ABCG2/BCRP expression modulates D-Luciferin based bioluminescence imaging

Bioluminescence imaging (BLI) is becoming indispensable to the study of transgene expression during development and, in many in vivo models of disease such as cancer, for high throughput drug screening in vitro. Because reaction of d-luciferin with firefly luciferase (fLuc) produces photons of sufficiently long wavelength to permit imaging in intact animals, use of this substrate and enzyme pair has become the method of choice for performing BLI in vivo. We now show that expression of the ATP-binding cassette (ABC) family transporter ABCG2/BCRP affects BLI signal output from the substrate d-luciferin. In vitro studies show that d-luciferin is a substrate for ABCG2/BCRP but not for the MDR1 P-glycoprotein (ABCB1/Pgp), multidrug resistance protein 1 (MRP1/ABCC1), or multidrug resistance protein 2 (MRP2/ABCC2). d-Luciferin uptake within cells is shown to be modulated by ABC transporter inhibitors, including the potent and selective ABCG2/BCRP inhibitor fumitremorgin C. Images of xenografts engineered to express transgenic ABCG2/BCRP, as well as xenografts derived from the human prostate cancer cell line 22Rv1 that naturally express ABCG2/BCRP, show that ABCG2/BCRP expression and function within regions of interest substantially influence d-luciferin-dependent bioluminescent output in vivo. These findings highlight the need to consider ABCG2/BCRP effects during d-luciferin-based BLI and suggest novel high throughput methods for identifying new ABCG2/BCRP inhibitors.     

Identification of candidate biomarkers for hepatocellular carcinoma through pre-cancerous expression analysis in an HBx transgenic mouse

The aim of this study is to identify candidate biomarkers for the detection of hepatocellular carcinoma (HCC) through pre-cancerous gene expression analysis in an HBx transgenic mouse model. The gene expression profiles of liver and tumor tissues from 6-, 12- and 18-month-old HBx transgenic and littermate control mice were monitored using DNA oligo microarrays. Data analysis revealed changes in 684 genes in the tumor tissues from HBx transgenic mice. Based on their pre-cancerous expression profiles, two separate gene groups, corresponding to HCC specific and non-specific groups respectively, were identified. Gene ontology analysis identified 47 genes encoding secretory or transmembrane proteins among 155 upregulated genes in the HCC-specific group. Among these, four genes encoding TFF3, IGF2, LPL and SPP1 were found to be promising biomarker candidates for the detection of HCC as compared with AFP. This work describes a new way to identify novel biomarker candidates for HCC diagnosis.     

Molecular discrimination between alpha-fetoprotein from patients with hepatocellular-carcinoma and nonneoplastic liver-diseases by their carbohydrate structures (review)

Serum alpha-fetoprotein (AFP) is a good marker of HCC. However, this protein also increases moderately in non-neoplastic liver diseases. The serum concentration of AFP in HCC at the time of initial diagnosis has become lower thanks to the advancement of imaging modalities. These clinical circumstances have lead to the need of molecular discrimination of AFP between HCC and benign liver diseases. This has been attained by taking advantage of the reactivity of AFP with various lectins. The relative amount of the Lens culinaris agglutinin (LCA)-reactive species of AFP is significantly greater in HCC than in benign liver diseases. Molecular basis of this variation is the fucosylation of sugar chain at innermost N-acetyl-glucosamine. On the other hand, the concanavalin A (Con A)-nonreactive species of AFP increases in AFP-producing gastrointestinal carcinoma as compared with HCC and benign liver diseases. Molecular basis of Con A-nonreactive variants is the N-acetylglucosaminylation of the mannose residue at the trimannosyl core, although the position to be modified can be different. Therefore, the terms 'fucosylation and glucosaminylation indices' have been introduced to express the percentages of LCA-reactive and Con A-nonreactive species of AFP, respectively. The reactivity of AFP to erythroagglutinating phytohemagglutinin of Phaseolus vulgaris (E-PHA) also provides useful information for discrimination between HCC and benign liver diseases. These indices together with the measurement of E-PHA molecular variants are useful to detect HCC even if the disease is at an early stage. Furthermore, they seem to serve the prediction of HCC in the follow-up course of chronic liver diseases. Thus, not only qualitative but also quantitative measurements of lectin-based molecular variants of AFP provide us valuable information for the differential diagnosis of various liver diseases.     

