NDRG1 as a biomarker for metastasis,recurrence and of poor prognosis in hepatocellular carcinoma

N-myc downstream-regulated gene 1 (NDRG1) has been reported to be a multifunctional protein associated with carcinogenesis, however, the cellular function of NDRG1 remains elusive in human cancers. Here, our proteomics profile analysis of HCC tissues with different metastatic capabilities revealed that NDRG1 was correlated with metastasis and recurrence in HCC patients after liver transplantation (LT). Immunohistochemical staining of 143 HCC patients after LT showed that NDRG1-positive expression had poor prognosis, either for shorter disease-free survival or overall survival (P < 0.001), compared with NDRG1-negative expression. Multivariate analysis confirmed NDRG1 as an independent prognostic value (P < 0.001). In addition, in vitro experiments HCC cells with small interfering RNA against NDRG1 significantly suppressed its proliferation, colony formation, invasion and migration ability. Microarray analysis revealed that NDRG1 modulated the expression of genes associated with transmembrane transporter activity, chemoattractant activity, immune response, cell adhesion and cell proliferation process. Taken together, these results suggested that NDRG1 was an important molecule in controlling HCC metastasis and thus suggested as a novel biomarker for predicting HCC recurrence after LT.     

热休克蛋白90α在不同转移潜能人肝癌细胞株及HBV相关性肝细胞癌患者血清中表达水平的临床研究

目的 探讨热休克蛋白90α(HSP90α)在不同转移潜能人肝癌细胞株及乙型肝炎病毒(HBV)相关性肝细胞癌患者血清中的表达水平.方法 收集HBV感染肝病患者60例和同期体检的健康受试者12例的血清样本,采用Western blot技术检测不同转移潜能人肝癌细胞株中HSP90α的表达水平,采用ELISA技术检测HBV相关性肝癌患者、良性肝病患者、健康受试者血清中HSP90α的表达水平,采用统计学方法分析HSP90α的表达水平与HBV相关性肝细胞癌患者临床病理特征的关系.结果 高转移潜能人肝癌细胞株MHCC97H细胞中HSP90α的表达水平明显高于无转移潜能人肝癌细胞株HepG2细胞,差异有统计学意义(t=13.375,P=0.001);肝癌组与良性肝病组患者血清中HSP90α的表达水平均明显高于健康受试者,差异有统计学意义(F=11.357,P=0.002;F=9.633,P=0.005),但肝癌组与良性肝病组患者血清中HSP90α的表达水平比较,差异无统计学意义(F=0.859,P=0.983);42例伴HBV慢性感染肝癌患者血清中HSP90α的表达水平与性别有关,其中,女性患者血清中HSP90α的表达水平高于男性患者,差异有统计学意义(F=4.729,P=0.039),但患者年龄、HBeAg是否为阳性、甲胎蛋白(AFP)浓度、HBV DNA及临床分期之间比较,差异无统计学意义(P﹥0.05).结论 HSP90α可成为抗癌治疗的新靶点,并为肝病的早期诊断、肝癌的侵袭转移、预后评估及科学治疗提供理论依据.     

Tenascin-C expression is associated with poor prognosis in hepatocellular carcinoma (HCC) patients and the inflammatory cytokine TNF-α-induced TNC expression promotes migration in HCC cells

