Normal Hepatocellular Cells Research Articles

Single-cell fucosylation breakdown: Switching fucose to europium

SummaryFucosylation and its fucosidic linkage-specific motifs are believed to be essential to understand their distinct roles in cellular behavior, but their quantitative information has not yet been fully disclosed due to the requirements of ultra-sensitivity and selectivity. Herein, we report an approach that converts fucose (Fuc) to stable europium (Eu) isotopic mass signal on hard ionization inductively coupled plasma mass spectrometry (ICP-MS). Metabolically assembled azido-fucose on the cell surface allows us to tag them with an alkyne-customized Eu-crafted bacteriophage MS2 capsid nanoparticle for Eu signal multiplication, resulting in an ever lowest detection limit of 4.2 zmol Fuc. Quantitative breakdown of the linkage-specific fucosylation motifs in situ preserved on single cancerous HepG2 and paracancerous HL7702 cells can thus be realized on a single-cell ICP-MS platform, specifying their roles during the cancering process. This approach was further applied to the discrimination of normal hepatocellular cells and highly, moderately, and poorly differentiated hepatoma cells collected from real hepatocellular carcinoma tissues.

MicroRNA-383 inhibits proliferation, migration, and invasion in hepatocellular carcinoma cells by targeting PHF8.

BackgroundTo study the effect of microRNA‐383 (miR‐383) on cell proliferation, migration, and invasion of hepatocellular carcinoma (HCC) cells, and explore its mechanism.MethodsThe expressions of miR‐383 and plant homology domain that refers to protein 8 (PHF8) were detected in tissues and cells by quantitative real‐time polymerase chain reaction (qRT‐PCR) or western blot respectively. The miR‐383 group (transfected miR‐383 mimics), miR‐con group (transfected miR‐con), si‐con group (transfected si‐con), si‐PHF8 group (transfected si‐PHF8), miR‐383 + ctrl group (cotransfected miR‐383 mimics and pcDNA‐3.1), miR‐383 + PHF8 group (cotransfected miR‐383 mimics and pcDNA‐3.1‐PHF8) were transfected into HepG2 cells by liposome method. Cell proliferation, migration and invasion were measured by 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide (MTT) or trans‐well assays respectively. The luciferase activity of each group was detected by dual luciferase reporter gene assay.ResultsCompared with normal adjacent tissues, the expression of miR‐383 was significantly down‐regulated and the expression of PHF8 was significantly up‐regulated (p < .05). Compared with normal hepatocellular cell LO2, the expression of miR‐383 was significantly reduced (p < .05) in HCC cells. Moreover, overexpression of miR‐383 or silencing of PHF8 significantly inhibited the proliferation, migration, and invasion of HCC cells. In addition, PHF8 was targeted by miR‐383 and its restoration rescued the inhibitory effect of miR‐383 on cell proliferation, migration, and invasion of HCC cells.ConclusionmiR‐383 could inhibit the proliferation, migration, and invasion of HCC cells by targeting PHF8, which will provide a basis for miR‐383 targeted therapy for HCC.

DNAM1 and 2B4 Costimulatory Domains Enhance the Cytotoxicity of Anti-GPC3 Chimeric Antigen Receptor-Modified Natural Killer Cells Against Hepatocellular Cancer Cells in vitro.

