The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma

Backgroundc-Met, a high-affinity receptor for Hepatocyte Growth Factor (HGF), plays a critical role in tumor growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with activated HGF/c-Met signaling have a significantly worse prognosis. Targeted therapies using c-Met tyrosine kinase inhibitors are currently in clinical trials for HCC, although receptor tyrosine kinase inhibition in other cancers has demonstrated early success. Unfortunately, therapeutic effect is frequently not durable due to acquired resistance.MethodsWe utilized the human MHCC97-H c-Met positive (c-Met⁺) HCC cell line to explore the compensatory survival mechanisms that are acquired after c-Met inhibition. MHCC97-H cells with stable c-Met knockdown (MHCC97-H c-Met KD cells) were generated using a c-Met shRNA vector with puromycin selection and stably transfected scrambled shRNA as a control. Gene expression profiling was conducted, and protein expression was analyzed to characterize MHCC97-H cells after blockade of the c-Met oncogene. A high-throughput siRNA screen was performed to find putative compensatory survival proteins, which could drive HCC growth in the absence of c-Met. Findings from this screen were validated through subsequent analyses.ResultsWe have previously demonstrated that treatment of MHCC97-H cells with a c-Met inhibitor, PHA665752, results in stasis of tumor growth in vivo. MHCC97-H c-Met KD cells demonstrate slower growth kinetics, similar to c-Met inhibitor treated tumors. Using gene expression profiling and siRNA screening against 873 kinases and phosphatases, we identified ErbB3 and TGF-α as compensatory survival factors that are upregulated after c-Met inhibition. Suppressing these factors in c-Met KD MHCC97-H cells suppresses tumor growth in vitro. In addition, we found that the PI3K/Akt signaling pathway serves as a negative feedback signal responsible for the ErbB3 upregulation after c-Met inhibition. Furthermore, in vitro studies demonstrate that combination therapy with PHA665752 and Gefitinib (an EGFR inhibitor) significantly reduced cell viability and increased apoptosis compared with either PHA665752 or Gefitinib treatment alone.Conclusionc-Met inhibition monotherapy is not sufficient to eliminate c-Met⁺ HCC tumor growth. Inhibition of both c-Met and EGFR oncogenic pathways provides superior suppression of HCC tumor growth. Thus, combination of c-Met and EGFR inhibition may represent a superior therapeutic regimen for c-Met⁺ HCC.     

Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H and MHCC97-L, with different metastasis potentials

To better understand the mechanism underlying hepatocellular carcinoma (HCC) metastasis and to search for potential markers for HCC prognosis, differential proteome analysis on two HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional (2-D) gel electrophoresis followed by matrix-assisted laser desorption/time of flight mass spectrometry and liquid chromatography ion trap mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 56 protein spots showed significant differential expression in MHCC97-H and MHCC97-L cells (Student's t-test, P < 0.05) and 4 protein spots were only detected in MHCC97-H cells. Fourteen protein spots were further identified using in-gel tryptic digestion, peptide mass fingerprinting and tandem mass spectrometry. The expressions of pyruvate kinase M2, ubiquitin carboxy-terminal hydrolase L1, laminin receptor 67 kDa, S100 calcium-binding protein A4, thioredoxin and cytokeratin 19 were elevated in MHCC97-H cells. However, manganese superoxide dismutase, calreticulin precursor, cathepsin D, lactate dehydrogenase B, non-metastatic cell protein 1, cofilin 1 and calumenin precursor were down-regulated in MHCC97-H cells. Intriguingly, most of these identified proteins have been reported to be associated with tumor metastasis. The functional implications of alterations in the levels of these proteins are discussed.     