Although tenascin-c (TNC) in inflammatory microenvironment contributes to progression in some tumors, its role in hepatocellular carcinoma (HCC) in metastasis and the mechanism by which TNC expression is regulated in HCC cells are elusive. In this study, we examined TNC expression in 100 HCC tissue samples by immunohistochemistry and compared which between the groups with or without metastasis. TNC expression was higher in metastatic HCC tissues than that in the non-metastatic HCC tissues, which was associated with the Knodell inflammation scores. Importantly, high level of TNC expression was associated with lower survival rate and shorter survival time in the HCC patients. We then investigated the mechanism by which TNC expression is regulated in HCC cells with an in vitro cell culture system. The recombinant TNF-α and conditioned medium from macrophages induced TNC expression at both mRNA and protein levels in HepG2 cells. The induction of TNC expression by conditioned medium from macrophages was suppressed by a TNF-α neutralizing antibody. TNF-α-promoted cell migration was inhibited by a TNC siRNA. In addition, TNF-α-induced TNC expression was blocked by a NF-κB pathway inhibitor. These results suggest that TNF-α in the tumor microenvironment induces TNC expression in HCC cells through the NF-κB pathway, which in turn, promotes HCC cell migration. Thus, TNC may play an important role in promoting HCC metastasis and TNC expression could be a predictive factor for poor prognosis in HCC patients.     

Notch1-Snail1-E-cadherin pathway in metastatic hepatocellular carcinoma

Notch signaling, a critical pathway for tissue development, also contributes to tumorigenesis in many cancers, but its pathological function in liver cancer is not well defined. In our study, Notch1 expression and its clinicopathological parameters were evaluated in 82 human hepatocellular carcinoma (HCC) patients. Plasmid-based siNotch1 shRNA was transiently or stably transfected into metastatic HCC cells and subsequently evaluated for the effects on orthotopic liver tumor metastasis in a mouse model as well as the effects on downstream pathways. Aberrant high expression of Notch1 was significantly associated with metastatic disease parameters in HCC patients, such as tumor-node-metastasis Stages III-IV and tumor venous invasion. Knocking-down Notch1 reduced cell motility in vitro and orthotopic tumor metastasis from the liver to the lung in vivo in a mouse model. In metastatic HCC cells, abnormal expression of Notch1 was associated with increased expression of Snail1 and repressed expression of E-cadherin; the Notch1-Snail1-E-cadherin association can also be found in HCC patient tumors. Inhibition of Notch1 by shRNA abolished Snail1 expression, which further resulted in the re-establishment of repressed E-cadherin in metastatic HCC cells. Thus, abnormal Notch1 expression was strongly associated with HCC metastatic disease, which might be mediated through the Notch1-Snail1-E-cadherin pathway. Knock-down of Notch1 reversed HCC tumor metastasis in a mouse model. Therefore, these data suggest that effective targeting of Notch signaling might also inhibit tumor metastasis.     

Lunasin potentiates the effect of oxaliplatin preventing outgrowth of colon cancer metastasis, binds to α5β1 integrin and suppresses FAK/ERK/NF-κB signaling

The effect of lunasin on colon cancer metastasis was studied using three human colon cancer cell lines in vitro and a liver metastasis model of colon cancer in vivo. Lunasin bound with α₅β₁ integrin and internalized into the nucleus of KM12L4 human colon cancer cells. Lunasin (10 μM) inhibited the activation of focal adhesion kinase (FAK) by 28%, 39% and 60% in RKO, HCT-116 and KM12L4 human colon cancer cells, respectively. Lunasin caused an increase in the expression of the inhibitor of kappa B alpha (IκB-α), a decrease in nuclear p50 NF-κB and a reduction in the migration of cancer cells. Lunasin (4 mg/kg bw) inhibited metastasis and potentiated the effect of oxaliplatin by reducing the expression of proliferating cell nuclear antigen. Liver metastatic nodules were reduced from 28 (PBS) to 14 (lunasin, P = 0.047) while combination of lunasin and oxaliplatin to 5 (P = 0.004). The tumor burden was reduced from 0.13 (PBS) to 0.10 (lunasin, P = 0.039) to 0.04 (lunasin + oxaliplatin, P < 0.0001). Moreover, lunasin potentiated the effect of oxaliplatin in modifying expression of proteins involved in apoptosis and metastasis including Bax, Bcl-2, IKK-α and p-p65. Lunasin inhibited metastasis of human colon cancer cells by direct binding with α₅β₁ integrin suppressing FAK/ERK/NF-κB signaling, and potentiated the effect of oxaliplatin in preventing the outgrowth of metastasis.     

