Disulfiram Eradicates Tumor-Initiating Hepatocellular Carcinoma Cells in ROS-p38 MAPK Pathway-Dependent and -Independent Manners

Tumor-initiating cells (TICs) play a central role in tumor development, metastasis, and recurrence. In the present study, we investigated the effect of disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, toward tumor-initiating hepatocellular carcinoma (HCC) cells. DSF treatment suppressed the anchorage-independent sphere formation of both HCC cells. Flow cytometric analyses showed that DSF but not 5-fluorouracil (5-FU) drastically reduces the number of tumor-initiating HCC cells. The sphere formation assays of epithelial cell adhesion molecule (EpCAM)⁺ HCC cells co-treated with p38-specific inhibitor revealed that DSF suppresses self-renewal capability mainly through the activation of reactive oxygen species (ROS)-p38 MAPK pathway. Microarray experiments also revealed the enrichment of the gene set involved in p38 MAPK signaling in EpCAM⁺ cells treated with DSF but not 5-FU. In addition, DSF appeared to downregulate Glypican 3 (GPC3) in a manner independent of ROS-p38 MAPK pathway. GPC3 was co-expressed with EpCAM in HCC cell lines and primary HCC cells and GPC3-knockdown reduced the number of EpCAM⁺ cells by compromising their self-renewal capability and inducing the apoptosis. These results indicate that DSF impaired the tumorigenicity of tumor-initiating HCC cells through activation of ROS-p38 pathway and in part through the downregulation of GPC3. DSF might be a promising therapeutic agent for the eradication of tumor-initiating HCC cells.     

Hepatocellular Carcinoma Cells Deteriorate the Biophysical Properties of Dendritic Cells

Dendritic cells (DCs) are potent antigen-presenting cells and induce antigen-specific immune responses in the organism. The dysfunction of DCs has been implicated in tumor-bearing host. In order to elucidate the effects of tumor microenvironment on the functions of DCs from interdisciplinary aspects, we characterized the biophysical properties of DCs co-cultured with hepatocellular carcinoma cells (HCC). The results showed that the biophysical characteristics of immature and mature DCs were severely impaired by HCC compared with those under normal conditions, including the increased osmotic fragilities, decreased cell membrane fluidities, increased membrane viscoelastic properties, dysfunction and increased expression of cytoskeleton protein F-actin, as well as the deteriorated transendothelium migration. The impaired biophysical properties of DCs may be one of many aspects of the immune escape mechanisms of tumors. These results are clinically and instructionally significant with regard to how to enhance efficiency of the anti-tumor therapy based on DCs. Zhu Zeng and Weijuan Yao contributed equally to this work.     

Diversity of Gene Expression in Hepatocellular Carcinoma Cells

Understanding tumor diversity has been a long-lasting and challenging question for researchers in the field of cancer heterogeneity or tumor evolution. Studies have reported that compared to normal cells, there is a higher genetic diversity in tumor cells, while higher genetic diversity is associated with higher progression risks of tumor. We thus hypothesized that tumor diversity also holds true at the gene expression level. To test this hypothesis, we used t-test to compare the means of Simpson's diversity index for gene expression(SDIG) between tumor and non-tumor samples.We found that the mean SDIG in tumor tissues is significantly higher than that in the non-tumor or normal tissues(P 0.05) for most datasets. We also combined microarrays and next-generation sequencing data for validation. This cross-platform and cross-experimental validation greatly increased the reliability of our results.     