原发性肝癌患者肿瘤组织生长抑素受体表达与血AFP水平的相关性

背景与目的:生长抑素类似物(somatostatin analogue,SSTA)对部分原发性肝癌(hepatocellular carcinoma,HCC)患者具有一定疗效,但HCC患者肿瘤组织中生长抑素受体(somatostatin receptor,SSTR)表达状况不详,其与血甲胎蛋白(alphafetoprotein,AFP)的相关性也未见探讨.本研究拟了解肝癌组织中SSTR1、SSTR2、SSTR3、SSTR4及SSTR5亚型的表达状况及其与血AFP的相关性.方法:逆转录-聚合酶链反应及免疫组织化学法分别检测人肝癌及肝硬化组织(各40例)SSTR1、SSTR2、SSTR3、SSTR4及SSTR5亚型的mRNA及蛋白表达:酶联免疫吸附试验检测HCC患者肿瘤组织及外周血AFP水平.结果:肝癌组织中SSTR1、SSTR2、SSTR3、SSTR4及SSTR5蛋白阳性率(分别为47.5%、70.0%、50.0%、65.0%及67.5%)显著高于肝硬化组织(分别为55.0%、67.5%、52.5%、60.0%及47.5%).肝癌组织中SSTR1、SSTR2、SSTR3、SSTR4及SSTR5表达与患者血AFP水平呈二次曲线正相关(r依次为0.882、0.901、0.877、0.854、0.903,P＜0.05).血AFP水平在200～800 μg/L范围时,伴有肝癌组织SSTRs的高表达,过低或过高的血AFP水平伴有SSTR低表达.结论:约60%HCC患者的肿瘤组织表达了SSTRs,其表达量明显高于肝硬化组织;15 AFP水平在200-800μg/L的HCC患者伴有肝癌组织SSTRs的高表达,提示为SSTA治疗的适宜候选者.     

HBx drives alpha fetoprotein expression to promote initiation of liver cancer stem cells through activating PI3K/AKT signal pathway

Hepatitis B virus (HBV)‐X protein (HBx) plays critical role in inducing the malignant transformation of liver cells. Alpha fetoprotein (AFP) expression is closely related to hepatocarcinogenesis. We report that Oct4, Klf4, Sox2 and c‐myc expression positively associated with AFP(+)/HBV(+) hepatocellular carcinoma(HCC) tissues, and the expression of the stemness markers CD44, CD133 and EpCAM was significantly higher in AFP(+)/HBV(+) HCC tissues compared to normal liver tissues or AFP (?)/HBV(?) HCC tissues. AFP expression turned on prior to expression of Oct4, Klf4, Sox2 and c‐myc, and the stemness markers CD44, CD133 and EpCAM in the normal human liver L‐02 cell line or CHL cell lines upon transfection with MCV‐HBx vectors. Stem‐like cells generated more tumour colonies compared to primary cells, and xenografts induced tumourigenesis in nude mice. Expression of reprogramming‐related proteins was significantly enhanced in HLE cells while transfected with pcDNA3.1‐afp vectors. The specific PI3K inhibitor Ly294002 inhibited the effects of pcDNA3.1‐afp vectors. AFP‐siRNA vectors were able to inhibit tumour colony formation and reprogramming‐related gene expression. Altogether, HBx stimulates AFP expression to induce natural reprogramming of liver cells, and AFP plays a critical role in promoting the initiation of HCC progenitor/stem cells. AFP may be a potential novel biotarget for combating HBV‐induced hepatocarcinogenesis.     