PP1蛋白在转移及非转移性恶性黑色素瘤中的表达

目的 研究蛋白磷酸酶-1(PP1)对恶性黑色素瘤转移的作用.方法 采用Western blot检测PP1蛋白在非转移性恶性黑色素瘤细胞系-HMCB及转移性恶性黑色素瘤细胞系WM266-4中的表达.用免疫组化的方法检测32例未转移恶性黑色素瘤及20例转移性恶性黑色素瘤标本中PP1的表达.结果 本研究证明非转移性恶性黑色素瘤细胞及组织中PP1蛋白表达明显高于在转移性恶性黑色素瘤细胞及组织中的表达,差异有统计学意义(P<0.05).结论 PP1在转移及未转移恶性黑色素瘤细胞及组织中的差异表达为恶性黑色素瘤转移机制方面的研究开辟新的方向.     

Significance of serum hepatocyte growth factor levels in patients with hepatocellular carcinoma undergoing hepatic resection

Background

Hepatocyte growth factor (HGF) is a potent hepatocyte mitogen and may stimulate the proliferation and invasiveness of human hepatocellular carcinoma (HCC) cells through the c-met receptor. This study evaluates the significance of serum HGF levels in patients undergoing HCC resection.

Study design

The peripheral and portal sera and HCC and non-tumorous tissues of 40 HCC patients, with tumor TNM stage I (n = 12), II (n = 17), and III (n = 11) diseases, who underwent hepatic resection were prospectively collected. Serum HGF levels were determined by enzyme-linked immunosorbent assay. The c-met protein expressions were examined by immunohistochemistry. Median follow-up time was 69 months.

Results

The prehepatectomy portal HGF levels (median, 622 pg/mL) were significantly higher than peripheral HGF levels (564 pg/mL) (P = 0.026). The posthepatectomy portal HGF levels (699 pg/mL) were significantly higher than prehepatectomy portal HGF levels (P < 0.001). C-met expression was detected in 87.5% HCC and in 85.0% non-tumorous liver tissues. By Cox multivariate analysis, posthepatectomy portal HGF level >699 pg/mL (P < 0.001), multiple tumors (P = 0.042), and TNM stages II (P = 0.019) and III (P = 0.009) were independent factors related with survival. Patients with a posthepatectomy portal HCG level >699 pg/mL and with a positive c-met expression in HCC tissue have the worst survival.

Conclusions

In HCC patients, high peripheral and portal HGF serum levels related with poor prognosis after hepatic resection. Hepatocyte growth factor and c-met receptor can be targets of future HCC postoperative treatment.     

Programmed cell death 4 (PDCD4) suppresses metastastic potential of human hepatocellular carcinoma cells

Background

Hepatocellular carcinoma (HCC) is a lethal malignancy with high rate of metastasis and poor prognosis. There are no effective managements to block metastasis of HCC. Programmed cell death 4 (PDCD4) is found to be a tumor transformation suppressor. Among investigations on effects of PDCD4, little is about the metastatic potentials of HCC cells. This study was to investigate the role of PDCD4 on metastatic potential of human HCC cells.

Methods

We examined the expression of PDCD4 in three HCC cell lines with different metastatic potentials, MHCC-97H (high metastatic potential), MHCC-97L (low metastatic potential) and Hep3B (no metastatic potential). A plasmid encoding PDCD4 gene was constructed and then transfected into HCC cells with the lowest PDCD4 expression level. Effects of PDCD4 on cell proliferation, cell apoptosis, gene expression of metastasis tumor antigen 1 (MTA1) and in vitro migration and invasion capacity were assessed after transfection.

Results

Our results showed that the expression level of PDCD4 was inversely correlated to the metastatic potential of HCC cells. After transfection with the PDCD4 gene, HCC cell proliferation rate was significantly decreased, cell apoptosis rate was significantly increased, the expression of MTA1 gene, HCC cell migration and Matrigel invasion were also remarkably inhibited.

