3 months, respectively (p < 0.0001). Our new grading system shows that prognostic indexes could be improved by using more levels of classification and confirms the strength of biological prognostic factors.     

重组肺癌抑癌基因1蛋白的表达、纯化及其多克隆抗体的制备

目的表达、纯化重组人肺癌抑癌基因1(Tumorsuppressor in lung cancer1,TSLC1)蛋白,并制备其多克隆抗体。方法采用RT-PCR法扩增TSLC1基因全长编码区序列,克隆入原核表达质粒pQE30,转化大肠杆菌M15,IPTG诱导表达,表达的重组蛋白经Ni2+-NTA亲和层析纯化后,免疫家兔,ProteinA亲和层析纯化抗血清,并经Westernblot分析其反应原性。结果重组表达质粒pQE30-TSLC1经双酶切及测序鉴定证明构建正确。重组TSLC1蛋白的表达量约占菌体总蛋白的14%,主要以包涵体形式存在。纯化的重组蛋白纯度为93.4%,可与小鼠抗His-Tag单克隆抗体发生特异性反应。以其制备的多克隆抗体具有良好的抗原识别特异性。结论已成功制备TSLC1多克隆抗体,为深入研究TSLC1分子的生物学活性奠定了基础。     

肺癌抑癌基因1对鼻咽癌细胞株HNE-1增殖与侵袭能力的影响

目的探讨肺癌抑癌基因1(Tumor suppressor in lung cancer 1,TSLC1)对鼻咽癌细胞株HNE-1增殖与侵袭能力的影响。方法采用RT-PCR法从乳腺癌细胞株MCF-7中扩增TSLC1基因全长编码区序列,构建重组真核表达质粒pcDNA3.1-TSLC1,转染HNE-1细胞,RT-PCR及Western blot检测TSLC1基因mRNA和蛋白的表达水平。MTT和Transwell小室试验检测TSLC1基因过表达对HNE-1细胞增殖及侵袭能力的影响。结果重组表达质粒pcDNA3.1-TSLC1经双酶切及测序证明构建正确;稳定转染重组表达质粒的HNE-1细胞中TSLC1基因出现过表达;TSLC1基因过表达可显著抑制HNE-1细胞的增殖与侵袭能力。结论 TSLC1基因过表达对HNE-1细胞增殖与侵袭能力具有明显的抑制作用,为鼻咽癌的基因治疗提供了理想的分子靶点。     

Modulation of MDR1 and MRP3 Gene Expression in Lung Cancer Cells after Paclitaxel and Carboplatin Exposure

Carboplatin-paclitaxel is a reference regimen in the treatment of locally advanced or disseminated non-small cell lung cancer (NSCLC). This paper discusses the multidrug resistance developed with this drug combination, which is one of the major obstacles to successful treatment. In order to understand and overcome the drug resistance pattern of NSCLC after carboplatin plus paclitaxel exposure, levels of mRNA expression of multidrug resistance 1 (MDR1) and multidrug resistance-associated protein 3 (MRP3) were investigated in primary NSCLC cell lines (A-549 and A-427) and a metastasis-derived NSCLC cell line (NODO). Our results showed that exposure of the three NSCLC lines to plasma concentrations of paclitaxel (5 μM) produced an increase in MDR1 expression, while MRP3 showed no alteration in expression. By contrast, the same cells exposed to carboplatin plasma concentrations (30 μM) showed overexpression of MRP3. In these cells, MDR1 showed no expression changes. Interestingly, the combination of both paclitaxel and carboplatin caused increased expression of the MDR1 drug resistance gene rather than the individual treatments. These results suggest that carboplatin and paclitaxel may induce drug resistance mediated by MDR1 and MRP3, which may be enhanced by the simultaneous use of both drugs.     

