Overexpression of transketolase protein TKTL1 is associated with occurrence and progression in nasopharyngeal carcinoma: a potential therapeutic target in nasopharyngeal carcinoma

Over 80 years ago, Warburg identified a particular metabolic pathway in carcinomas characterised by the anaerobic degradation of glucose even in the presence of oxygen that leads to the production of large amounts of lactate (known as the Warburg effect). Now, widespread clinical use of positron-emission tomography (PET) has confirmed that there exists enhanced glucose degradation in tumors. Recent research demonstrated that pentose phosphate pathway (PPP) was augmented in some tumors, especially non-oxidative part of PPP. The non-oxidative part of PPP is controlled by transketolase enzyme reactions. The present study designed to evaluate the effect of transketolase activity on nasopharyngeal carcinoma. It was found that the transketolase activity was significantly stronger in human nasopharyngeal carcinoma tissues than those in human chronic nasopharyngitis tissues. There is a strong upregulation of the transketolase-like-1 (TKTL1) in human nasopharyngeal carcinoma tissues and cell line (CNE), whereas transketolase (TKT) and transketolase-like-2 (TKTL2) were not upregulated. After inhibited the expression of (TKTL1) by RNAi, we found that total transketolase activity was dramatically downregulated and the proliferation of cancer cells was significantly inhibited in CNE cells. These results indicate that TKTL1 gene influences total transketolase activity and cell proliferation in human nasopharyngeal carcinoma cells, suggesting that TKTL1 gene plays an important role on glycometabolism in tumors and it might become a novel target for tumor gene therapy.     

Transketolase-like protein 1 confers resistance to serum withdrawal in vitro

Transketolase-like protein 1 (TKTL1) is a member of the family of transketolase enzymes of which the founder member transketolase (TKT) is known to play a central role in the non-oxidative part of the pentose phosphate pathway. According to several publications TKTL1 is the only family member, whose expression is substantially de-regulated in a variety of solid tumours. Over-expression of TKTL1 correlates with poor prognosis of cancer patients and TKTL1 itself represents a potential therapeutic target owing to its possible involvement in the regulation of the proliferation and metabolism of cancer cells. We show that exogenously expressed TKTL1 provides HEK293 cells with moderate growth advantages under standard culture conditions, while protecting cells from growth factor withdrawal-induced apoptosis. Importantly, we identified TKTL1 with the JFC12T10 antibody as a 65kDa protein, which was however absent in most tumour cell lines tested. Primary head and neck squamous cell carcinomas of various localisations were characterised by a focal pattern with single cells strongly expressing TKTL1, rather than by a homogeneous expression pattern within the tumour mass.     

结肠癌及癌旁组织中TKTL1 的表达

 目的　探讨转酮酶样基因TKTL1 在人类结肠癌发生发展中的作用。方法　用实时定量PCR 检测结肠癌及癌旁组织中转酮酶样基因TKTL1 mRNA 表达水平,连续监测法检测各组织中总转酮酶 活性。结果　TKTL1 mRNA 在结肠癌组织中比癌旁组织中的表达明显增强,且侵袭性结肠癌比非侵 袭性结肠癌表达增强。同样,结肠癌组织中总转酮酶活性比癌旁组织中高( P < 0. 01) ,侵袭性结肠癌较 非侵袭性结肠癌中转酮酶活性增高( P < 0. 01) 。结论　TKTL1 mRNA 表达与人类结肠癌的发生发展 密切相关。     

Transketolase‐like protein 1 (TKTL1) is required for rapid cell growth and full viability of human tumor cells

