酸性环境诱导肝癌HepG2细胞EMT

目的 研究酸性环境对肝癌HepG2细胞上皮细胞间质转化(EMT)的影响。方法分别在酸性和碱性条件下培养HepG2细胞,观察细胞形态的区别。划痕实验检测两组细胞的迁移能力,Matrigel侵袭实验检测细胞的侵袭能力。RT-PCR检测两组细胞EMT相关基因mRNA的表达水平,Western blot检测两组细胞EMT相关基因蛋白的表达水平。结果 酸性条件下培养的HepG2细胞形态明显从上皮型转变为间质型;酸性条件下HepG2细胞的迁移能力明显强于碱性条件下;酸性条件下HepG2细胞穿越Matrigel的数量明显多于碱性条件下;酸性条件下HepG2细胞EMT相关基因Vimentin、Slug、Snail及Zeb1的mRNA表达以及EMT相关蛋白Vimentin和MMP9的表达强度均明显高于碱性条件下。结论 酸性环境能诱导肝癌HepG2细胞EMT的发生。     

Cancer stem cells and EMT in carcinoma

The majority of deaths from carcinoma are caused by secondary growths that result from tumour invasion and metastasis. The importance of epithelial-to-mesenchymal transition (EMT) as a driver of invasion and metastasis is increasingly recognised, and recent evidence has highlighted a link between EMT and the cancer stem cells that initiate and maintain tumours and have also been implicated in invasion and metastasis. Here, we review cancer stem cells and their link with EMT, and explore the importance of this link in metastasis and therapeutic resistance of tumours. We also discuss new evidence from our laboratory demonstrating that cancer stem cells display a remarkable phenotypic plasticity that enables them to switch between an epithelial phenotype that drives tumour growth and an EMT phenotype that drives metastasis. As successful therapies must eradicate cancer stem cells in all their guises, the identification of sub-types of cancer stem cells that display therapeutic resistance and phenotypic plasticity has important implications for the future design of therapeutic strategies. The ability to assay the responses of different cancer stem cell phenotypes in vitro holds promise for the rapid development of a new generation of targeted therapies that fulfil this objective.     

类泛素化蛋白酶1基因的表达与肝癌细胞株侵袭转移的关系

研究类泛素化蛋白酶-1（sentrin-specific protease-1,SENP-1）基因在不同侵袭潜能肝细胞肝癌细胞株及人永生化肝细胞中mRNA和蛋白的表达水平及其与肝癌细胞侵袭能力的关系。方法：使用RT-PCR、Real-time PCR、Western blotting和免疫荧光方法对肝癌细胞株MHCC97L（低侵袭性）、MHCC97H和HCCLM3（高侵袭性）、SMMC-7721和HepG2（无明显侵袭性）SENP-1 mRNA和蛋白表达水平进行检测,以人永生化肝细胞株HL-7702为对照,分析SENP-1的表达与肝癌细胞株侵袭转移能力的关系。结果：RT-PCR和Real-time PCR检测结果显示,SENP-1 mRNA（F=5.658;P=0.042）和蛋白（F=88909,P=0.000）在肝癌细胞株中的表达随着肝癌细胞株侵袭潜能的增高而增高;Western blotting检测结果显示,5种肝癌细胞株SENP-1蛋白水平均显著高于人永生化肝细胞株;免疫荧光法观察发现,SENP-1蛋白在肝癌细胞株内主要为胞质表达,部分胞核表达。结论：SENP-1基因在肝癌细胞株中的高表达可能具有促进肝癌细胞侵袭转移的作用。     

BMP4 augments the survival of hepatocellular carcinoma (HCC) cells under hypoxia and hypoglycemia conditions by promoting the glycolysis pathway