Depletion of C3orf1/TIMMDC1 Inhibits Migration and Proliferation in 95D Lung Carcinoma Cells

In our previous study, we identified an association of high expression of c3orf1, also known as TIMMDC1 (translocase of inner mitochondrial membrane domain-containing protein 1), with metastatic characteristics in lung carcinoma cells. To investigate the preliminary function and mechanism of this mitochondrial protein, we depleted C3orf1 expression by introducing siRNA into 95D lung carcinoma cells. We demonstrated that C3orf1 depletion significantly suppressed 95D cell growth and migration. We confirmed C3orf1 localization in the inner mitochondrial membrane and showed that mitochondrial viability, membrane potential, and ATPase activity were remarkably reduced upon depletion of C3orf1. Microarray data indicated that genes involved in regulation of cell death, migration, and cell-cycle arrest were significantly altered after C3orf1 depletion for 48 h. The expression of genes involved in focal adhesion, ECM-receptor interaction, and p53-signaling pathways were notably altered. Furthermore, cell-cycle arrest genes such as CCNG2 and PTEN as well as genes involved in cell migration inhibition, such as TIMP3 and COL3A1, were upregulated after C3orf1 depletion in 95D cells. Concurrently, expression of the migration-promoting gene NUPR1 was markedly reduced, as confirmed by real-time PCR. We conclude that C3orf1 is critical for mitochondrial function, migration, and proliferation in 95D lung carcinoma cells. Depletion of C3orf1 inhibited cell migration and cell proliferation in association with upregulation of genes involved in cell-cycle arrest and cell migration inhibition. These results suggest that C3orf1 (TIMMDC1) may be a viable treatment target for lung carcinoma, and that further study of the role of this protein in lung carcinoma pathogenesis is justified.     

A Functional Polymorphism in the 3'-UTR of PXR Interacts with Smoking to Increase Lung Cancer Risk in Southern and Eastern Chinese Smoker

Pregnane X receptor (PXR) is an important member of the nuclear receptor superfamily that copes with various endobiotic and xenobiotic stimuli, such as carcinogens by regulating an array of environmental response genes. Low PXR expression has been shown to promote tumor initiation and metastasis. The aim of the current study was to investigate whether the single nucleotide polymorphisms (SNPs) of PXR could alter lung cancer susceptibility in Chinese by affecting the function or expression of PXR. We genotyped three putatively functional SNPs of PXR (i.e., rs3814055C>T, rs3732360C>T, and rs3814058C>T) and analyzed their associations with lung cancer risk in a two-stage case-control study with a total of 1559 lung cancer cases and 1679 controls in the southern and eastern Chinese population. We found that in comparison to the rs3814058CC common genotype, the rs3814058T variants (TC/TT) which is located in the 3''-untranslated region (3''-UTR) of PXR conferred a consistently increased risk of lung cancer in both the southern Chinese (odd ratios (OR) = 1.24, 95% confidence interval (CI) = 1.03−1.49) and the eastern Chinese (OR = 1.33, 95% CI = 1.02−1.75). The variants also significantly interacted with smoking on increasing cancer risk (p = 0.023). Moreover, lung cancer tissues with the rs3814058T variants showed significantly lower PXR expression than those with rs3814058CC genotype in the smokers (p = 0.041). These results suggested that the rs3814058C>T polymorphism of PXR interacts with smoking on increasing lung cancer risk in Chinese smokers, which might be a functional genetic biomarker for lung cancer.     

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。     

可溶性Fas与VEGF及TIMP-1表达在乳腺癌淋巴结转移中的相互关系

目的通过对乳腺癌可溶性Fas浓度与血管内皮生长因子(VEGF)及金属蛋白酶抑制物(TIMP-1)表达之间关系的分析,探讨患者血清中sFas浓度改变对乳腺癌细胞生长、浸润和转移的影响。方法通过S-P免疫组化法和ELISA法,分别检测乳腺癌细胞VEGF和TIMP-1表达及血清sFas浓度,结合VEGF、TIMP-1与乳腺癌临床病理参数,分析浸润、淋巴结转移乳腺癌中VEGF、TIMP-1表达失衡与血清sFas浓度之间的关系。结果①VEGF阳性表达与乳腺癌淋巴结转移呈显著相关,VEGF阳性的肿瘤有淋巴结转移率为56%,明显高于VEGF阳性的肿瘤无淋巴结转移率(7%)。随着乳腺癌TNM分期和组织学分级的进展,VEGF阳性表达率逐渐增加,呈正相关。TIMP-1阳性表达与肿瘤淋巴结转移明显相关。随着TNM分期和组织学分级的进展,TIMP-1阳性表达率逐渐降低,呈负相关。②VEGF阳性表达而TIMP-1阴性表达组发生乳腺癌浸润和淋巴结转移率及血清sFas浓度最高,VEGF阴性表达而TIMP-1阳性表达组发生淋巴结转移率及血清sFas浓度最低,两组比较差异有极显著意义。结论sFas与VEGF及TIMP-1表达之间存在着反馈机理以及相互激活的密切关系。sFas浓度可以间接地反映基质金属蛋白酶(MMPs)和VEGF的表达情况,sFas在乳腺癌中的浓度变化可以反映乳腺癌的生长、局部浸润和转移。     