Conclusion

PDCD4 expression is inversely correlated to the metastatic potential of HCC cells. PDCD4 can effectively suppress the metastatic potential of HCC cells.     

Determination of metastasis-associated proteins in non-small cell lung cancer by comparative proteomic analysis

The development of metastasis is the leading cause of death and an enormous therapeutic challenge in cases of non-small cell lung cancer. To better understand the molecular mechanisms underlying the metastasis process and to discover novel potential clinical markers for non-small cell lung cancer, comparative proteomic analysis of two non-small cell lung cancer cell lines with different metastatic potentials, the non-metastatic CL1-0 and highly metastatic CL1-5 cell lines, was carried out using two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and tandem mass spectrometry. Thirty-three differentially expressed proteins were identified unambiguously, among which 16 proteins were significantly upregulated and 17 proteins were downregulated in highly metastatic CL1-5 cells compared with non-metastatic CL1-0 cells. Subsequently, 8 of 33 identified proteins were selected for further validation at the mRNA level using real-time quantitative polymerase chain reaction, and three identified proteins, S100A11, PGP 9.5 and HSP27, were confirmed by western blotting. The protein S100A11 displaying significant differential expression at both the protein and mRNA levels was further analyzed by immunohistochemical staining in 65 primary non-small cell lung cancer tissues and 10 matched local positive lymph node specimens to explore its relationship with metastasis. The results indicated that the upregulation of S100A11 expression in non-small cell lung cancer tissues was significantly associated with higher tumor-node-metastasis stage (P = 0.001) and positive lymph node status (P = 0.011), implying that S100A11 might be an important regulatory molecule in promoting invasion and metastasis of non-small cell lung cancer.     

Serological investigation of the clinical significance of fascin in non-small-cell lung cancer

Objectives

Fascin, a conserved actin bundling protein family member, is frequently up-regulated in various cancer types, including non-small-cell lung cancer (NSCLC), and it plays increasingly important roles in the progression of tumor invasion and metastasis. However, variations in the serum fascin level in tumor patients are usually neglected.

Materials and Methods

In the present study, serum samples from 501 stage I-IV NSCLC patients and 109 healthy volunteers were investigated by an enzyme-linked immunosorbent assay.

Results

The serum fascin level was markedly increased in the NSCLC patients (P < 0.05), particularly in advanced cases (P < 0.01), compared with that in the healthy controls. We also found that the serum fascin level was significantly correlated with female sex (P = 0.02), non-smoking status (P = 0.044), and adenocarcinoma histology (P < 0.001), with a higher positive rate relative to each counterpart. Furthermore, our results suggested that the serum fascin level reflects the aggressive progress of both lymphatic (P < 0.001) and distant (P < 0.001) metastases in NSCLC. A survival analysis of 283 eligible patients who underwent a follow-up examination after 3 years revealed that the patients in the serum fascin-positive group had a significantly lower overall survival rate compared with those in the negative group for 134 non-distant metastatic (stage M0) cases (P = 0.044). A subsequent Cox regression analysis revealed that the serum fascin level was an independent prognostic factor for M0-stage NSCLC (univariate, P = 0.001; multivariate, P = 0.038).

Conclusion

Our study suggests that the serum fascin level is an effective indicator of tumor aggressiveness, and that it plays an important role in the prognosis of NSCLC, particularly for non-distant metastatic patients.     