Cancer cells display high rates of aerobic glycolysis, a phenomenon known as the Warburg effect. Lactate and pyruvate, the end products of glycolysis, are overproduced by cancer cells even in the presence of oxygen. The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis, glucose degradation to lactate, and regeneration of redox equivalents. The nonoxidative part of the PPP is controlled by transketolase (TKT) enzymes. One TKT isoform, the transketolase‐like protein 1 (TKTL1) is specifically upregulated in different human cancers and its overexpression predicts a poor patient's survival. This finding implicates that an increased TKTL1 expression may activate the PPP leading to enhanced cancer cell growth and survival. To analyze the functional role of TKTL1 in malignant progression, we inhibited TKTL1 by RNAi technologies in human HCT116 colon carcinoma cells. TKTL1 suppression resulted in a significantly slowed cell growth, glucose consumption and lactate production. In TKTL1 knockdown‐cells, the intracellular reactive oxygen species levels were not significantly increased, whereas the sensitivity towards oxidative stress‐induced apoptosis was clearly enhanced. These data provide new clues on the importance of TKTL1 dys‐regulation in tumor cells and indicate that TKTL1 overexpression may be considered not only as a new tumor marker but also as a good target for anticancer therapy. © 2008 Wiley‐Liss, Inc.     

干扰PRMT2基因对肝癌细胞系生物学行为的影响

目的:研究抑制蛋白质精氨酸甲基转移酶2(protein arginine methyltransferase 2,PRMT2)基因的表达对肝癌细胞系生物学行为的影响。方法:qRT-PCR检测人肝癌细胞系(HepG2和SMMC-7721)和人正常肝细胞(LO2)中PRMT2的表达情况;设计并合成针对PRMT2基因的3对小干扰RNA,体外通过脂质体Lipofectamine 3000转染到相对高表达PRMT2的人肝癌细胞系HepG2和SMMC-7721中抑制PRMT2基因的表达,并设置阴性对照组（NC组）和空白对照组。qRT-PCR、Western blot检测转染后细胞PRMT2 mRNA水平和蛋白水平的变化。CCK-8法检测转染后细胞增殖能力,Transwell小室检测细胞体外迁移及侵袭能力。结果:与人正常肝细胞LO2相比,PRMT2基因在人肝癌细胞系HepG2和SMMC-7721中相对高表达。将siPRMT2转染HepG2和SMMC-7721细胞后,与NC组相比,siPRMT2-1和siPRMT2-2组的PRMT2 mRNA和蛋白表达均明显降低（P均<0.05）,NC组与空白对照组无明显差异。与NC组相比,将siPRMT2-1和siPRMT2-2转染进HepG2和SMMC-7721细胞后,细胞增殖速度减慢、迁移和侵袭能力显著减弱（P均<0.05）,NC组与空白对照组无明显差异。结论:RNA干扰抑制PRMT2表达后,抑制肝癌细胞的增殖、迁移和侵袭,PRMT2可能影响肝癌的预后。     

Experimental therapy of hepatoma with artemisinin and its derivatives: in vitro and in vivo activity, chemosensitization, and mechanisms of action

PURPOSE: ART and its derivatives, clinically used antimalarial agents, have recently shown antitumor activities. However, the mechanisms underlying these activities remain unclear. This study was designed to determine their antitumor efficacy and underlying mechanisms of action in human hepatoma cells. EXPERIMENTAL DESIGN: The in vitro cytotoxicities of ART, DHA, artemether, and artesunate were compared in human hepatoma cells, HepG2 (p53 wild-type), Huh-7 and BEL-7404 (p53 mutant), and Hep3B (p53 null), and a normal human liver cell line, 7702. Based on their activity and specificity, ART and DHA were further investigated for their in vitro and in vivo antitumor effects and their effects on the protein expression of genes associated with cell proliferation and apoptosis. RESULTS: ART and DHA exerted the greatest cytotoxicity to hepatoma cells but significantly lower cytotoxicity to normal liver cells. The compounds inhibited cell proliferation, induced G(1)-phase arrest, decreased the levels of cyclin D1, cyclin E, cyclin-dependent kinase 2, cyclin-dependent kinase 4, and E2F1, and increased the levels of Cip1/p21 and Kip1/p27. They induced apoptosis, activated caspase-3, increased the Bax/Bcl-2 ratio and poly(ADP-ribose) polymerase, and down-regulated MDM2. In mice bearing HepG2 and Hep3B xenograft tumors, ART and DHA inhibited tumor growth and modulated tumor gene expression consistent with in vitro observations. DHA increased the efficacy of the chemotherapeutic agent gemcitabine. CONCLUSIONS: ART and DHA have significant anticancer effects against human hepatoma cells, regardless of p53 status, with minimal effects on normal cells, indicating that they are promising therapeutics for human hepatoma used alone or in combination with other therapies.     