肝癌患者围手术期外周血甲胎蛋白mRNA表达与术后复发的关系

背景与目的:甲胎蛋白(alpha-fetoprotein,AFP)mRNA是检测肝癌患者外周血中癌细胞常用的标志物。本研究探讨肝癌患者围手术期的外周血中AFPmRNA表达与术后复发的关系。方法:应用巢式PCR和Taq Man MGB探针法PCR定量技术,检测56例肝细胞癌患者术前和术后外周血、15例良性肝占位性病变合并肝硬化患者外周血以及30例健康志愿者外周血AFP mRNA的表达情况。结果:肝癌患者术前外周血AFP mRNA的阳性率为42.9%(24/56),高于良性肝占位合并肝硬化患者的13.3%(2/15)和健康对照的10.0%(3/30),其差异均有统计学意义(P=0.035,P=0.002)。术前AFP mRNA的表达与肿瘤有无肉眼及镜下血管侵犯的相关性有统计学意义(P=0.029,P<0.001)。AFP mRNA阴性患者1、2、3年生存率及无瘤生存率均比AFP mRNA阳性患者术后高(P=0.003,P=0.039)。Cox多因素分析结果显示术前外周血AFP mRNA阳性是预测术后复发的独立因素(P=0.018)。术后患者外周血AFP mRNA的阳性率为37.5%(21/56),术后AFP mRNA的表达与患者各项临床病理特征、预后以及术前AFP mRNA水平的相关性均无统计学意义。结论:肝癌患者术前外周血中AFP mRNA阳性者比AFP mRNA阳性者肿瘤侵袭性更强,术后可能更易复发。     

外周血AFP mRNA表达与原发性肝癌转移的关系

目的 通过检测手术或介入治疗前后的肝癌患者外周血甲胎蛋白信使核糖核酸（ＡＦＰ ｍＲＮＡ）的表达,了解原发生性肝癌的血行播散情况及肝癌综合治疗的病理生理基础。方法 采用巢式逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）检测技术,对５４例原发性吕患者外周血样本ＡＦＰｍＲＮＡ的表达进行了研究。结果 ５４例样本中,２６例（４８．１％）ＡＦＰ ｍＲＮＡ阳性,２０例正常人及１０例良性肝病患者分别只有１例阳性。１６例伴     

Analysis of alpha-fetoprotein gene expression in hepatocellular carcinoma and liver cirrhosis by in situ hybridization

The expression of the alpha-fetoprotein (AFP) gene in hepatocellular carcinoma (HCC) and liver cirrhosis by in situ hybridization analysis of AFP mRNA in cryostat and paraffin-embedded tissue sections was studied. In HCC sections the majority of tumor cells showed positive hybridization with a 3H-labeled AFP complementary DNA probe. The number of radioactive hybrid grains detected in HCC sections generally paralleled the level of serum AFP in the patient. In liver cirrhosis sections a small number of cells showed positive hybridization. These cells were dispersed in the tissue and morphologically indistinguishable from surrounding hepatocytes. Each of these cells contained a high level of hybridization signals to permit easy identification. In situ hybridization analysis of AFP mRNA may be of use in detecting preneoplastic cells in liver cirrhosis that cannot be defined on the morphologic and immunohistochemical basis.     

Clinical Utility of Protein Induced by Vitamin K Absence-II in Patients with Hepatocellular Carcinoma

Objective: Despite moderate sensitivity, alpha fetoprotein (AFP) is widely used in screening and prognostication for hepatocellular carcinoma (HCC). The objective of the current study was to assess clinical utility of Prothrombin induced by Vitamin K absence-II (PIVKAII) in addition to AFP in patients with HCC. Methods: We retrospectively reviewed 244 patients with documented AFP, PIVKA II and dynamic imaging of the liver. Using ROC curves, cutoff values for AFP and PIVKAII for HCC detection, tumor grade and microvascular invasion (MVI) were assessed. In patients who underwent liver transplantation (LT) for HCC, survival was determined using Kaplan Meier curves. Results: The median PIVKAII in healthy living donors was 28.6mAU/ml (15.9-55). In cirrhotics, the sensitivity of an AFP cutoff of 7.6 ng/ml or PIVKAII  cutoff of 250 mAU/ml for HCC detection was 91.7% (176/192) and specificity was 62.9%(68/108) (p <0.0001). In patients with HCC, PIVKAII values were significantly elevated with tumor size > 5 cm (P < 0.0001), tumor nodules > 3(P=0.01), and macrovascular invasion(p <0.0001).  The high risk group (patients with AFP ≥ 40 ng/ml + PIVKAII ≥ 350 mAU/ml), had a sensitivity of (23/33) 69.6% and specificity of (22/22)100% for MVI (P <0.001). The estimated 3 year RFS after LT in the low risk group (AFP     