PurposeHepatocellular cancer (HCC) is the sixth most prevalent cancer and the third leading cause of cancer-related death worldwide. Cellular immunotherapy against glypican 3 (GPC3) has recently been used in the treatment of HCC, following the success of chimeric antigen receptor (CAR)-T therapy in treatment of B cell malignancy. However, CAR-T cells are not “off-the-shelf” and always cause cytokine release syndrome, which can be eliminated by using natural killer (NK) cells as effector cells. Since a costimulatory signal is necessary for the activation, persistence, or cytotoxicity of CAR-T cells, we speculated that the costimulatory signal is also required for CAR-NK cells in HCC treatment.MethodsFive anti-GPC3 CAR plasmids containing different costimulatory domains were constructed. They included Z (only the CD3ζ domain, no costimulatory domain), CD28.Z (T-cell costimulatory domain CD28), DNAM1/2B4.Z (NK-cell-associated costimulatory domain DNAM1 or 2B4), and DNAM1.2B4.Z (both NK-cell-associated costimulatory domains). Respective CAR-NK-92 cells were generated. The MTT viability assay was performed to evaluate the effect of the different costimulatory domains on CAR-NK-cell proliferation. The effect on persistence was analyzed using an apoptosis assay and flow cytometry. Special cytotoxicity against normal hepatocellular cells and GPC3+ malignant cells was investigated in vitro. The concentration of cytokines (TNF-α and IFN-γ) released by CAR-NK-92 cells was also measured by ELISA.ResultsNK-cell-associated costimulatory signal was necessary for CAR-NK-92 cells. CAR-NK-92 cells with DNAM1 and/or 2B4 expanded more quickly and persisted with a lower apoptotic ratio, compared to the presence of CD28 or no costimulatory signal. All CAR-NK-92 cells showed special cellular cytotoxicity in vitro. CAR-NK-92 cells with NK-cell-associated costimulatory domains exhibited higher cytotoxic ability compared with those without any costimulatory domain or with T-cell costimulatory domain. CAR-NK-92 cells with both DNAM1 and 2B4 displayed the highest cytotoxicity. The cytokine release assay results were consistent with those of the cytotoxicity assay.ConclusionWe provided the first evidence supporting a strategy using DNAM1 and 2B4 costimulatory domains to generate anti-GPC3 CAR-NK-92 cells, which exhibits enhanced cytotoxicity against hepatocellular cancer cells in vitro.

MEK inhibition by cobimetinib suppresses hepatocellular carcinoma and angiogenesis in vitro and in vivo

Hepatocellular carcinoma which is featured with the extensive vascularization is the third most frequent cause of cancer-related deaths with limited therapeutic options, particularly for advanced disease. Cobimetinib, a MEK inhibitor, has been approved for the treatment of melanomas with a BRAF mutation. In this work, we investigated the efficacy of cobimetinib in sensitive and resistant HCC cells. Using a panel of HCC cell lines and normal hepatocellular cells as control, we showed that cobimetinib is active against HCC cells and spare normal hepatocellular cells. Cobimetinib at nanomolar concentration inhibited proliferation and induced apoptosis in sorafenib-resistant HCC cells (Hep3B-r), suggesting its ability to overcome HCC resistance to standard of care. This was further demonstrated by our results that cobimetinib significantly augmented the inhibitory effects of sorafenib and doxorubicin in HCC cells. Notably, cobimetinib dose-dependently inhibited tumor angiogenesis by inhibiting HCC endothelial cell (HCCEC) growth, survival and capillary network work formation. Cobimetinib suppressed ERK/RSK without affecting JNK or p38 signaling pathways in Hep3B-r and HCCEC cells. In addition, cobimetinib negatively influenced the apoptosis pathways by increasing pro-apoptotic protein Bim and decreasing anti-apoptotic proteins Mcl-1 and Bcl-2. In addition, we validated the in vitro findings in HCC xenograft mouse model and demonstrated that cobimetinib inhibited ERK signaling, promoted apoptosis, and was active against resistant HCC growth and angiogenesis in vivo, without causing significant toxicity in mice. Our findings support the clinical trials of cobimetinib for HCC treatment and highlight the therapeutic value of inhibiting MEK/ERK/RSK to overcome HCC resistance.

Hepatocellular carcinoma cell-derived exosomes regulates Treg cells via cyclic adenosine monophosphate response element binding protein/forkhead/winged helix transcription factor P3 pathway