lncRNA PVT1调控肝癌细胞放射敏感性的分子机制

目的:探讨lncRNA PVT1调控肝癌细胞对放射照射增殖、凋亡的敏感性及机制。方法:运用qRT-PCR检测人肝癌细胞HepG2、人正常肝细胞L02中PVT1的表达。si-NC组（转染si-NC）、si-PVT1组（转染si-PVT1）、IR+si-NC组（转染si-NC后放射照射）、IR+si-PVT1组（转染si-PVT1后放射照射）均用脂质体法转染至HepG2细胞。MTT法检测各组细胞增殖;流式细胞术检测各组细胞凋亡;Western blot检测各组细胞中p-ATM、p-p53、p-Chk2、Bcl-2、Bax的蛋白表达。结果:与人正常肝细胞L02相比,人肝癌细胞HepG2中PVT1的表达显著升高（P<0.05）;敲减PVT1联合放射照射可明显抑制HepG2细胞增殖,促进凋亡且可下调p-ATM、p-p53、p-Chk2、Bcl-2蛋白表达,上调Bax蛋白表达。结论:敲减lncRNA PVT1可通过抑制HepG2细胞增殖,促进凋亡,增强其对放射照射的敏感性,为提高肝癌的治疗效率提供新靶点。     

Down-regulation of c-myc and Cyclin D1 genes by antisense oligodeoxy nucleotides inhibits the expression of E2F1 and in vitro growth of HepG2 and Morris 5123 liver cancer cells

A number of genetic interactions are involved in the control of cell cycle, but their role and nature have not been completely clarified. The knowledge of the behavior of these interactions in hepatocellular carcinoma, could optimize preventive and therapeutic strategies based on cell cycle restraint. We studied downstream events following c-MYC and CYCLIN D1 gene inhibition, by lipoplex-delivered MYC and CYCLIN D1 antisense oligodeoxy nucleotides (aODNM, aODND1), in in vitro cultured human HepG2 and rat Morris 5123 hepatoma cells. 0.5-20 micro M aODN(M) and aODND1 inhibited in vitro growth of both cell types. Scramble oligomer (SCR) and sense ODNs had no or relatively poor effect. Ten micromolar aODNM and aODND1, but not SCR, also induced a significant increase in the apoptotic index of HepG2 and 5123 cells, and inhibited colony formation in soft agar by HepG2 cells. Treatment of the cells with aODNM plus aODND1 had no additive effect on growth and apoptosis. aODNM and aODND1 induced >50% decrease in c-MYC and CYCLIN D1 gene expression, respectively, at both mRNA and protein level. The inhibition of gene expression by aODNs was highly specific, and SCR was without effect. The reduction in c-MYC and CYCLIN D1 expression by aODNs, was associated with a >50% decrease in E2F1 mRNA and protein production, without changes in CYCLIN A and CYCLIN E expression. These results suggest the involvement of both c-MYC and CYCLIN D1 on E2F1 gene function, and indicate that aODNM and aODND1 may inhibit hepatoma cell growth through down-regulation of the E2F1 gene. The inhibition of E2F1 gene expression by E2F1 aODN, was associated with strong growth restraint of HepG2 cells. Thus, interactions of c-MYC and CYCLIN D1 with E2F1 gene are essential for cell cycle activity in hepatoma cells, and their inhibition may have a therapeutic effect.     