Bub1基因对人肝癌MHCC97-H细胞增殖、周期和凋亡的影响

目的:研究Bub1基因在肝癌中的表达以及对肝癌细胞系MHCC97-H增殖、周期和凋亡的影响。方法:利用RNA干扰技术下调肝癌细胞系MHCC97-H中Bub1的表达;qRT-PCR和Western Blot分别检测Bub1在mRNA和蛋白水平表达的变化;CCK-8实验检测肿瘤细胞增殖能力的改变;流式细胞术检测细胞周期和凋亡的变化。结果:qRT-PCR和Western Blot结果显示si-Bub1能够成功下调Bub1的表达;下调Bub1后肝癌MHCC97-H细胞的增殖能力下降(P0.05),细胞的凋亡比例升高(P0.05),细胞发生S期阻滞。结论:Bub1基因在肝癌中高表达,下调Bub1的表达后能够降低肝癌细胞的增殖能力,促进细胞凋亡,诱导细胞发生S期阻滞。     

From proteomic analysis to clinical significance: overexpression of cytokeratin 19 correlates with hepatocellular carcinoma metastasis

To better understand the mechanism underlying the hepatocellular carcinoma (HCC) metastasis and to search potential markers for HCC prognosis, differential proteomic analysis on two well-established HCC cell strains with high and low metastatic potentials, MHCC97-H and MHCC97-L, was conducted using two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/time-of-flight mass spectrometry. Cytokeratin 19 (CK19) was identified and found to be overexpressed in MHCC97-H as compared with MHCC97-L. This result was further confirmed by two-dimensional Western blot analysis and immunofluorescence assay. Furthermore, one-dimensional Western blot analysis showed consistently increased CK19 expression in progressively more metastatic cells. Immunohistochemical study on 102 human HCC specimens revealed that more patients in the CK19-positive group had overt intrahepatic metastases (satellite nodules, p < 0.05; vascular tumor emboli, p < 0.001; tumor node metastatis staging, p < 0.001). CK19 fragment CYFRA 21-1 levels measured in sera from nude mice model of human HCC metastasis with radioimmunoassay increased in parallel with tumor progression and rose remarkably when pulmonary metastases occurred. The results demonstrated that overexpression of CK19 in HCC cells is related to metastatic behavior. Serum CK19 level might reflect the pathological progression in some HCC and may be a useful marker for predicting tumor metastasis and a therapeutic target for the treatment of HCC patients with metastases.     

Derepression of c‐Fos caused by MicroRNA‐139 down‐regulation contributes to the metastasis of human hepatocellular carcinoma

This study investigates whether the anti‐metastasis effect of microRNA‐139 (miR‐139) on hepatocellular carcinoma (HCC) is mediated through regulating c‐fos expression. The expression levels of miR‐139 and c‐fos in human HCC cell sublines with high (MHCC97H) and low (MHCC97L) spontaneous metastatic potentials were quantified using QPCR or Western blot. miR‐139 mimics was transfected into MHCC97H cells to overexpress miR‐139, and miR‐139 inhibitor was transfected into MHCC97L cells to down‐express miR‐139. The effect of overexpression or down‐expression of miR‐139 on c‐fos expression of MHCC97H and MHCC97L cells was evaluated using QPCR and Western blot. The 3′ untranslated region segments of FOS containing the miR‐139 binding sites were amplified by PCR, and the luciferase activity in the transfected cells was assayed. In comparison with the expression level of miR‐139 in MHCC97L cells, the expression level in MHCC97H cells was significantly decreased, whereas c‐Fos was significantly up‐regulated in MHCC97H. The overexpression of miR‐139 significantly inhibited the expression of c‐fos in MHCC97H cells, and the down‐expression of miR‐139 significantly promoted the expression of c‐fos in MHCC97L cells. miR‐139 suppressed the luciferase activity of the pGL‐FOS by approximately 40% compared with the negative control. In vitro cell migration analysis demonstrated that depletion of c‐fos or overexpression of miR‐139 in MHCC97H cells reduced cell migration, whereas overexpression of c‐fos or depletion of miR‐139 in MHCC97L cells increased cell migration. Thus, we got the conclusion that miR‐139 expression is down‐regulated in human HCC cell sublines with high spontaneous metastatic potentials (MHCC97H). Derepression of c‐Fos caused by miR‐139 down‐regulation contributes to the metastasis of HCC. Copyright © 2012 John Wiley & Sons, Ltd.     