Expression of E-selectin on endothelial cells of small veins in human colorectal cancer

E-selectin is an adhesion molecule of endothelial cells that binds to cancer cells mediated by sialyl Lewis A (sLe^a) or sialyl Lewis X (sLe^x). It is suspected to be involved in hematogenous metastasis of tumors. Therefore, it is worth examining E-selectin expression in human colorectal cancer and its hepatic metastasis. In the present study, E-selectin was clearly revealed on the endothelial cells of small vessels adjacent to cancer nests both in primary and in metastatic nests in immunohistochemistry. In these tissues, E-selectin was observed on the endothelial cells lining the lumen of small vessels. Its expression adjacent to cancer nests appears to be induced through some stimuli by cancer cells, since its degree of expression is inversely correlated to the distance of the blood vessels from the cancer nests (p < 0.001). Endothelial cells adjacent to the metastatic lesion expressed E-selectin more extensively than those adjacent to the primary foci. This is also in line with the finding on serum E-selectin levels which were significantly elevated in the metastatic group as compared with the non-metastatic group. The serum E-selectin level may provide useful information in the diagnosis for hepatic metastasis of colorectal cancer, although the results are still tentative. © 1995 Wiley-Liss, Inc.     

Genetic variation in Transaldolase 1 and risk of squamous cell carcinoma of the head and neck

Background: The Pentose Phosphate Pathway (PPP) is involved in the body's protection against oxidative stress and resistance/susceptibility to apoptosis and thus has been implicated in tumor development and progression. Here we present data examining the association of genetic variation in one of the key enzymes of the PPP, Transaldolase 1 (TALDO1) with squamous cell carcinoma of the head and neck (SCCHN). Methods: We performed sequencing analysis to identify common genetic variations in TALDO1 and then investigated their association with SCCHN using samples from a population-based case/control study with both European American (EA) and African American (AA) former and current smokers. Results: We identified three polymorphisms in TALDO1 that were associated with SCCHN risk in our EA study population. Specifically the 5′ upstream variant −490C > G or T (rs10794338), which we identified as tri-allelic, showed a reduced risk compared with any presence of the common allele, odds ratio (OR) [95% confidence interval (95% CI)]: 0.57 (0.38-0.86). Additionally two intronic high frequency polymorphisms demonstrated a positive association with disease, with the presence of the variant IVS1 + 1874T > A (rs3901233), 1.76 (1.19-2.61) and IVS4 + 2187A > C (rs4963163), 1.71 (1.16-2.53). Conclusion: These results provide preliminary evidence that genetic polymorphisms in TALDO1 are associated with SCCHN.     

复发转移大肠癌患者血清PTEN、COX2蛋白表达的研究

目的 分析血清PTEN、COX2蛋白表达与大肠癌复发转移的相关性,为临床治疗提供参考.方法选择160例复发转移大肠癌患者作为复发转移组,100例同期健康体检者作为对照组,120例初发大肠癌患者作为初发大肠癌组,120例复发未转移大肠癌患者作为复发未转移组.比较分析各组患者的血清PTEN、CD105、VEGF、COX2、PI3K、AKT蛋白表达水平,以及基质金属蛋白酶MMP1、MMP3、MMP9及其抑制剂TIMP1、TIMP2、TIMP7水平.结果复发转移组患者的血清PTEN蛋白表达水平低于对照组、初发大肠癌组及复发未转移组(P﹤0.05),而CD105、VEGF、COX2、PI3K、AKT、基质金属蛋白酶及其抑制剂水平高于对照组、初发大肠癌组及复发未转移组(P﹤0.05).血清PTEN水平与复发转移大肠癌患者的预后呈正相关(r=0.9076,P﹤0.05),血清COX2水平与复发转移大肠癌患者的预后呈负相关(r=-0.9022,P﹤0.05).结论大肠癌复发转移与血清PTEN水平降低及COX2水平升高有关,血清PTEN和COX2有望成为判断大肠癌复发转移及预后的重要生物学指标.     