肠道病毒71型P1和3CD基因双顺反子重组腺病毒的构建及表达

目的构建肠道病毒71型(EV71)P1和3CD基因双顺反子重组腺病毒,并检测3CD蛋白酶的切割作用和表达产物的抗原性。方法采用RT-PCR法从阜阳分离的EV71中扩增P1和3CD基因,构建穿梭质粒pShuttle-CMV-P1-3CD、pShuttle-CMV-P1和pShuttle-CMV-3CD;经同源重组获得重组腺病毒rAd-P1-3CD、rAd-P1和rAd-3CD,分别转染293细胞,RT-PCR法检测目的基因的转录,免疫荧光法检测目的蛋白的表达。结果3种重组腺病毒经PCR和酶切鉴定表明构建正确;RT-PCR检测表明,3个重组腺病毒均已转录目的基因mRNA;免疫荧光检测仅观察到rAd-P1-3CD表达产物具有特异性,而rAd-P1、rAd-3CD未能检测到特异性表达产物。结论已成功构建EV71P1和3CD基因双顺反子重组腺病毒,表达产物具有EV71特异抗原性,提示P1产物只有在被3CD蛋白酶切割后才体现抗原性。     

Quantum Dots-Based Immunofluorescent Imaging of Stromal Fibroblasts Caveolin-1 and Light Chain 3B Expression and Identification of Their Clinical Significance in Human Gastric Cancer

Caveolin-1 (Cav-1) expression deficiency and autophagy in tumor stromal fibroblasts (hereafter fibroblasts) are involved in tumor proliferation and progression, particularly in breast and prostate cancer. The aim of this study was to detect the expression of fibroblastic Cav-1 and LC3B, markers of autophagy, in gastric cancer (GC) and to analyze their clinical significances. Furthermore, because Epstein-Barr virus (EBV)-associated GC (EBVaGC) is a unique subtype of GC; we compared the differential expression of fibroblastic Cav-1 and LC3B in EBVaGC and non-EBVaGC. Quantum dots (QDs)-based immunofluorescence histochemistry was used to examine the expression of fibroblastic Cav-1 and LC3B in 118 cases of GC with adequate stroma. QDs-based double immunofluorescence labeling was performed to detect the coexpression of Cav-1 and LC3B proteins. EBV-encoded small RNA was detected by QDs-based fluorescence in situ hybridization to identify EBVaGC. Multivariate analysis indicated that low fibroblastic Cav-1 level was an independent prognosticator (p = 0.029) that predicted poorer survival of GC patients. Positive fibroblastic LC3B was correlated with lower invasion (p = 0.032) and was positively associated with Cav-1 expression (r = 0.432, p < 0.001). EBV infection did not affect fibroblastic Cav-1 and LC3B expression. In conclusion, positive fibroblastic LC3B correlates with lower invasion, and low expression of fibroblastic Cav-1 is a novel predictor of poor GC prognosis.     