低氧条件下血红素加氧酶1过表达对人肝癌细胞的保护作用

目的 探讨低氧条件下人肝癌细胞(HepG2)中血红素加氧酶1(HO-1)过表达对细胞的保护作用,及其与低氧诱导因子1α(HIF-1α)的关系.方法 应用低氧气体(0.5%O2、94.5%N2、5%CO2)和化学模拟低氧(250 μmol/L CoCl2)方法 刺激HepG2细胞.采用逆转录聚合酶链反应(RT-PCR)方法 ,从mRNA水平上检测低氧条件下HO-1和HIF-1α的表达.采用Western bolt方法 ,从蛋白水平上检测低氧条件下HO-1和HIF-1α的表达及其相关性.采用四甲基偶氮唑蓝(MTT)方法 ,分析低氧刺激后细胞的存活率,以及低氧刺激同时抑制HO-1蛋白表达后细胞的存活率.采用超氧化物歧化酶(SOD)试剂盒,检测低氧刺激细胞后总SOD的活性,以及抑制HO-1蛋白表达后细胞中总SOD的活性.结果 低氧诱导的HepG2细胞中,HO-1 mRNA和蛋白过表达.用锌原卟啉IX(ZnPPIX)抑制低氧条件下HO-1蛋白过表达,可导致HepG2细胞在低氧应激条件下存活率明显降低(P<0.01).低氧条件下,HepG2细胞中总SOD的活性显著升高(P<0.05);而在抑制低氧条件下HO-1蛋白过表达后,HepG2细胞中总SOD活性明显降低(P<0.01).低氧条件下,HIF-1α蛋白表达显著升高,抑制HIF-1α蛋白表达后,HO-1蛋白表达明显降低;而抑制低氧条件下HO-1的过表达后,HIF-1α蛋白表达没有明显改变.结论 低氧诱导HepG2细胞中HO-1蛋白的过表达依赖或部分依赖HIF-1α;HO-1的过表达对HepG2细胞抵抗低氧应激发挥保护作用;HO-1的过表达对处于低氧应激条件下细胞的保护作用可能源于SOD.     

Hepatitis B virus X protein upregulates expression of SMYD3 and C-MYC in HepG2 cells

The carcinogenic role of Hepatitis B X (HBX) in hepatocellular carcinoma (HCC) remains largely unknown. Histone H3 lysine 4 methyltransferase SMYD3 was found to be over-expressed and have a pro-carcinogenic effect in HCC. The role of HBX in regulating SMYD3 activity and the corresponding C-MYC gene in HCC carcinogenesis was investigated. SMYD3 and C-MYC expression in HBV-negative HepG2 and HBV-positive HepG2.2.15 were detected by real time PCR and Western blot. After transfection of HBX into HepG2, SMYD3 and C-MYC protein expression was detected and the apoptosis and proliferation of hepatoma cells were assayed. After SMYD3 expression in HepG2 with HBX transfection downregulated by siRNA, the corresponding C-MYC expression, cellular apoptosis, and proliferation were assayed by FACS. SMYD3 mRNA and protein and C-MYC protein were significantly higher in HepG2.2.15 than in HepG2. HBX transfection resulted in enhanced SMYD3 and C-MYC expressions, decreased cell apoptosis, and increased cell proliferation in HepG2 cells. Knocking down of SMYD3 in HepG2 with HBX transfection inhibited C-MYC expression and promoted apoptosis. These results suggest that HBX upregulates SMYD3 expression in HepG2, which may promote hepatoma development and progress. C-MYC may act as a down-stream gene in HBX-SMYD3-related hepatocarcinogenesis.     