Cytotoxicity of three naturally occurring flavonoid derived compounds (artocarpesin,cycloartocarpesin and isobavachalcone) towards multi-factorial drug-resistant cancer cells

IntroductionCancer remains an aggressive deadly disease, if drug resistance develops. This problem is aggravated by the fact that multiple rather than single mechanisms are involved in resistance and that multidrug resistance (MDR) phenomena cause inefficacy of many clinical established anticancer drugs. We are seeking for novel cytotoxic phytochemicals to combat drug-resistant tumour cells.MethodsIn the present study, we investigated the cytotoxicity of three naturally occurring flavonoids including two flavones artocarpesin (1) and cycloartocarpesin (2) and one chalcone, isobavachalcone (3) against 9 drug-sensitive and MDR cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analysed via flow cytometry.ResultsFlavones 1 and 2 as well as chalcone 3 displayed cytotoxic effects at various extent on all the 9 tested cancer cell lines with IC₅₀ values respectively below 106 µM, 50 µM and 25 µM. The IC₅₀ values for the three investigational flavonoids ranged from 23.95 µM (towards hepatocarcinoma HepG2 cells) to 105 µM [towards colon carcinoma HCT116 (p53^−/−) cells] for 1, from 15.51 µM (towards leukemia CCRF-CEM cells) to 49.83 µM [towards glioblastoma U87MG.ΔEGFR cells] for 2 and from 2.30 µM (towards CCRF-CEM cells) to 23.80 µM [towards colon carcinoma HCT116 (p53^+/+) cells] for 3 and from 0.20 µM (towards CCRF-CEM cells) to 195.12 µM (towards leukemia CEM/ADR5000 cells) for doxorubicin. Compounds 2 and 3 induced apoptosis in CCRF-CEM leukemia cells, mediated by caspase activation and the disruption of MMP.ConclusionsThe three tested flavonoids and mostly chalcone 3 are potential cytotoxic natural products that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug-resistant cancers.     

A garlic derivative, S-allylcysteine (SAC), suppresses proliferation and metastasis of hepatocellular carcinoma

Background

Hepatocellular carcinoma (HCC) is highly malignant and metastatic. Currently, there is no effective chemotherapy for patients with advanced HCC leading to an urgent need to seek for novel therapeutic options. We aimed to investigate the effect of a garlic derivative, S-allylcysteine (SAC), on the proliferation and metastasis of HCC.

Methodology/Principal Findings

A series of in vitro experiments including MTT, colony-forming, wound-healing, invasion, apoptosis and cell cycle assays were performed to examine the anti-proliferative and anti-metastatic effects of SAC on a metastatic HCC cell line MHCC97L. The therapeutic values of SAC single and combined with cisplatin treatments were examined in an in vivo orthotopic xenograft liver tumor model. The result showed that the proliferation rate and colony-forming abilities of MHCC97L cells were suppressed by SAC together with significant suppression of the expressions of proliferation markers, Ki-67 and proliferating cell nuclear antigen (PCNA). Moreover, SAC hindered the migration and invasion of MHCC97L cells corresponding with up-regulation of E-cadherin and down-regulation of VEGF. Furthermore, SAC significantly induced apoptosis and necrosis of MHCC97L cells through suppressing Bcl-xL and Bcl-2 as well as activating caspase-3 and caspase-9. In addition, SAC could significantly induce the S phase arrest of MHCC97L cells together with down-regulation of cdc25c, cdc2 and cyclin B1. In vivo xenograft liver tumor model demonstrated that SAC single or combined with cisplatin treatment inhibited the progression and metastasis of HCC tumor.

Conclusions/Significance

Our data demonstrate the anti-proliferative and anti-metastatic effects of SAC on HCC cells and suggest that SAC may be a potential therapeutic agent for the treatment of HCC patients.     