散发性结直肠癌组织中ezrin mRNA的表达及临床意义

目的研究散发性结直肠癌（SCRC）组织中ezrin mRNA的表达，探讨其在SCRC发生发展中的作用以及与ezrin蛋白表达部位、临床病理参数之间的关系。方法运用TaqMan实时逆转录-聚合酶链反应（real—time RT—PCR）技术检测132例SCRC患者的肿瘤组织及43例患者的癌旁正常组织中ezrinmRNA的表达；免疫组化法检测相应组织中ezrin蛋白的表达。结果SCRC组织中ezrinmRNA含量和ezrin蛋白表达阳性率均显著高于癌旁正常组织（P〈0．05）；转移组ezrinmRNA含量和ezrin蛋白表达阳性率均高于未转移组（P〈0．05）。转移组胞膜为主型ezrin蛋白表达阳性率（31．1％）显著高于未转移组（6．9％，P=0．0006）；转移组胞浆为主型ezrin蛋白表达阳性率（55．4％）与未转移组（63．8％）的差异无统计学意义（P〉0．05）；胞膜为主型组ezrinmRNA含量与胞浆为主型组的差异无统计学意义（P〉0．05）。年龄〈50岁组ezrinmRNA含量高于年龄≥50岁组（P=0．016）；右半结肠组胞膜为主型ezrin蛋白表达阳性率显著高于左半结肠和直肠（P=0．0003），左半结肠组胞浆为主型ezrin蛋白表达阳性率显著高于右半结肠和直肠（P=0．004）。ezrinmRNA、ezrin蛋自在SCRC中的表达与性别、肿瘤大小、侵袭程度、分化程度均无关（P〉0．05）。结论ezrinmRNA和ezrin蛋白表达的增高与SCRC的发生发展密切相关；ezrin蛋白表达从胞浆到胞膜的移位在肿瘤转移中可能起着重要作用；ezrinmRNA含量可能与患者年龄相关，ezrin表达部位可能与SCRC发生部位相关。ezrinmRNA、ezrin蛋白水平及表达部位可能成为临床判断SCRC预后的重要参考指标。     

肝细胞癌化疗栓塞后血清血管内皮细胞生长因子与转移关系的研究

目的　前瞻性评估肝细胞癌 (HCC)经导管化疗栓塞 (TACE)前后血清血管内皮细胞生长因子 (VEGF)表达变化与复发转移的关系。方法　采用酶联免疫夹心法 (ELISA)对 3 0例HCC患者分别于术前、术后 3d和 4周测量血清VEGF水平 ,TACE术后 3个月评估复发转移发生情况。结果　 3 0例HCC患者术前血清VEGF表达范围为 154.47± 90 .17pg/ml ,术后血清VEGF表达较术前增高 (P <0 .0 5)。在碘油分布不均匀及门静脉癌栓组中 ,血清VEGF表达增高。追踪半年后 ,血清VEGF水平升高者中有 74%复发 ,而血清VEGF表达下降者无一例复发。结论　HCC患者TACE后血清VEGF表达增加 ,与复发转移发生有关     

The importance of microenvironment: the role of CCL8 in metastasis formation of melanoma

We have attempted to characterize the changes occurring on the host side during the progression of human melanoma. To investigate the role of tumor microenvironment, we set up such an animal model, which was able to isolate the host related factors playing central role in metastasis formation. One of these ‘factors’, CCL12, was consequently selected and its behavior was examined alongside its human homologue (CCL8). In our animal model, metastasis forming primary melanoma in the host exhibited increased level of CCL12 mRNA expression. In clinical samples, when examining the tumor and the host together, the cumulative (tumor and host) CCL8 expression was lower in the group in which human primary melanoma formed lung metastasis compared to non-metastatic primary tumors. We could not detect significant difference in CCL8 receptor (CCR1) expression between the two groups. Increased migration of the examined tumor cell lines was observed when CCL8 was applied as a chemoattractant. The tumor cells and their interactions can be influenced the expression of CCL8 by dermal fibroblasts, as a significant change in the metastatic microenvironment. Furthermore, we examined changes in miRNA profile resulted by CCL8 and miR146a appears to be a promising prognostic marker for following this process.     

RELATIONSHIP OF THE EXPRESSION OF MDR1 GENE PRODUCT IN HEPATO CELLULAR CARCINOMA TO INVASION AND METASTASIS

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