Enhanced proliferation of human hepatoma cells by PAR-2 agonists via the ERK/AP-1 pathway

To investigate the expression and role of PAR-2 in the proliferation of the human hepatoma cell line HepG2, PAR-2 protein and mRNA expression were evaluated by immuno-histochemistry, immunofluorescence and RT-PCR analysis. The signaling pathways downstream of PAR-2 activation that lead to hepatoma cell proliferation were analyzed. The results showed that PAR-2 is expressed in human hepatoma cells and PAR-2 mRNA expression was found to be upregulated in cells treated with trypsin or SLIGKV-NH2 (P<0.001). The proliferation rate of HepG2 cells treated with trypsin or SLIGKV-NH2 was significantly increased (P<0.001). The percentage of S?phase, G2/M phase and the proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH2 were significantly elevated (P<0.001). The proliferative responses of HepG2 to trypsin and SLIGKV-NH2 were associated with the upregulation of c-fos and PCNA, which were significantly blocked by PD98059 pretreatment. In conclusion, our results indicate that PAR-2 enhances proliferation of human hepatoma cells possibly via the ERK/AP-1 pathway.     

Inhibited proliferation of cyclooxygenase-2 expressing human hepatoma cells by NS-398, a selective COX-2 inhibitor

Hepatocellular carcinoma (HCC) is a growing human health problem worldwide. Limited treatment and poor prognosis of this disease emphasize the importance in developing an effective chemoprevention. Overexpression of cyclooxygenase-2 (COX-2) has been associated with hepatocarcinogenesis. Although COX-2 inhibitors have been tested for chemoprevention of colon cancer, it remains unknown whether these agents possess anti-HCC effects as well. The present study assessed the effects of a selective COX-2 inhibitor, NS-398, on proliferation of human hepatoma cells in association with COX-2 expression, and the possible mechanisms. In four tested human hepatoma cell lines, overexpression of COX-2 was confirmed in HepG2, HuH7, and Chang liver cells, but not in PLC/PRF/5 cells. Addition of 50 micro M NS-398 resulted in both dose-dependent and time-course inhibition of HepG2 proliferation. In contrast, addition of 50 micro M NS-398 to COX-2 non-expressing PLC/PRF/5 cells resulted in only a mild reduction of cell proliferation. Consistent with this, a 48-h culture of HepG2 cells with 50 micro M NS-398 caused a significant decrease of prostaglandin E2 (PGE2) production. While, the same NS-398 treatment showed only a mild suppression of PGE2 production in COX-2 non-expressing PLC/PRF/5 cells. These findings indicate that NS-398-induced suppression of HepG2 proliferation appears mediated by decreased COX-2/prostaglandin (PG) production. We also found that NS-398-induced inhibition of HepG2 proliferation was associated with decreased 5-bromo-2'-deoxyuridine (BrdU) uptake, suggesting a reduced cell cycle progression in G1-S transition. NS-398 treatment also enhanced the apoptotic rate in COX-2 expressing HepG2 cells, but not in COX-2 non-expressing PLC/PRF/5 cells. Our findings confirmed an effective inhibitory effect of NS-398 on proliferation of COX-2 expressing human hepatoma cells through a decreased COX-2/PG activity that is associated with altered cell cycle progression and apoptotic rate.     

TLR4基因多态性对肝癌细胞HepG2增殖、迁移及凋亡的影响

目的:研究TLR4基因多态性对人肝癌细胞HepG2细胞增殖、迁移及凋亡的影响,并探讨其相关分子机制.方法:构建TLR4野生型(WT)、1196(C/T)位点及896(A/G)位点突变型质粒并将其稳转HepG2细胞株中;CCK-8法检测、Annexin V-PE/7-AAD流式细胞仪及Transwell小室实验检测TLR4基因多态性对HepG2细胞增殖、迁移及凋亡的影响;Western blot检测NF-κB p65(nuclear factorκB p65)表达水平的差异.结果:Western blot结果表明三组TLR4过表达细胞株中TLR4的含量显著高于正常组.与正常HepG2细胞相比,三组TLR4过表达细胞株增殖能力、迁移能力及p65表达量均增加;与TLR4野生型组相比,两组突变型细胞株增殖能力、迁移能力及p65表达量均减弱,且凋亡率增加,差异具有统计学意义(P<0.05).结论:TLR4可能通过影响NF-κB p65相关蛋白的表达促进肝癌细胞HepG2的增殖及迁移,其基因1196(C/T)位点及896(A/G)位点的突变会减弱肝癌细胞的增殖及迁移能力.     