PARP-1抑制剂3-AB对肝癌细胞增殖及凋亡的影响*

目的:探讨PARP-1抑制剂3-AB对肝癌细胞系MHCC97-H和SMMC7721及正常肝细胞系L02的增殖与凋亡的影响。方法:细胞增殖试验观察不同浓度3-AB对三种不同细胞系细胞的增殖作用。Annexin V荧光探针标记,流式细胞学检查观察不同浓度3-AB对不同细胞系细胞凋亡的影响。结果:当3-AB浓度分别为5 mM、10 mM与20 mM时,与对照组(0 mM)相比,在培养第6天时开始出现增殖明显减慢,出现统计学差异(p0.05),第九天差异明显(p0.05)。随着浓度增加,其对肿瘤细胞系MHCC97-H和SMMC7721细胞增殖的抑制程度增加,细胞数均逐渐减少;而同样浓度梯度3-AB对人类肝细胞系L02生长则无明显的抑制作用。进一步实验发现,当3-AB浓度为5mM、10 mM与20 mM时,均可诱导肝癌细胞株MHCC97-H和SMMC7721凋亡,与对照组(0 mM)比较均有统计学差异(p0.05),且细胞凋亡率与3-AB的药物浓度相关:浓度越高,凋亡越明显。而同等浓度3-AB对肝脏细胞系L02无明显的促进凋亡作用。结论:3-AB可以抑制肝癌肿瘤细胞的增殖,促进肿瘤细胞的凋亡,对正常肝脏细胞无明显毒害作用,具有治疗肝癌的的潜在应用价值。     

miR-221在肝癌干细胞亚群中的差异表达

目的：分离肝癌细胞系MHCC97中肝癌干细胞并分析肝癌细胞高表达miR-221在肝癌干细胞和非干细胞亚群中的表达差异情况,探讨miR-221表达水平与肝癌干细胞分化之间的关系。方法：利用流式细胞荧光激活分选法从肝癌细胞系MHCC97中分选出肝癌干细胞（hepatocareinoma stem cells,HSCs）和非干细胞（non-hepatocareinoma stem cells,non-HSCs）两个亚群。采用实时荧光定量RT-PCR（Real-time RT-PCR）检测miR-221在两个不同肝癌细胞亚群中的表达。结果：HSC亚群肝癌细胞仅占细胞总体的2.59%;HSC亚群细胞中miR-221的表达明显高于non-HSC亚群（P〈0.01）。结论：miR-221在HSC亚群肝癌细胞中的明显高表达,提示miR-221可能在维持HSC亚群肝癌细胞的干细胞特性方面具有重要意义。通过调控肝癌干细胞中miR-221的表达,可以促进其分化成熟,从而为肝癌治疗提供新的思路。     

Hepatoma‐derived growth factor promotes growth and metastasis of hepatocellular carcinoma cells

We aimed to elucidate the effects of hepatoma‐derived growth factor (HDGF) on growth and metastasis of hepatocellular carcinoma (HCC) cells. Tissue microarrays with 236 HCC specimens and 18 extrahepatic metastases were utilized to detect the HDGF expression by immunohistochemistry. Meanwhile, HDGF expressions in HCC cell lines with different metastatic potentials were examined using immunofluorescence staining, real‐time PCR and western blotting. After HDGF silencing, the growth and metastatic potentials of HCC cells were evaluated by soft agar assay, invasion assay, together with tumorigenicity assay in nude mice. The gelatin zymography was performed by detecting MMP‐2 and MMP‐9 levels. Additionally, western blotting was conducted to determine the levels of total and phosphorylated ERK1/2, JNK, p38 and Akt. The results showed that HDGF was overexpressed in HCC metastasis tumour, and the expression increased with the differentiation degree of tumours (Grade I 44.0%, Grade II 48.4% and Grade III 65.6%). Consistently, HDGF levels were positively associated with the metastatic capability of HCC cells (MHCC97L < MHCC97H < HCCLM3). The growth and metastasis were suppressed by HDGF‐siRNA. Gelatinolytic activities were enhanced in the three metastatic HCC cell lines, but had no significant difference among them. The tumourigenicity and metastatic capability of HCCLM3 cells in nude mice were inhibited after silencing HDGF. Meanwhile, HDGF‐siRNA specifically suppressed the total and phosphorylated protein levels of ERK1/2, while not JNK, p38 and Akt. In conclusion, HDGF was overexpressed in HCC patients and cells, and HDGF might be closely correlated with HCC metastasis via regulating ERK signalling pathway. Copyright © 2016 John Wiley & Sons, Ltd.     