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide although its pathogenic mechanism remains to be fully understood. Unlike normal cells, most cancer cells rely on aerobic glycolysis and are more adaptable to the microenvironment of hypoxia and hypoglycemia. Bone Morphogenetic Protein 4 (BMP4) plays important roles in regulating proliferation, differentiation, invasion and migration of HCC cells. We have recently shown that BMP4 plays an important role in regulating glucose metabolism although the effect of BMP4 on glucose metabolic reprogramming of HCC is poorly understood. In this study, we found that BMP4 was highly expressed in HCC tumor tissues, as well as HCC cell lines that were tolerant to hypoxia and hypoglycemia. Mechanistically, we demonstrated that BMP4 protected HCC cells from hypoxia and hypoglycemia by promoting glycolysis since BMP4 up-regulated glucose uptake, the lactic acid production, the ATP level, and the activities of rate limiting enzymes of glycolysis (including HK2, PFK and PK). Furthermore, we demonstrated that BMP4 up-regulated HK2, PFKFB3 and PKM2 through the canonical Smad signal pathway as SMAD5 directly bound to the promoter of PKM. Collectively, our findings shown that BMP4 may play an important role in regulating glycolysis of HCC cells under hypoxia and hypoglycemia condition, indicating that novel therapeutics may be developed to target BMP4-regulated glucose metabolic reprogramming in HCC.     

长链非编码RNA通过上皮 间质转化对射频消融术后肝癌复发的影响

目的探讨长链非编码RNA（LncRNA）通过上皮 间质转化（EMT）对射频消融术后肝癌复发的影响。方法通过47 ℃水浴热诱导Huh 7细胞得到Huh 7h细胞,体外模拟不完全射频消融肝癌细胞。Western blot检测EMT分子标记;Transwell实验检测细胞迁移和侵袭能力;CCK 8实验检测细胞增殖能力;使用LncPathTM芯片筛选Huh 7与Huh 7h细胞差异表达的EMT相关LncRNA,并通过RT PCR验证。结果Huh 7h细胞上皮表型标志物E 钙黏蛋白相对表达量低于Huh 7细胞,细胞间质表现标志物N 钙黏蛋白、波形蛋白相对表达量高于Huh 7细胞（P<005）。Transwell细胞迁移实验中Huh 7h穿膜细胞数高于Huh 7穿膜细胞数（P<005）;细胞侵袭实验中Huh 7h穿膜细胞数高于Huh 7穿膜细胞数（P<005）。CCK 8实验显示,培养24 h、48 h、72 h时Huh 7h细胞吸光度值高于Huh 7细胞（P<005）。使用LncPathTM芯片最终筛选出与差异表达相关的2个下调LncRNA,分别为Fundc2p4（ENST00000552318）、Rpl27p7（ENST00000552432）;1个上调LncRNA为Mtnd4lp14（ENST00000538558）。经RT PCR验证,Huh 7h细胞中Fundc2p4相对表达量低于Huh 7细胞（P<005）。临床标本RT PCR验证,LncRNAFundc2p4在癌旁组织中的相对表达量>手术切除的肝癌组织>射频消融术后残余肝癌组织,差异有统计学意义（P<005）。结论射频消融不完全的肝癌细胞通过低表达EMT相关的LncRNA Fundc2p4基因来促进EMT,增强癌细胞的迁移和侵袭能力,提升肝癌复发的风险。     

放疗诱导人肝细胞癌上皮-间质转化

Objective

Epithelial-mesenchymal transition (EMT) is a critical early event for the invasion and metastasis of many carcinomas. In the present study, we examined EMT markers in the residual cancer cells of hepatocellular carcinoma (HCC) after radiotherapy.

Methods

Eight patients with large HCC who underwent hepatectomy with preoperative radiotherapy were studied. The expressions of E-cadherin and vimentin were determined immunohistochemically in the residual cancer cells of HCC following radiotherapy, and also in the pre-radiotherapy biopsy cancer cells.

Results

Histological analysis showed that some residual cancer cells of HCC displayed an elongated spindle or fibroblast-like shape. The expression of Ecadherin was markedly reduced or negative in the spindle residual cancer cells, but the expression of vimentin significantly induced. However, the above changes were not found in the pre-radiotherapy biopsy cancer cells.