Construction of a recombinant eukaryotic expression vector containing PHD3 gene and its expression in HepG2 cells

ABSTRACT: Prolyl hydroxylase domain 3 (PHD3) is a hypoxia inducible factor-alpha (HIFalpha) regulator; it degrades HIFalpha in the presence of oxygen. Recently, there have been an increasing number of studies about the role of PHD3 in proliferation and apoptosis of cancer cells. However, most of the evidence for the role of PHD3 is observational, and little is known of the molecular mechanism. In our current study, we constructed a recombinant eukaryotic expression vector containing the PHD3 gene and detected its biological activity in human hepatoma cell line (HepG2 cells). We successfully constructed a recombinant pcDNA 3.1(+)-PHD3 plasmid; the results showed that PHD3 overexpression could inhibit the proliferation of HepG2 cells and induce apoptosis by activating caspase-3 activity. Our study has provided preliminary materials and data for further investigation of the effect of PHD3 on HepG2 cells.     

Influence of exogenous RAR alpha gene on MDR1 expression and P-glycoprotein function in human and rodent cell lines.

The goal of our study was to obtain direct evidence of co-ordinated regulation of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) and differentiation in tumour cells and to study some signalling pathways involved in joint regulation of these two cell phenotypes. The sublines of human melanoma (mS) and hepatoma (human HepG2 and rat McA RH 7777) cell lines were obtained by retroviral infection of the wild-type cells with the cDNA of the human retinoic acid receptor alpha (RAR alpha). The resulting sublines stably overexpressed exogenous RAR alpha gene. The infectants became more differentiated than the parental cells as determined by a decrease in the synthesis of the embryo-specific alpha-fetoprotein in HepG2 and McA RH 7777 hepatoma cells and by an increase in melanin synthesis in mS cells. The differentiation of human cells was accompanied by an increase in the amounts of MDR1 mRNA but not by an increase in P-gp activity as a drug transporter, in contrast, in the rat RAR alpha overexpressing cells P-gp functional activity was elevated. Treatment with cytotoxic drug (colchicine) or retinoic acid (RA) resulted in a slight increase in P-gp activity in the parental and RAR alpha-infected melanoma cells, whereas the increase in P-gp function in the infected hepatoma cells (both human and rat) was very prominent. Thus, we provide new evidence that cell differentiation caused by the overexpression of the gene participating in the differentiation programme leads to overexpression of MDR1 gene and drug resistance and that this effect is tissue and species specific. These data imply that the activation of the RA-controlled signalling pathway up-regulates MDR1 gene expression.     

氨基葡萄糖对人肝癌HepG2细胞增殖及凋亡的影响

目的:研究氨基葡萄糖对人肝癌HepG2细胞增殖及凋亡的影响,并初步探讨其分子机制。方法:CCK-8法检测人肝癌HepG2细胞的增殖情况。流式细胞术检测细胞周期及细胞凋亡。Western blot检测ERK及P-ERK蛋白的表达水平。结果:氨基葡萄糖可以抑制人肝癌HepG2细胞的增殖,且该作用随着药物浓度的增加而逐渐增强。1.0mmol/L氨基葡萄糖作用72h可以使G0/G1期细胞比例增加,S期细胞比例降低。同时,2.0mmol/L及4.0mmol/L氨基葡萄糖处理72h可以使人肝癌HepG2细胞发生凋亡。另外,随着药物浓度的增加,人肝癌HepG2细胞中ERK蛋白的磷酸化水平逐渐降低。结论:氨基葡萄糖可能是通过降低ERK1/2蛋白的磷酸化水平影响细胞周期,从而抑制人肝癌HepG2细胞的增殖并诱导其发生凋亡。