组织hTERT基因及GGTmRNA-H亚型与血清AFP对小肝癌早期诊断意义的比较

目的 :研究小肝癌组织中端粒酶逆转录酶hTERT基因及谷氨酰转移酶GGTmRNA H亚型表达 ,通过与小肝癌血清AFP的比较 ,探讨其在小肝癌早期诊断中的意义。方法 :应用RT PCR法检测 65例原发性肝癌患者癌组织hTERT与GGTmRNA H亚型的表达 ,同时通过放免法检测血清AFP阳性表达情况。结果 :在 65例原发性肝癌中 ,癌组织hTERT基因的阳性例数 5 6例 ( 86 2 % ) ,GGTmRNA H亚型的阳性例数为 61例 ( 93 8% ) ,肝组织血清AFP阳性 40例 ( 61 5 % ) ,P <0 0 5 ;hTERT基因在2 0例小肝癌癌组织阳性表达例数为 16例 ,阳性率为 80 0 % ;GGTmRNA H亚型阳性表达例数为 18例 ,阳性率为 90 0 % ;与血清AFP组比较 ,差异有统计学意义 ,P <0 0 5。结论 :肝癌组织中hTERT基因、GGTmRNA H亚型联合检测可弥补AFP测定的不足 ,对提高肝癌早期诊断具有重要意义。hTERT基因及GGTmRNA H亚型的检测比AFP更敏感、特异性更高 ,有望成为小肝癌早期诊断的理想指标     

GPC3基因在原发性肝癌中的表达意义

目的分析GPC3基因在肝癌组织和癌周组织中的表达及对原发性肝癌的诊断价值。方法从20例原发性肝癌及相对应的癌周组织提取总RNA,利用RT-PCR方法对AFP、GPC3基因进行半定量分析。结果AFP、GPC3基因在癌组织和癌周组织的表达存在显著差异,在癌组织的表达率分别为70.0%和75.0%,联合应用达80.0%,在癌周组织的表达率均为10.0%,组织AFP mRNA的表达与血清AFP相关,而GPC3 mRNA的表达不受血清AFP值影响,在血清AFP≤20 ug/l的肝癌组织中,GPC3 mRNA表达率为40.0%（4/8）。结论GPC3 mRNA在肝癌癌组织中的表达明显高于癌周组织的表达,GPC3基因对血清AFP阴性肝癌有一定的辅助诊断意义,可能是肝癌的一种新的潜在的肿瘤特异标志物。     

Evaluation of Osteopontin as a Biomarker in Hepatocellular Carcinomas in Egyptian Patients with Chronic HCV Cirrhosis

Background: Hepatocellular carcinoma (HCC) is a high incidence disease in Egypt with a poor prognosis andsurvival. Biomarkers are important for diagnosis of HCC at an early stage. Osteopontin (OPN), a glycoprotein secreted bymacrophages, osteoblasts, and T cells, is also highly expressed in a variety of tumors, such as examples in the breast, colon,and stomach. The present study aimed to correlate the serum level of OPN in HCV-positive hepatocellular carcinomapatients, with OPN expression in tumor and non-tumor liver tissues in order to identify its efficacy as a biomarkerfor diagnosis. Material and Methods: Out of total of 146 patients, 80 were selected for inclusion in the study. Bloodsamples as well as specimens of tumor and non-tumor liver tissue were collected. In addition, blood samples from 20healthy volunteers were obtained as controls. Serum OPN and alpha-fetoprotein (AFP) were evaluated by ELISA forHCC and control groups. OPN and AFP gene expression were examined by real-time PCR, after homogenization andDNA extraction from serum samples and liver tissues. Results: It was found that serum OPN levels were significantlyhigher in the HCC group compared to normal group (P=0.009), with a strong positive correlation with AFP expression.However, there was no significant difference between OPN expression in tumor and non-tumor liver tissue. Conclusion:Serum OPN is highly suggested to be a professional candidate for HCC early diagnosis, with a diagnostic ability andaccuracy equal or higher than for AFP.     