Conclusion

EMT is induced in the residual cancer cells of HCC following radiotherapy, which may facilitate the systemic dissemination of cancer cells.     

肝脏树突状细胞与肝细胞癌

近年来随着对肝脏DC免疫生物学研究的不断深入，人们更深入地了解了肝脏DC的免疫学特性及其与肝脏疾病尤其是肝细胞癌（hepatocellular carcinoma, HCC）的关系，并据此设计有针对性的治疗策略。在肝脏微环境的作用下，肝脏DC处于非成熟状态，具有数量相对不足、显著的异质性、抗原摄取能力及T细胞活化能力低下等特点，它可表达特殊的CC和CCR。上述因素使肝脏DC在肝脏免疫调节中起重要作用，主要体现在大量肝脏非成熟DC无法活化T细胞，尤其在肝细胞癌的状态下，非成熟肝脏DC进一步增加，伴随着免疫活化因子IL12分泌的减少，不利于机体抗肿瘤免疫的产生，而肝细胞癌自身可通过AFP、IL10、IL8及HBV/HCV的作用抑制肝脏DC。但也有观点认为，肝脏DC为成熟状态，可通过调节T细胞活化的途径最终引起肝脏免疫耐受的产生。     

树突状细胞与肝细胞癌免疫治疗

树突状细胞（DC）是体内功能最强的抗原提呈细胞（APC）,由DC激活的T细胞免疫在抗肿瘤过程中起着主导作用。关于DC肿瘤疫苗的基础与临床研究结果显示,DC疫苗在恶性肿瘤治疗中具有良好的应用前景。     

Secretion of albumin and alpha-foetoprotein by dimethylsulphoxide-stimulated hepatocellular carcinoma cells.

Exposure of BW77-1 and BW77-2 mouse hepatic tumour cells to the polar solvent dimethylsulphoxide (DMSO) altered extracellular accumulation of albumin and alpha-foetoprotein (AFP) and perturbed their cell cycle kinetics. The amount of albumin secreted into the culture growth medium was dependent on the concentration of DMSO used. Hepatic tumour cells cultured in 1 and 2% DMSO accumulated 50% and 111% more albumin, respectively, than non-DMSO-stimulated cells during the final 24 h of a 4-day exposure to the polar solvent. Commitment of mouse hepatoma cells to increased albumin secretion was temporally dependent, requiring a minimum of 48 h in the presence of DMSO. The AFP level in 1% DMSO-treated cultures was also significantly increased, compared with control cells. Unlike albumin secretion, however, exposure of hepatic tumour cells to 2% DMSO did not further increase (but slightly decreased) extracellular AFP accumulation. Treatment of BW77-1 cells with DMSO resulted in a gradual decline in the percentage of 2C DNA content cells (diploid G1 population) and in a corresponding increase in the proportion of cells with a 4C DNA content (generation of either a G2 or tetraploid G1 population). The extent of this shift directly reflected the concentration of polar solvent in the medium and paralleled the DMSO-induced stimulation in albumin secretion. DMSO-stimulated hepatic tumour cells, therefore, may prove useful in the elucidation of specific regulatory events underlying control of gene expression during the hepatocyte cell cycle.     

Indomethacin and SC236 enhance the cytotoxicity of doxorubicin in human hepatocellular carcinoma cells via inhibiting P-glycoprotein and MRP1 expression