TRFIA螯合剂中间体2,6-二(3′-溴甲基-1′-吡唑基)-4-溴吡啶的合成、分离与表征

以2,6-二（3-甲基-1-H-吡唑基）-4-溴吡啶为原料,经重氮化、溴化合成了新型时间分辨荧光免疫分析（TR-FIA）双功能螯合剂中间体2,6-二（3＇-溴甲基-1＇-吡唑基）-4-溴吡啶,通过IR、GC-MS1、HNMR和元素分析等对其结构进行了确认,探讨了合成条件及反应机理。同时,通过GC-MS1、HNMR和元素分析等对第一副产物4-溴-2-（3＇-溴甲基-1＇-吡唑基）-6-（3＇-甲基-1＇-吡唑基）吡啶的结构也进行了确认,以其为原料可继续合成目标化合物,大幅提高总产率。     

CLA Reduces Inflammatory Mediators from A427 Human Lung Cancer Cells and A427 Conditioned Medium Promotes Differentiation of C2C12 Murine Muscle Cells

Conjugated linoleic acid (CLA) is thought to have anti-proliferative and anti-inflammatory properties, but its effect on cancer cachexia is unknown. Two effects were here investigated: that of CLA on inflammatory mediator production in human lung cancer cells, and that of reduced mediators on the myogenic differentiation of murine muscle C2C12 cells. The latter cells were grown in medium conditioned by human lung cancer A427 cells, with or without CLA, to mimic only the effect of molecules released from the tumor “in vivo”, excluding the effect of host-produced cachectic factors. The results obtained show that CLA was found to reduce the production of tumor necrosis factor-α, interleukin (IL)-1β and prostaglandin E2 (PGE2), but had no effect on IL-6 production. The mechanisms underlying the effect of CLA on cytokine or PGE2 release in A427 cells are probably mediated by activation of peroxisome proliferator-activated receptor (PPAR)α, which increased at 24 h CLA treatment. In turn, the reduced content of inflammatory mediators in medium conditioned by A427 cells, in the presence of CLA, allowed muscle cells to proliferate, again by inducing PPAR. The involvement of PPARα was demonstrated by treatment with the antagonist MK-886. The findings demonstrate the anti-inflammatory and myogenic action of CLA and point to its possible application as a novel dietary supplement and therapeutic agent in inflammatory disease states, such as cachexia.     

Co-Expression and Co-Localization of Cartilage Glycoproteins CHI3L1 and Lubricin in Osteoarthritic Cartilage: Morphological,Immunohistochemical and Gene Expression Profiles

Osteoarthritis is the most common human arthritis characterized by degeneration of articular cartilage. Several studies reported that levels of human cartilage glycoprotein chitinase 3-like-1 (CHI3L1) are known as a potential marker for the activation of chondrocytes and the progression of Osteoarthritis (OA), whereas lubricin appears to be chondroprotective. The aim of this study was to investigate the co-expression and co-localization of CHI3L1 and lubricin in normal and osteoarthritic rat articular cartilage to correlate their modified expression to a specific grade of OA. Samples of normal and osteoarthritic rat articular cartilage were analyzed by the Kellgren–Lawrence OA severity scores, the Kraus’ modified Mankin score and the Histopathology Osteoarthritis Research Society International (OARSI) system for histomorphometric evaluations, and through CHI3L1 and lubricin gene expression, immunohistochemistry and double immuno-staining analysis. The immunoexpression and the mRNA levels of lubricin increased in normal cartilage and decreased in OA cartilage (normal vs. OA, p < 0.01). By contrast, the immunoexpression and the mRNA levels of CHI3L1 increased in OA cartilage and decreased in normal cartilage (normal vs. OA, p < 0.01). Our findings are consistent with reports suggesting that these two glycoproteins are functionally associated with the development of OA and in particular with grade 2/3 of OA, suggesting that in the future they could be helpful to stage the severity and progression of the disease.     

2,3-二甲氧基-5-甲基-6-(3-甲基-2-烯-1-丁基)-1,4-苯酚的合成及其副产物的研究

2,3-二甲氧基-5-甲基-1,4-苯酚与2-甲基丁烯醇在BF3OEt2的催化下进行傅-克烷基化反应合成2,3-二甲氧基-5-甲基-6-(3-甲基-2-烯-1-丁基)-1,4-苯酚。对鲜见文献报道的副产物2,2,5-三甲基-3-氢-6-羟基-7,8-二甲氧基-苯并吡喃进行了结构鉴定并推测了其形成机理,在原料辅酶Q0、甲基丁烯醇和BR的配比为1：1.5：0.19(摩尔比),反应温度45℃,滴加时间30min,反应时间2h的条件下收率达90.24％。     