Hepatic miR‐126 is a potential plasma biomarker for detection of hepatitis B virus infected hepatocellular carcinoma

Controversies about the origin of circulating miRNAs have encouraged us to identify organ specific circulating miRNAs as disease biomarkers. To identify liver‐specific miRNAs for hepatocellular carcinoma (HCC), global expression profiling of miRNAs in liver tissue of HBV‐HCC and HBV‐control with no or mild fibrosis was evaluated. A total of 40 differentially expressed miRNAs were identified in HCC. Among ten highly altered miRNAs, six miRNAs were successfully validated in tissues, whereas only two miRNAs, miR‐126 and miR‐142‐3p showed increased expression in plasma of HBV‐HCC compared to HBV‐non‐HCC patients. Subsequently, ROC curve analysis revealed that neither miR‐126 nor miR‐142‐3p performed better than AFP in discriminating HCC from non‐HCC while combination of each with AFP showed significantly higher efficiency rather than AFP alone (AUC: 0.922, 0.908 vs. 0.88; sensitivity: 0.84, 0.86 vs. 0.82 and specificity: 0.92, 0.94 vs. 0.86 respectively). Interestingly, triple combination of markers (miR‐126 + miR‐142‐3p + AFP) showed no additive effect on efficiency (AUC: 0.925) over the dual combination. Again, the expression of only miR‐126 was noticed significantly higher in HBV‐HCC patients with low‐AFP [<250 ng/ml] compared to either non‐HCC or liver cirrhosis (AUC: 0.77, 0.64, respectively). Furthermore, no alteration in expression of mir‐126 in HCV‐HCC or non‐viral‐HCC revealed that miR‐126 + AFP might be specific to HBV‐HCC. To understand the physiological role of these two miRNAs in hepato‐carcinogenesis, target genes related to cancer pathways (APAF1, APC2, CDKN2A, IRS1, CRKL, LIFR, EGR2) were verified. Thus, combination of circulating miR‐126 + AFP is a promising noninvasive diagnostic biomarker for HBV‐HCC and may be useful in the management of HCC patients.     

Spontaneous Regression of Hepatocellular Carcinoma: Its Imaging Course Leading to Complete Disappearance

Spontaneous regression of hepatocellular carcinoma (HCC) is a rare phenomenon. We followed a detailed clinical course of a spontaneous and complete disappearance of HCC during a short interval. A 73-year-old man with hepatitis B virus infection presented with a 15-mm mass in the right anterior superior segment of the liver. The mass was diagnosed as HCC by imaging findings. We found an elevated serum α-fetoprotein (AFP) level of 748 ng/ml. The tumor regressed to 6 mm on imaging examination, and the AFP serum level decreased to 87.8 ng/ml 1 month after the diagnostic hepatic angiography. Therefore, the patient was followed up without any treatment for HCC. The tumor disappeared 5 months later when the AFP serum level was 5.0 ng/ml. The diagnostic hepatic angiography might have had some effect on the spontaneous regression of HCC in the present case.Key Words: Spontaneous regression, Hepatocellular carcinoma, Imaging course, Complete disappearance, Hepatic angiography, Ischemia     

GP73在肝细胞癌诊断中的价值

目的:探讨GP73(Golgi protein-73)在肝细胞癌诊断中的价值.方法:收集外周血血清504例,其中肝细胞癌144例、肝硬化50例、乙型病毒性肝炎100例、乙型肝炎病毒携带者84例、其他恶性肿瘤50例、肝良性肿瘤26例和健康志愿者50例;应用双抗体夹心酶联免疫定量测定方法和电化学发光法检测血清中GP73和甲胎蛋白(α-fetoprotein,AFP)的表达水平.结果:肝细胞癌组血清GP73的表达水平显著高于其他各组(P＜0.05);受试者工作特征(receiver operating characteristic,ROC)曲线设定GP73临界值为64 ng/mL时,GP73诊断肝细胞癌的灵敏度和特异度分别为83.3％和88.3％,显著高于AFP(72.2％和76.7％),差异有统计学意义(P＜0.05);血清GP73联合AFP检测诊断肝细胞癌的灵敏度可达94.4％.结论:血清GP73的表达水平用于诊断肝细胞癌的灵敏度和特异度优于AFP,血清GP73联合AFP检测可提高肝细胞癌的诊断率.     

Clinical Value of Hepatoma-Specific Alpha-Fetoprotein in the Diagnosis of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide and the number 2 cause of cancer mortality in Chi- na.[1] It often develops in cases of liver cirrhosis and chronic hepati- tis. [1-3] Advanced imaging procedures including utrasonograph…