Doxorubicin is a chemotherapeutic drug widely used for the treatment of hepatocellular carcinoma but its efficacy is restricted by multidrug resistance. Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX)-2-selective inhibitors exhibit anti-cancer properties as well as abilities to overcome drug resistance. In the present study, indomethacin (a NSAID) and SC236 (a COX-2-selective inhibitor) enhanced the cytotoxicity of doxorubicin in the hepatocellular carcinoma cell line HepG2 and its drug-resistant sub-line R-HepG2. Both drugs increased the intracellular accumulation and retention of doxorubicin in vitro. The effects were not reversed by prostaglandin E(2), implicating a COX-independent mechanism. Indomethacin and SC236 partially reversed the increase in expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) induced by doxorubicin in R-HepG2 cells. In conclusion, indomethacin and SC236 increased the intracellular accumulation and retention of doxorubicin and thus its cytotoxicity in HepG2 and drug-resistant HepG2 cells. These effects, mediated through decrease in P-gp and MRP1 expression and/or direct inhibition of P-gp activity, may improve multidrug resistant-cancer chemotherapy.     

HBV感染与肝癌组织CDKN2A基因甲基化关系的研究

目的:探讨HBV感染和CDKN2A异常甲基化之间的潜在关联及其相互作用在肝癌形成过程中的作用。方法:对44例肝癌患者癌组织及癌旁组织CDKN2A甲基化状况、血清HBV感染标志物等到进行了检测分析。血清HBV、HCV感染标志物采用全自动化学发光分析仪进行检测。采用酚、氯仿法抽提组织DNA,用CDKN2A甲基化特异性引物进行PCR扩增,并检测特异性产物。患者按HBV感染情况进行分组,统计分析。结果:CDKN2A甲基化产物在肝癌组织中的总检出率为70.5%(31/44)。其中,HBV相关肝癌组和非病毒相关肝癌组CDKN2A甲基化率为90.6%(29/32)和16.7%(2/12),P=0.000。对HBV相关肝癌进一步分组分析,HBsAg /HBeAg 、HBsAg /HBeAg-及HBcAb /HBcAb-三组,三组之间差异无统计学意义(P=0.568)。经非参数相关和logistic回归分析,发现:CDKN2A甲基化与性别、肿瘤分化程度无明显相关(R2=0.11,P=0.650),而与HBV感染,特别是HBsAg密切相关(R2=0.37,P=0.02)。结论:在肝癌形成过程中,HBV可能通过促进抑癌基因,如CDKN2A等的异常甲基化,使CDKN2A失活,肝细胞正常的生长调控机制失常,形成肝癌,这也可能是HBV导致肝癌形成的机制之一。     

MARK2在肝癌中的表达及其对肝癌细胞迁移的影响

背景与目的：肝细胞癌是我国常见恶性肿瘤之一,其发生机制目前尚未明确。该研究旨在探讨微管亲和性调节激酶2（microtubule affinity-regulating kinase 2,MARK2）在肝癌中的表达及其对肝癌细胞迁移能力的影响。方法：通过组织芯片和免疫组织化学法对肝癌组织中MARK2的表达进行分析;将MARK2基因真核表达载体及其空载体转染至肝癌HepG2细胞中,采用蛋白质印迹法（Western blot）检测转染后MARK2蛋白表达水平;采用Transwell实验及细胞划痕实验检测MARK2对HepG2细胞迁移能力的影响;通过癌症基因组图谱（the Cancer Genome Atlas,TCGA）分析MARK2在肝癌组织和正常组织中的表达差异,及其与肝癌患者临床病理特征的关系;运用网络在线生存期分析软件Kaplan-Meier plotter分析MARK2表达与肝癌患者生存预后的关系。结果：MARK2在肝癌组织中表达明显高于癌旁组织,肝癌组织中MARK2高表达与TNM分期及病理分级显著相关（P<0.001）;生存分析结果表明,MARK2高表达患者5年生存率明显低于MARK2低表达组（P<0.01）;将MARK2基因真核表达载体转染肝癌HepG2细胞后,MARK2蛋白表达上调,肝癌细胞的迁移能力增强。结论：MARK2在肝癌组织中高表达,并促进肝癌细胞的迁移。     

The potassium channel KCa3.1 promotes cell proliferation by activating SKP2 and metastasis through the EMT pathway in hepatocellular carcinoma