过表达微小RNA miR-125b抑制肝癌细胞上皮-间质转换及促进细胞凋亡

微小RNA-125b（miR-125b）在许多恶性肿瘤的增殖、分化和凋亡等过程中具有很重要的作用,但miR-125b是否涉及肝癌的上皮 间质转换过程（EMT）还有待进一步研究。本研究通过构建过表达miR-125b的肝癌稳转细胞株,初步检测miR-125b对于肝癌的EMT过程和相关的TGF-β信号通路的影响,以及对于肝癌细胞凋亡的影响。以慢病毒载体pHRS-1cla EGFP 构建过表达miR-125b的载体质粒(pHRS-1cla-miR125b-CMV-EGFP),并对上述载体进行NheⅠ、XbaⅠ双酶切和测序鉴定,鉴定正确后,在293T细胞中进行慢病毒包装,浓缩病毒后,对MHCC97-H进行慢病毒感染并采用流式分选GFP阳性的细胞。实时定量PCR检测表明肝癌细胞稳转株MHCC97-H-PHRS-miR-125b-EGFP的miR-125b表达量是空载体转染组的6倍。Western印迹检测发现,与空载体对照组相比,MHCC97-H-PHRS-miR-125b-EGFP细胞中间质细胞标志α-SMA表达显著下调,上皮细胞标志E-cadherin表达显著上调,同样的,用Western印迹检测也发现MHCC97-H-PHRS-miR-125b-EGFP细胞中TGF-β信号通路关键下游分子Smad2和Smad4的表达显著下调,细胞凋亡检测结果表明,与对照组相比,过表达miR-125b的稳转株凋亡率增加到19.66%,加入TGF-β1后,过表达miR-125b的稳转株凋亡率进一步增加到74.7%。同样的,在体内治疗实验中,我们采用商品化的体内核酸转染试剂,在皮下肿瘤组织中过表达miR-125b mimics,结果表明miR-125b的过表达与肿瘤组织的凋亡成正相关性（r=0.83463,P < 0.01）,且免疫组化结果也表明,miR-125b过表达后,E-cadherin表达显著上调,α-SMA及Smad2和Smad4的表达显著下调。上述结果表明,我们成功构建了过表达miR-125b的肝癌细胞稳转株,并成功建立了肿瘤组织中过表达miR-125b mimics的动物模型,在体内外均观察到过表达miR-125b后对肝癌细胞EMT过程的抑制作用和对细胞凋亡的促进作用。相关研究结果加深了我们对miR-125b在肝癌中抑制肝癌发展作用机制的理解,及其作为潜在的治疗肝癌的新靶点的重要性。     

Cytotoxicity of a naturally occurring furoquinoline alkaloid and four acridone alkaloids towards multi-factorial drug-resistant cancer cells

IntroductionChemotherapy is one of the preferred mode of treatment of malignancies, but is complicated by the expression of diverse resistance mechanisms of cancer cells.MethodsIn the present study, we investigated the cytotoxicity of five alkaloids including a furoquinoline montrofoline (1) and four acridones namely 1-hydroxy-4-methoxy-10-methylacridone (2), norevoxanthine (3), evoxanthine (4), 1,3-dimethoxy-10-methylacridone (5) against 9 drug-sensitive and multidrug-resistant (MDR) cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analyzed via flow cytometry.ResultsFuroquinoline 1 as well as the acridone alkaloids 2–5 displayed cytotoxic effects with IC₅₀ values below 138 µM on all the 9 tested cancer cell lines. The IC₅₀ values ranged from 41.56 µM (towards hepatocarinoma HepG2 cells) to 90.66 µM [towards colon carcinoma HCT116 (p53^−/−) cells] for 1, from 6.78 µM [towards HCT116 (p53^−/−) cells) to 106.47 µM [towards breast adenocarcinoma MDA-MB-231-pcDNA cells] for 2, from 5.72 µM (towards gliobastoma U87MG.ΔEGFR cells) to 137.62 µM (towards leukemia CCRF-CEM cells] for 3, from 6.11 µM [towards HCT116 (p53^+/+) cells] to 80.99 µM (towards HepG2 cells] for 4, from 3.38 µM (towards MDA-MB-231-BCRP cells) to 58.10 µM (towards leukemia CEM/ADR5000 cells] for 5 and from 0.20 µM (against CCRF-CEM cells) to 195.12 µM (against CEM/ADR5000 cells) for doxorubicin. Acridone alkaloid 5 induced apoptosis in CCRF-CEM leukemia cells, mediated by increased ROS production.ConclusionsThe five tested alkaloids and mostly acridone 5 are potential cytotoxic natural products that deserve more investigations to develop novel cytotoxic compounds against multifactorial drug-resistant cancers.     