Giant Lysosomes as a Chemotherapy Resistance Mechanism in Hepatocellular Carcinoma Cells

Despite continuous improvements in therapeutic protocols, cancer-related mortality is still one of the main problems facing public health. The main cause of treatment failure is multi-drug resistance (MDR: simultaneous insensitivity to different anti-cancer agents), the underlying molecular and biological mechanisms of which include the activity of ATP binding cassette (ABC) proteins and drug compartmentalisation in cell organelles. We investigated the expression of the main ABC proteins and the role of cytoplasmic vacuoles in the MDR of six hepatocellular carcinoma (HCC) cell lines, and confirmed the accumulation of the yellow anti-cancer drug sunitinib in giant (four lines) and small cytoplasmic vacuoles of lysosomal origin (two lines). ABC expression analyses showed that the main ABC protein harboured by all of the cell lines was PGP, whose expression was not limited to the cell membrane but was also found on lysosomes. MTT assays showed that the cell lines with giant lysosomes were more resistant to sorafenib treatment than those with small lysosomes (p<0.01), and that verapamil incubation can revert this resistance, especially if it is administered after drug pre-incubation. The findings of this study demonstrate the involvement of PGP-positive lysosomes in drug sequestration and MDR in HCC cell lines. The possibility of modulating this mechanism using PGP inhibitors could lead to the development of new targeted strategies to enhance HCC treatment.     

人肝癌细胞与肝星形细胞裸鼠皮下共培养体系的建立

目的:建立裸鼠皮下共培养人肝癌细胞与肝星形细胞模型,观察人肝癌细胞与肝星形细胞间相互作用后超微结构的改变.方法:将16只裸鼠分为两组,肝癌细胞单独培养组和癌细胞与肝星形细胞共培养组,40天后将荷瘤组织切片行光镜及透射电镜观察.结果:肝癌细胞单独培养组中可观察到肝癌细胞的胞质液化及早期细胞凋亡现象,而共培养组中可见肝星形细胞时肝癌细胞的趋化现象,可观察到肝癌细胞结构完整且有增殖趋势.结论:裸鼠皮下荷瘤三维立体模型建立成功,该模型能够模拟肝癌微环境中肝癌细胞与肝星形细胞问的作用,为进一步研究肝癌细胞与肝星形细胞间的相互作用奠定了基础.     

BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

TGF-β family members play a relevant role in tumorigenic processes, including hepatocellular carcinoma (HCC), but a specific implication of the Bone Morphogenetic Protein (BMP) subfamily is still unknown. Although originally isolated from fetal liver, little is known about BMP9, a BMP family member, and its role in liver physiology and pathology. Our results show that BMP9 promotes growth in HCC cells, but not in immortalized human hepatocytes. In the liver cancer cell line HepG2, BMP9 triggers Smad1,5,8 phosphorylation and inhibitor of DNA binding 1 (Id1) expression up- regulation. Importantly, by using chemical inhibitors, ligand trap and gene silencing approaches we demonstrate that HepG2 cells autocrinely produce BMP9 that supports their proliferation and anchorage independent growth. Additionally, our data reveal that in HepG2 cells BMP9 triggers cell cycle progression, and strikingly, completely abolishes the increase in the percentage of apoptotic cells induced by long-term incubation in low serum. Collectively, our data unveil a dual role for BMP9, both promoting a proliferative response and exerting a remarkable anti-apoptotic function in HepG2 cells, which result in a robust BMP9 effect on liver cancer cell growth. Finally, we show that BMP9 expression is increased in 40% of human HCC tissues compared with normal human liver as revealed by immunohistochemistry analysis, suggesting that BMP9 signaling may be relevant during hepatocarcinogenesis in vivo. Our findings provide new clues for a better understanding of BMPs contribution, and in particular BMP9, in HCC pathogenesis that may result in the development of effective and targeted therapeutic interventions.     

KMT6在肝癌细胞转移中的作用及对肝癌预后的影响

目的:探讨组蛋白赖氨酸甲基转移酶6(histone lysine methyltransferase 6,KMT6)对人肝癌细胞转移的调控作用及对肝癌患者预后的影响。方法:采用RNA干涉技术合成靶向KMT6的小干扰RNA(KMT6 siRNA)片段,瞬时转染至人肝癌细胞系Huh-7。随后,分别采用细胞黏附实验、细胞侵袭实验以及划痕迁移实验检测肝癌细胞的黏附、侵袭和迁移等肿瘤细胞转移能力。运用Oncomine数据库和cBioportal肝癌数据库分析KMT6的表达情况及对肝癌预后的影响。结果:靶向KMT6的小干扰RNA(KMT6 siRNA)片段瞬时转染至人肝癌细胞系Huh-7后,KMT6蛋白的表达显著下降,且黏附率、侵袭率和划痕修复率均显著降低(P<0.05)。Oncomine肝癌数据库分析表明肝癌组织KMT6的表达增加,cBioportal肝癌数据库分析表明KMT6基因改变与肝癌患者预后不良密切相关。结论:KMT6分子可促进肝癌细胞黏附、侵袭和迁移,其基因改变与肝癌患者预后不良显著相关。KMT6分子有望成为肝癌治疗的新靶点。     