Objective To investigate whether hepatocellular carcinoma cell line HepG2 derived exosomes regulates Treg cells via cyclic adenosine monophosphate response element binding protein/forkhead/winged helix transcription factor P3 (CREB/Foxp3) pathway. Methods Ultracentrifugation was used to isolate exosomes of hepatocellular carcinoma cell line HepG2 and normal hepatocellular cell line L02. Transmission electron microscopy was used to determine the morphology of exosomes and Western blotting was used to detect the protein markers of exosomes and CREB/Foxp3 expression. HepG2-exo and L02-exo were co-cultured with peripheral blood mononuclear cells. The percent of Treg cells were determined by flow cytometry. Results Exosomes was about 30-100 nm with a circular or oval shape vesicular structure. CD81 and CD63 were highly expressed in exosomal proteins, while not expressed in cell proteins derived from HepG2 or L02 cells. Compared to L02-exo [(1.87±0.18)%] and blank control group [(1.43±0.12)%], HepG2-exo strongly increased the percent of Treg cells [(6.87±0.87)%], with P value of 0.005 and 0.004 respectively. Phosphorylation of CREB in HepG2-exo group was 1.74 and 1.63 fold higher than it in L02-exo and blank control group, and both of the P=0.001. Phosphorylation of Foxp3 in HepG2-exo group was 1.93 and 1.78 fold higher than it in L02-exo and blank control group, and both of the P=0.001. Conclusion These results provided evidence that hepatocellular carcinoma cell HepG2 derived exosomes could regulates Treg cells via CREB/Foxp3 pathway. Key words: Exsomes; Treg cell; Cyclic adenosine monophosphate response element binding protein; Forkhead/winged helix transcription factor P3; Hepatocellular carcinoma

Glycoproteomic Analysis of Human Hepatoblastoma Cell Lines Using Glycopeptide Capture and Mass Spectrometry

Hepatoblastoma (HB) is the most common form of liver tumour in infants and children. The cancer cell line HepG2 and normal hepatocellular cell line L02 are valuable cell models and are already widely used in the world while their glycopeoteome profiles are still unknown. This study focuses on N-linked glycoproteomic analysis of these two cell lines using mass spectrometry. Using two complementary approaches, Hydrazide reaction and hydrophilic affinity solid phase extraction methods, almost 400 glycosylation sites were identified from the two cell lines. Functional annotation suggests that N-glycoproteins with molecular binding ability were enriched in HepG2 cells compared to normal liver cells.

MicroRNA-590-5p regulates proliferation and invasion in human hepatocellular carcinoma cells by targeting TGF-β RII.

MicroRNAs (miRNAs) are regulatory small non-coding RNAs that can regulate gene expression by binding to gene elements, such as the gene promotor 5'UTR, mainly in the 3'UTR of mRNA. One miRNA targets many mRNAs, which can be regulated by many miRNAs, leading to a complex metabolic network. In our study, we found that the expression level of miR-590-5p is higher in the human hepatocellular carcinoma cell line HepG2 than in the normal hepatocellular cell line L02. Downregulation of miR-590-5p inhibited proliferation and invasion of hepatocellular carcinoma cells (HCCs). We also showed that expression of TGF-beta RII, which has been regarded as a regulator of tumor proliferation, invasion, and migration in hepatocellular carcinoma, is regulated by miRNA-590-5p. In addition, miR-590-5p downregulated the expression of TGF-beta RII by targeting the 3'UTR of mRNA. We also found that downregulation of miR-590-5p was associated with an elevation of TGF-beta RII and inhibition of proliferation and invasion in HepG2 cells. Furthermore, overexpression of miR-590-5p was associated with upregulation of TGF-beta RII and could promote proliferation and invasion in L02 cells. In conclusion, we determined that TGF-beta RII is a novel target of miRNA-590-5p. Thus, the role of TGF-beta RII in regulating proliferation and invasion of human HCCs is controlled by miR-590-5p. In other words, miR-590-5p promotes proliferation and invasion in human HCCs by directly targeting TGF-beta RII.