Trans-chalcone induces death by autophagy mediated by p53 up-regulation and β-catenin down-regulation on human hepatocellular carcinoma HuH7.5 cell line

BackgroundHepatocellular Carcinoma (HCC) is extremely aggressive and presents low rates of response to the available chemotherapeutic agents. Many studies have focused on the search for alternative low-cost natural compounds with antiproliferative potential that selectively respond to liver cancer cells.PurposeThis study assessed the in vitro direct action of trans-chalcone (TC) on cells of the human HCC HuH7.5 cell line.MethodsWe subjected the HuH7.5 tumor cells to TC treatment at increasing concentrations (12.5–100 µM) for 24 and 48 h. Cell viability was verified through MTT and 50% inhibitory concentration of cells (IC₅₀ 23.66 µM) was determined within 48 h. We quantified trypan blue proliferation and light microscopy, ROS production, mitochondrial depolarization and autophagy, cell cycle analysis, and apoptosis using Muse® cell analyzer and immunocytochemical markings of p53 and β-catenin.ResultsData showed an effective dose- and time-dependent TC-cytotoxic action at low micromolar concentrations without causing toxicity to non-cancerous cells, such as erythrocytes. TC-treatment caused mitochondrial membrane damage and cell cycle G0/G1 phase arrest, increasing the presence of the p53 protein and decreasing β-catenin, in addition, to inducing cell death by autophagy. Additionally, TC decreased the metastatic capacity of HuH7.5, which affected the migration/invasion of this type of cell.ConclusionIn vitro TC activity in the human HCC HuH7.5 tumor cell line is shown to be a potential molecule to develop new therapies to repair the p53 pathway and prevent the overexpression of Wnt/β-catenin tumor development inducing autophagy cell death and decreasing metastatic capacity of HuH7.5 cell line.     

Cytotoxicity of compounds from Xylopia aethiopica towards multi-factorial drug-resistant cancer cells

IntroductionMultidrug resistance (MDR) in cancer represent a major hurdle in chemotherapy. Previously, the methanol extract of the medicinal spice Xylopia aethiopica displayed considerable cytotoxicity against multidrug resistant (MDR) cancer cell lines.MethodsThe present study was designed to assess the cytotoxicity of compounds, 16α-hydroxy-ent-kauran-19-oic acid (2), 3,4′,5-trihydroxy-6″,6″-dimethylpyrano[2,3-g]flavone (3), isotetrandrine (5) and trans-tiliroside (6) derived from the methanol crude extract of Xylopia aethiopica against 9 drug-sensitive and -resistant cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analyzed via flow cytometry.ResultsFlavonoid 3 and alkaloid 5 also displayed IC₅₀ values ranging from 2.61 µM (towards leukemia CCRF-CEM cells) to 18.60 µM (towards gliobastoma multiforme U87MG.ΔEGFR cells) and from 1.45 µM (towards HepG2 cells) to 7.28 µM (towards MDA-MB-231-pcDNA cells), respectively. IC₅₀ values ranged from 0.20 µM (against CCRF-CEM cells) to 195.12 µM (against CEM/ADR5000 cells) for doxorubicin. Compound 3 induced apoptosis in leukemia CCRF-CEM cells mediated by the disruption of the MMP, whilst 5 induced apoptosis mediated by ROS production.ConclusionsCompounds 2 and 5 represent potential cytotoxic phytochemicals that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug-resistant cancers.     