肝细胞生长因子诱导人肝癌细胞上皮间质化

上皮间质转化（epithelial-mesenchymal transition,EMT）与肿瘤侵袭转移密切相关.虽然肝细胞生长因子（hepatocyte growth factor,HGF）已被证实为肿瘤EMT的主要诱导剂,但是HGF诱导肿瘤EMT发生的分子机制尚不完全清楚.本研究旨在探讨Snail在HGF诱导肝癌细胞上皮间质转化中的作用.用HGF处理肝癌HepG2和Hep3B细胞,显微镜观察细胞形态变化,划痕试验及Transwell试验检测细胞迁移能力,Western印迹检测Met,AKT的磷酸化及蛋白质表达的变化,Western印迹与real-time RT-PCR检测上皮细胞表面标志E-Cadherin和间质细胞表面标志N-Cadherin、Fibronectin的表达变化,以及EMT相关转录因子的表达变化.经HGF处理的HepG2、Hep3B细胞,Met和AKT的磷酸化水平显著增强;相差倒置显微镜下观察细胞形态向间质型细胞形态转化;细胞划痕和Transwell试验检测细胞的迁移能力较对照组显著增强;Real-time RT-PCR和Western印迹实验显示HGF的诱导能上调间质标记蛋白的表达及下调上皮型标志蛋白的表达.进一步发现,HGF能上调转录因子Snail的表达,干扰Snail能逆转HGF对HepG2和Hep 3B细胞EMT发生的诱导作用.由此可见,HGF可能通过诱导Snail的表达促进肝癌细胞EMT的发生.这为阐明肝癌细胞侵袭转移机制,以及肝癌的防治提供新线索.     

Metformin Treatment for Diabetes Mellitus Correlates with Progression and Survival in Colorectal Carcinoma

BACKGROUND: Diabetes mellitus is unfavorably associated with cancer risk. The purpose of this multidisciplinary project was to evaluate a possible association of diabetes mellitus and other comorbidities and their treatment with progression of colorectal cancer. PATIENTS AND METHODS: We investigated the correlation between pathological characteristics and clinical course, including comorbidities in 1004 Czech patients diagnosed and surgically treated for colorectal adenocarcinoma (CRC) between 1999 and 2016. RESULTS: In our data set, CRC patients treated with metformin due to coexisting diabetes mellitus type 2 (T2DM) developed fewer distant metastases which clinically correlates with slower CRC progression. Survival in metformin subgroup was longer, particularly in men with CRC. Osteoporosis may be a negative factor of survival in CRC patients. CONCLUSIONS: Our findings also indicate that aging, higher tumor grade and TNM stage, coexistence of selected endocrine disorders, and metabolic abnormalities may change the tumor microenvironment and impact survival in colorectal cancer, although mechanism of these observations yet to be explained. Patients with diabetes mellitus type 2 treated with metformin may represent the altered microenvironment with specifically tuned metabolic molecular responses and with various epigenetic characteristics. More awareness and increased understanding of the mechanisms underlying the positive effect of metformin on patients' survival could offer insight into new treatment methods and permit more individualized treatment plans.     