Effect of hypoxanthine-3(N)-oxide and hypoxanthine-1(N)-oxide on central nervous excitation of the black tetraGymnocorymbus ternetzi (Characidae,Ostariophysi, Pisces) indicated by dorsal light response

The change of state in the central nervous system ofGymnocorymbus ternetzi after detection of hypoxanthine-l(N)-oxide, hypoxanthine-3(N)-oxide, and of the alarm substance from conspecifics was measured quantitatively by means of the fishes' equilibrium behavior. The fish swam freely in a tiny cage, illuminated horizontally from one side. The change of the angle of inclination of the dorsoventral axis of the fish was registered by means of a videorecorder. The recordings were later measured on the monitor in single frames at 0.2-sec intervals where the equilibrium position of the fish could be accurately determined ± 1 °. Various substances were presented to the fish, and their effects upon equilibrium position were recorded. An enhanced optical alertness shown by an increase in the fishes' inclination was generally produced with alarm substance. Without any additional stimulation, the factorU, representing quantitatively the degree of the change of central state, varied slightly within the experimental period of 1 min; however, this factor never exceededU= 1.0 ± 0.15 in control fish. The increase ofU usually exceeded considerably the value 1.15 when skin extract from conspecifics or 7–8 g of hypoxanthine-3(N)-oxide were given. However, when hypoxanthine-l (N)-oxide was presented,U generally did not exceed 1.15. The difference between hypoxanthine-3(N)-oxide and hypoxanthine-l(N)-oxide was highly significant. This result is in accordance with the findings on fish schools ofDanio malabaricus, where hypoxanthine-3(N)-oxide elicited the fright reaction, but hypoxanthine-1(N)-oxide was ineffective. The results support the hypothesis that the alarm substance from the skin ofPhoxinus phoxinus is identical with hypoxanthine-3(N)-oxide. The results with alarm substance or hypoxanthine-3(N)-oxide did not show any adaptation. This was also true in fish that were stimulated repeatedly at intervals of a couple of minutes only. InGymnocorymbus, which has compensated for removal of the otolith of one utriculus, conspecific skin extract triggers the typical postoperative phenomenon, i.e., rotation around the fishes' long axis towards the operated side. Whereas such a decompensation could be elicited by hypoxanthine-3(N)-oxide as well, hypoxanthine-l(N)-oxide had no effect. This finding is interpreted as an effect of the alarm substance and of hypoxanthine-3(N)-oxide on the centers of equilibrium.     

Combustion synthesis of (Ti1−xNbx)2AlC solid solutions from elemental and Nb2O5/Al4C3-containing powder compacts

Preparation of the (Ti_1−xNb_x)₂AlC solid solution (formed from the M_n+1AX_n or MAX carbides, where n = 1, 2, or 3, M is an early transition metal, A is an A-group element, and X is C) with x = 0.2-0.8 was investigated by self-propagating high-temperature synthesis (SHS). Nearly single-phase (Ti,Nb)₂AlC was produced through direct combustion of constituent elements. Due to the decrease of reaction exothermicity, the combustion temperature and reaction front velocity decreased with increasing Nb content of (Ti_1−xNb_x)₂AlC formed from the elemental powder compacts. In addition, the samples composed of Ti, Al, Nb₂O₅, and Al₄C₃ were adopted for the in situ formation of Al₂O₃-added (Ti,Nb)₂AlC. The SHS process of the Nb₂O₅/Al₄C₃-containing sample involved aluminothermic reduction of Nb₂O₅, which not only enhanced the reaction exothermicity but also facilitated the evolution of (Ti,Nb)₂AlC. Based upon the XRD analysis, two intermediates, TiC and Nb₂Al, were detected in the (Ti,Nb)₂AlC/Al₂O₃ composite and their amounts were reduced by increasing the extent of thermite reduction involved in the SHS process. The laminated microstructure characteristic of the MAX carbide was observed for both monolithic and Al₂O₃-added (Ti,Nb)₂AlC solid solutions synthesized in this study.