8,8-bis-(Dihydroconiferyl)-diferulate displayed impressive cytotoxicity towards a panel of human and animal cancer cells

BackgroundRecalcitrant cancers appear as a major obstacle to chemotherapy, prompting scientists to intensify the search for novel drugs to tackle the cell lines expressing multi-drug resistant (MDR) phenotypes.PurposeThe purpose of this study was to evaluate the antiproliferative potential of a ferrulic acid derivative, 8,8-bis-(dihydroconiferyl)-diferulate (DHCF2) on a panel of 18 cancer cell lines, including various sensitive and drug-resistant phenotypes, belonging to human and animals. The mode of induction of cell death by this compound was further studied.MethodsThe antiproliferative activity, autophagy, ferroptotic and necroptotic cell death were evaluated by the resazurin reduction assay (RRA). CCRF-CEM leukemia cells were used for all mechanistic studies. A caspase-Glo assay was applied to evaluate the activity of caspases. Cell cycle analysis (PI staining), apoptosis (annexin V/PI staining), mitochondrial membrane potential (MMP) (JC-1) and reactive oxygen species (ROS) (H₂DCFH-DA) were assessed by flow cytometry.ResultsDHCF2 demonstrated impressive cytotoxic effects towards the 18 cancer cell lines tested, with IC₅₀ values all below 6.5 µM. The obtained IC₅₀ values were in the range of 1.17 µM (towards CCRF-CEM leukemia cells) to 6.34 µM (towards drug-resistant HCT116 p53^−/− human colon adenocarcinoma cells) for DHCF2 and from 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against multidrug-resistant CEM/ADR5000 leukemia cells) for the reference drug, doxorubicin. DHCF2 had IC₅₀ values lower than those of doxorubicin, against CEM/ADR5000 cells and on some melanoma cell lines, such as MaMel-80a cells, Mel-2a cells, MV3 cells and SKMel-505 cells. DHCF2 induced autophagy as well as apoptosis in CCRF-CEM cells though caspases activation, MMP alteration and increase of ROS production.ConclusionThe studied diferulic acid, DHCF2, is a promising antiproliferative compound. It deserves further indepth investigations with the ultimate aim to develop a novel drug to fight cancer drug resistance.     

Notch信号通路对肝癌细胞迁移能力及E-cadherin，COX-2表达的影响

目的:探讨Notch信号通路对肝癌细胞迁移能力及钙粘附蛋白E(E-cadherin)、环氧化酶-2(COX-2)表达的影响。方法:体外培养肝癌细胞系(SMMC-7721、MHCC97H)、正常非肿瘤肝细胞系(HL-7702),Transwell小室用于测定细胞的迁移侵袭能力,Western blot蛋白印迹法用于测定Notch1、E-cadherin、COX-2蛋白的表达水平,并采用DAPT阻断Notch信号通路,比较肝癌细胞系与正常非肿瘤肝细胞系的迁移侵袭能力及肝癌细胞中E-cadherin、COX-2蛋白的表达水平的改变。结果:SMMC-7721细胞、MHCC97H细胞的迁移能力强于HL-7702细胞,差异有统计学意义(P0.05);相比于HL-7702细胞,MHCC97H细胞、SMMC-7721细胞中的Notch1、COX-2表达水平均显著升高,E-cadherin的表达水平明显降低(P0.05);DAPT处理后,SMMC-7721细胞、MHCC97H细胞发生迁移的能力均弱于对照组,差异有统计意义(P0.05);DAPT处理后,SMMC-7721细胞、MHCC97H细胞内COX-2、Notch1的表达量明显降低,而E-cadherin的表达水平升高(P0.05)。结论:Notch信号通路参与肝癌细胞迁移过程,其机制可能与E-cadherin、COX-2的表达相关。     

Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels

Background  

The expression level of cyclin D1 plays a vital role in the control of proliferation. This protein is reported to be degraded following phosphorylation by glycogen synthase kinase 3 (GSK3) on Thr-286. We recently showed that phosphorylation of Thr-286 is responsible for a decline in cyclin D1 levels during S phase, an event required for efficient DNA synthesis. These studies were undertaken to test the possibility that phosphorylation by GSK3 is responsible for the S phase specific decline in cyclin D1 levels, and that this event is regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway which controls GSK3.     