二氢青蒿素通过诱导铁死亡抑制肝癌细胞生长

二氢青蒿素（dihydroartemisinin,DHA）是青蒿素的一种衍生物,在多种肿瘤中表现出明显的抗肿瘤活性,但其具体机制不详。本文探讨了DHA对肝癌细胞的毒性作用机制。利用CCK-8试剂检测DHA对肝癌细胞株活力的影响,通过荧光探针染色及流式细胞术分析细胞内ROS及脂质过氧化物水平的变化;通过谷胱甘肽测定试剂盒检测细胞内还原型谷胱甘肽含量的变化,并通过免疫印迹分析DHA作用下细胞内铁死亡通路蛋白质中GPX4的变化。结果发现,DHA能显著抑制SMMC-7721及Huh-7细胞活力,其半数抑制浓度分别为23.74 μmol/L及26.92 μmol/L。 在35 μmol/L DHA 处理下,SMMC-7721及Huh-7细胞内ROS分别升高2.6倍和2.1倍,脂质过氧化物升高2.3倍和1.7倍。DHA可诱导细胞内GSH含量下降,并能下调铁死亡相关蛋白质GPX4蛋白水平。通过利用小分子抑制剂进行功能恢复实验发现,ROS抑制剂、铁螯合剂及铁死亡抑制剂都可不同程度恢复DHA引起的细胞活力下降。进一步检测发现,铁死亡抑制剂可抑制DHA诱导的脂质过氧化,并恢复GSH含量及GPX4蛋白水平。结果表明,在肝癌细胞中,DHA可通过诱导细胞发生铁死亡抑制肝癌细胞生长。     

二氢青蒿素通过诱导铁死亡抑制肝癌细胞生长

二氢青蒿素（dihydroartemisinin,DHA）是青蒿素的一种衍生物,在多种肿瘤中表现出明显的抗肿瘤活性,但其具体机制不详。本文探讨了DHA对肝癌细胞的毒性作用机制。利用CCK-8试剂检测DHA对肝癌细胞株活力的影响,通过荧光探针染色及流式细胞术分析细胞内ROS及脂质过氧化物水平的变化;通过谷胱甘肽测定试剂盒检测细胞内还原型谷胱甘肽含量的变化,并通过免疫印迹分析DHA作用下细胞内铁死亡通路蛋白质中GPX4的变化。结果发现,DHA能显著抑制SMMC-7721及Huh-7细胞活力,其半数抑制浓度分别为23.74 μmol/L及26.92 μmol/L。 在35 μmol/L DHA 处理下,SMMC-7721及Huh-7细胞内ROS分别升高2.6倍和2.1倍,脂质过氧化物升高2.3倍和1.7倍。DHA可诱导细胞内GSH含量下降,并能下调铁死亡相关蛋白质GPX4蛋白水平。通过利用小分子抑制剂进行功能恢复实验发现,ROS抑制剂、铁螯合剂及铁死亡抑制剂都可不同程度恢复DHA引起的细胞活力下降。进一步检测发现,铁死亡抑制剂可抑制DHA诱导的脂质过氧化,并恢复GSH含量及GPX4蛋白水平。结果表明,在肝癌细胞中,DHA可通过诱导细胞发生铁死亡抑制肝癌细胞生长。     

岩藻糖链与肝癌细胞发生和转移的相关性研究

研究观察了大鼠诱发肝癌过程中,与UEA、LCA凝集素相结合的含岩藻糖糖蛋白尤其是80 ku蛋白的动态变化．在肝癌病人标本中,也观察到了高转移性肝癌细胞比低转移性肝癌细胞表达更多的UEA、LCA相结合的岩藻糖蛋白．岩藻糖寡糖可以构成一些非常重要的黏附分子的结构,如Lewis抗原．继而进一步观察了不同转移潜能的肝癌细胞中Lewis抗原的表达差异,发现高转移性肝癌细胞 (HMCC97H) 比低转移性肝癌细胞(HMCC97L)表达更高的Lewis x 和 b．在肝癌转移动物模型中,转移灶组织中的Lewis抗原合成关键酶α1,3/1,2以及 α1,6 岩藻糖转移酶活性远比对照组高．当肝癌细胞在维甲酸作用以后,细胞表面的Lewis x 或 b 的水平显著下降,α1,3/1,2岩藻糖转移酶活性也显著下降．同时我们观察到Lewis x可以存在于表皮细胞生长因子受体(EGFR)分子上,在维甲酸作用以后,EGFR上的Lewis x抗原和磷酸化水平都显著性下降．上述结果提示岩藻糖化的糖链如Lewis x在肝癌细胞的发生和转移过程中起重要的作用．     