The intermediate conductance calcium-activated potassium channel (KCa3.1) plays an important role in maintaining intracellular calcium homeostasis and is involved in the tumorigenesis of many human cancers. However, it is unknown whether KCa3.1 plays a role in the genesis of hepatocellular carcinoma (HCC), one of the most common malignant tumors worldwide with a very poor prognosis. In our study, we found that the expression of KCa3.1 was significantly elevated in poorly differentiated HCC tissues compared to adjacent noncancerous tissues. In vitro and in vivo experiments showed that KCa3.1 could promote cell proliferation, migration, and invasion of HCC. Mechanistically, KCa3.1 promoted cell cycle progression and migration and invasion of HCC cells by activating S-phase protein kinase 2 (SKP2) to trigger the degradation of p21 and p27 and targeting Reelin (RELN) to induce epithelial-mesenchymal transition (EMT), respectively. Taken together, our results demonstrate that KCa3.1 plays an important role in the genesis and progression of HCC, implying that it might be a promising therapeutic target in HCC.     

Epirubicin may enhance the inhibition of hepatocellular carcinoma induced by iodine-125 seeds through downregulating WNT pathway

Aim

To investigate the role of iodine-125 (¹²⁵I) combined with epirubicin (EPI) in inhibiting hepatocellular carcinoma (HCC) growth and promoting apoptosis.

Methods

Both in vivo and in vitro experiments were conducted. CCK-8 assay was performed to determine the cells viability after EPI treatment. HepG2 and SMMC7721 cells were treated with EPI or ¹²⁵I or in combination. Colony formation assays were performed to verify the antiproliferation effect. Annexin V–FITC/PI, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, and western blotting were performed to analyze cellular apoptosis. Scratch wound healing assays and transwell assays were used to examine migration following different treatments. An isobaric tag for relative and absolute quantitation analysis was used to detect changes in protein expression after ¹²⁵I treatment, identifying the potential mediating protein cathelicidin (LL-37). LL-37 protein and WNT pathway-related proteins were detected by western blotting in SMMC7721 and HepG2 cells. Mice were treated with ¹²⁵I and EPI to evaluate whether EPI enhanced the antitumor effect of ¹²⁵I.

Results

EPI promoted ¹²⁵I-induced apoptosis and reduced the proliferation of HepG2 and SMMC7721 cells. EPI also prevented the migration of HepG2 and SMMC7721 cells. EPI combined with ¹²⁵I may interfere with the WNT signaling pathway by decreasing LL-37 to inhibit HCC development. The antitumor effects of EPI with ¹²⁵I were verified in mice.

Conclusion

EPI combined with ¹²⁵I induced apoptosis and inhibited the proliferation of HCC cells by LL-37 downregulating the WNT pathway.     

Twist promotes the migration of hepatocellular carcinoma cells

OBJECTIVE To study the effect of the Twist gene on the migration of human hepatocellular carcinoma cells and the possible mechanisms involved. METHODS RT-PCR was used to detect expression of the Twist gene in primary （Hep11） and recurrent （Hep12） cell lines from the same HCC patient. Hep11 cells were stably transfected with Twist-cDNA, and Hep12 cells were transiently transfected with Twist RNAi plasmid. Cell migration assays were performed on Twist up-regulated Hep11 cells and Twist RNAi Hep12 cells. RT- PCR and Western blot were used to test the expression of EMT markers. RESULTS Twist was expressed higher level and had increased migration capability in recurrent Hep12 cells than those in primary Hep11 cells. Cell models （Twist-Hep11） in which Twist protein was steadily and highly expressed were obtained. Compared with pcDNA3-Hep11 cells, migration of Twist-Hep11 cells was clearly increased. However, migration of Twist RNAi （Si-Twist-Hep12） Hep12 cells were reduced. Overexpression of Twist in Hep11 cells promoted expression of N-cad and vimentin. CONCLUSION These results indicate that Twist promotes the migration of hepatocellular carcinoma cells in vitro and may play an important role in the upregulation of mesenchymal markers.