Pristimerin synergistically sensitizes conditionally reprogrammed patient derived-primary hepatocellular carcinoma cells to sorafenib through endoplasmic reticulum stress and ROS generation by modulating Akt/FoxO1/p27kip1 signaling pathway

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. Sorafenib (SORA), as a first-line therapeutic drug, has been used to treat HCC, but resistance poses a major limitation on the efficacy of SORA chemotherapy. Pristimerin (PRIS), a natural bioactive component isolated from various plant species in the Celastraceae and Hippocrateaceae families, has been reported to exhibit outstanding antitumor effects in several types of cells in vitro.PurposeThe aim of this study was to investigate whether PRIS can exert synergistic anti-tumor effects with the combination of SORA, and if so, through what mechanism.MethodsConditionally reprogrammed patient derived-primary hepatocellular carcinoma cells (CRHCs) were isolated from human liver cancer tissues and treated with SORA and PRIS. Cell proliferation, apoptosis, migration and tube formation ability were detected by DNA content quantification, flow cytometry, transwell assay and Matrigel-based angiogenesis assay. Gene and protein expression were assessed by qRT-PCR and Western blot respectively.ResultsInitially, we observed that the combination of the two drugs had a much stronger inhibitory effect on CRHCs growth than either drug alone. Moreover, the combination of 2 µM SORA and 1 µM PRIS exhibited a significant anti‑migrative and anti-invaded effect on CRHCs, and remarkably inhibited capillary structure formation of Human Umbilical Vein Endothelial Cells (HUVECs). Furthermore, the combined treatment with SORA and PRIS synergistically induced intrinsic apoptosis in CRHCs, involving a caspase-4‐dependent mechanism paralleled by an increased Bax/Bcl‐xL ratio. These activities were mediated through ROS generation and the induction of endoplasmic reticulum (ER) stress and mitochondrial dysfunction. GRP78 silencing or ER stress inhibitor 4-phenylbutyric acid administration was revealed to abolish the anticancer effects of PRIS, indicating the critical role of GRP78 in mediating the bioactivity of PRIS. The present study also provides mechanistic evidence that PRIS modulated the Akt/FoxO1/p27^kip1 signaling pathway, which is required for mitochondrial-mediated intrinsic apoptosis, activation of ER stress, and stimulation of caspase-4 induced by PRIS, and, consequently resulting in suppressed cell viability, migration and angiogenesis co-treated with SORA in CRHCs.ConclusionOur results suggest the use of PRIS as sensitizers of chemotherapy paving the way for innovative and promising targeted chemotherapy-based therapeutic strategies in human HCC.     

Polycomb chromobox 4 enhances migration and pulmonary metastasis of hepatocellular carcinoma cell line MHCC97L

We recently report that the expression of polycomb chromobox 4(Cbx4)is significantly correlated with the overall survival of a great cohort of hepatocellular carcinoma(HCC)patients and it enhances hypoxia-induced vascular endothelial growth factor(VEGF)expression and angiogenesis in HCC cells through enhancing sumoylation of hypoxia inducible factor-1alpha(HIF-1α).Here we continue to investigate the potential effects of Cbx4 on the migration and metastasis of the metastatic HCC cell line MHCC97L.Our results show that Cbx4 overexpression in the cell line increases the in vitro vessel formation of vascular endothelial cells in its SUMO interaction motifs-dependent manner,and promotes the in vitro migration of the cancer cell,which can be effectively abrogated by anti-VEGF antibody.Although Cbx4 expression does not impact the in vitro growth of MHCC97L cells,it still promotes the progression and metastasis of orthotopically transplanted tumors in nude mice.These results further support the role of Cbx4 as a SUMO E3 ligase in the progression and metastasis of HCC.