BCRP/ABCG2 Inhibition Sensitizes Hepatocellular Carcinoma Cells to Sorafenib

The multikinase inhibitor, sorafenib (Nexavar®, BAY43-9006), which inhibits both the Raf/MEK/ERK pathway and several receptor tyrosine kinases (RTKs), has shown significantly therapeutic benefits in advanced hepatocellular carcinoma (HCC). However, not all HCC patients respond to sorafenib well and new therapeutic strategies to optimize the efficacy of sorafenib are urgently required. Overexpression of breast cancer resistance protein (BCRP/ABCG2) mediates the drug-efflux of several tyrosine kinase inhibitors (TKIs) to attenuate their efficacy. This study aimed to investigate the role of BCRP/ABCG2 in the sensitivity of HCC to sorafenib. Our data showed that BCRP/ABCG2 mediated the efflux of sorafenib. Co-treatment with a BCRP/ABCG2 inhibitor greatly augmented the cytotoxicity of sorafenib in HCC cells. Similar results were also achieved by the competitive inhibitor of BCRP/ABCG2, gefitinib, in combination with sorafenib. These results suggest not only that BCRP/ABCG2 is a potential predictor for the sorafenib sensitivity in HCC, but also that blockage of BCRP/ABCG2 may be a potential strategy to increase the response of HCC cells to sorafenib.     

p16 Stimulates CDC42-Dependent Migration of Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. Tumor dissemination to the extra-hepatic region of the portal vein, lymph nodes, lungs or bones contributes to the high mortality seen in HCC; yet, the molecular mechanisms responsible for HCC metastasis remain unclear. Prior studies have suggested a potential link between accumulated cytoplasm-localized p16 and tumor progression. Here we report that p16 enhances metastasis-associated phenotypes in HCC cells – ectopic p16 expression increased cell migration in vitro, and lung colonization after intravenous injection, whereas knockdown of endogenous p16 reduced cell migration. Interestingly, analysis of p16 mutants indicated that the Cdk4 interaction domain is required for stimulation of HCC cell migration; however, knockdown of Cdk4 and Cdk6 showed that these proteins are dispensable for this phenomenon. Intriguingly, we found that in p16-positive HCC samples, p16 protein is predominantly localized in the cytoplasm. In addition, we identified a potential role for nuclear-cytoplasmic shuttling in p16-stimulated migration, consistent with the predominantly cytoplasmic localization of p16 in IHC-positive HCC samples. Finally, we determined that p16-stimulated cell migration requires the Cdc42 GTPase. Our results demonstrate for the first time a pro-migratory role for p16, and suggest a potential mechanism for the observed association between cytoplasmic p16 and tumor progression in diverse tumor types.     

Anti-Tumor Effects of Metformin in Animal Models of Hepatocellular Carcinoma: A Systematic Review and Meta-Analysis

Background

Several studies have reported that metformin can reduce the risk of hepatocellular carcinoma (HCC) in diabetes patients. However, the direct anti-HCC effects of metformin have hardly been studied in patients, but have been extensively investigated in animal models of HCC. We therefore performed a systematic review and meta-analysis of animal studies evaluating the effects of metformin on HCC.

Methods

We collected the relevant studies by searching EMBASE, Medline (OvidSP), Web of Science, Scopus, PubMed Publisher, and Google Scholar. Studies were included according to the following inclusion criteria: HCC, animal study, and metformin intervention. Study quality was assessed using SYRCLE’s risk of bias tool. A meta-analysis was performed for the outcome measures: tumor growth (tumor volume, weight and size), tumor number and incidence.

Results

The search resulted in 573 references, of which 13 could be included in the review and 12 included in the meta-analysis. The study characteristics of the included studies varied considerably. Two studies used rats, while the others used mice. Only one study used female animals, nine used male, and three studies didn’t mention the gender of animals in their experiments. The quality of the included studies was low to moderate based on the assessment of their risk of bias. The meta-analysis showed that metformin significantly inhibited the growth of HCC tumour (SMD -2.20[-2.96,-1.43]; n=16), but no significant effect on the number of tumors (SMD-1.05[-2.13,0.03]; n=5) or the incidence of HCC was observed (RR 0.62[0.33,1.16]; n=6). To investigate the potential sources of significant heterogeneities found in outcome of tumor growth (I²=81%), subgroup analyses of scales of growth measures and of types of animal models used were performed.

Conclusion

Metformin appears to have a direct anti-HCC effect in animal models. Although the intrinsic limitations of animal studies, this systematic review could provide an important reference for future preclinical animal trials of good quality and clinical development.     

三元基序家族蛋白15敲低诱导肝癌细胞周期阻滞

三元基序家族蛋白15 (tripartite motif-containing protein 15,TRIM15)是TRIM家族成员,该家族是一类具有E3泛素连接酶活性的蛋白质.TRIM15在肿瘤中的功能鲜有报导.本研究意在阐释TRIM15在肝细胞癌(hepatocellular carcinoma,HCC)中的作用...     

MicroRNA-122 Inhibits Tumorigenic Properties of Hepatocellular Carcinoma Cells and Sensitizes These Cells to Sorafenib

MicroRNAs are negative regulators of protein coding genes. The liver-specific microRNA-122 (miR-122) is frequently suppressed in primary hepatocellular carcinomas (HCCs). In situ hybridization demonstrated that miR-122 is abundantly expressed in hepatocytes but barely detectable in primary human HCCs. Ectopic expression of miR-122 in nonexpressing HepG2, Hep3B, and SK-Hep-1 cells reversed their tumorigenic properties such as growth, replication potential, clonogenic survival, anchorage-independent growth, migration, invasion, and tumor formation in nude mice. Further, miR-122-expressing HCC cells retained an epithelial phenotype that correlated with reduced Vimentin expression. ADAM10 (a distintegrin and metalloprotease family 10), serum response factor (SRF), and insulin-like growth factor 1 receptor (Igf1R) that promote tumorigenesis were validated as targets of miR-122 and were repressed by the microRNA. Conversely, depletion of the endogenous miR-122 in Huh-7 cells facilitated their tumorigenic properties with concomitant up-regulation of these targets. Expression of SRF or Igf1R partially reversed tumor suppressor function of miR-122. Further, miR-122 impeded angiogenic properties of endothelial cells in vitro. Notably, ADAM10, SRF, and Igf1R were up-regulated in primary human HCCs compared with the matching liver tissue. Co-labeling studies demonstrated exclusive localization of miR-122 in the benign livers, whereas SRF predominantly expressed in HCC. More importantly, growth and clonogenic survival of miR-122-expressing HCC cells were significantly reduced upon treatment with sorafenib, a multi-kinase inhibitor clinically effective against HCC. Collectively, these results suggest that the loss of multifunctional miR-122 contributes to the malignant phenotype of HCC cells, and miR-122 mimetic alone or in combination with anticancer drugs can be a promising therapeutic regimen against liver cancer.     

一种新型的阳离子型姜黄素纳米粒对肝细胞癌增殖的影响

姜黄药用的主要有效成分是姜黄素,曾被认为是理想的抗癌化学治疗药物之一,然而,姜黄素在水中的溶解度低,体内吸收少,生物利用度低,极大地限制了它的应用。采用乳化聚合的方法,成功地制备了粒径在250nm左右的表面带正电荷的聚氰基丙烯酸正丁酯包载的姜黄素纳米粒,该纳米姜黄素仍然保留了姜黄素本身的生物活性,可抑制人肝癌细胞株HepG2细胞的生长,阻滞细胞周期于G₂/M期,对HepG2细胞有抗增殖作用,能诱导细胞凋亡,下调在肿瘤血管生长中起重要作用的血管内皮生长因子和调控血管内皮生长因子的环氧合酶-2的表达。