Enhancement of cytotoxic and pro-apoptotic effects of 2-aminophenoxazine-3-one on the rat hepatocellular carcinoma cell line dRLh-84, the human hepatocellular carcinoma cell line HepG2, and the rat normal hepatocellular cell line RLN-10 in combination with 2-deoxy-D-glucose

The cytotoxic and pro-apoptotic effects of a single dose of 2-aminophenoxazine-3-one (Phx-3) or 2-deoxyglucose (2-DG) or of a combined dose of Phx-3 and 2-DG were studied in the rat hepatocellular carcinoma cell line dRLh-84, the human hepatocellular carcinoma cell line HepG2 and the rat normal hepatocellular cell line RLN-10. The number of viable cells decreased in a dose-dependent manner, when dRLh-84, HepG2 or RLN-10 cells were treated with 2-DG (0.5-20mM) or Phx-3 (1-50µM) alone at 37˚C for 48h. When these cells were treated with 10mM 2-DG and different concentrations of Phx-3, the number of viable cells decreased dose-dependently and in an additive manner for these agents. A single dose of 2 or 10µM Phx-3 induced apoptotic morphology characterized by nuclear condensation and cell shrinkage in dRLh-84, HepG2 and RLN-10 cells, while a single dose of 10mM 2-DG did not. When Phx-3 (2 or 10µM) treatment was combined with 2-DG (10mM) treatment in these three cell lines, the cells with apoptotic morphology increased extensively, which was confirmed by flow cytometric analysis. In addition, autophagic morphology characterized by cytosolic vacuole formation was significantly increased in the hepatocellular carcinoma cell lines dRLh-84 and HepG2 but not in the normal hepatocellular cell line RLN-10 after a single dose of Phx-3 or 2-DG or a combined dose of Phx-3 and 2-DG. Furthermore, when dRLh-84 and HepG2 cells were treated with Phx-3 alone or a combined dose of Phx-3 and 2-DG, depolarization of the mitochondria was extensive, but that of the normal cell line RLN-10 was not. These results may imply that the mechanism for the apoptosis of hepatocellular carcinoma cells caused by Phx-3 alone or a combined dose of Phx-3 and 2-DG differs from that of the normal cell line RLN-10. The present results demonstrate that Phx-3 alone may be beneficial for targeting liver cancer and that its anticancer activity may be enhanced by 2-DG. However, a combined dose of Phx-3 and 2-DG may exert adverse effects on normal liver cells, as evidenced by the cytotoxic and pro-apoptotic effects of the combined treatment in the rat normal hepatocellular cell line RLN-10.

Synthesis and Reversal Activity of Multidrug Resistance of Hepatocellular Carcinoma of (−)-Epigallocatechin Gallate Analogues

Aim To investigate the reversal activity and structure-activity relationship of (−)-epigallocatechin gallate (EGCG) analogues on the multidrug-resistant tumor cells. Methods (−)-Epigallocatechin gallate analogues were synthesized and confirmed by 1H, 13C NMR and 2DNMR data. MTT method was used to evaluate their cytotoxicities against human normal hepatocellular cells LO2, hepatic carcinoma cells BEL-7402, and the hepatic carcinoma 5-FU-resiatant cells BEL-7402/5-FU. Results 5, 7, 3′, 4′, 5′, 3″, 4″, 5″-octa-acetyl-EGCG (AcEGCG), 5, 7, 3′, 4′, 3″, 4″, 5″-hepta-acetyl-ECG (AcECG), 7, 3′, 4′, 5′, 3″, 4″, 5″-hepta-acetyl-EGCG (Ac-A), and 3′, 4′, 5′, 3″, 4″, 5″-hexa-acetyl-EGCG (Ac-B) were synthesized. Compounds Ac-A and Ac-B were reported for the first time. IC 50 of AcECG, Ac-A, and Ac-B against the BEL-7402 and BEL-7402/5-FU cells appeared to be lower than EGCG, whereas that of AcEGCG appeared to be lower than EGCG against the BEL-7402 cells, but much higher against the BEL-7402/5-FU cells. Although the peracetates of EGCG and AcEGCG significantly reversed the 5-FU resistant BEL-7402/5-FU cells, partly acetated EGCG, Ac-A and Ac-B showed weaker reversal effect, indicating that after the acetyl (Ac) was introduced to EGCG, the introduction of more Ac units will enhance the reversal activity. AcEGCG and AcECG, especially AcEGCG, showed significant reversal activity in BEL-7402/5-FU cells, while EGC showed only weak reversal effect. Conclusion The galloyl in EGCG could be necessary to its reversal activity. AcEGCG significantly increased the sensitivity of the BEL-7402/5-FU cells towards 5-FU and could be used as a potential reversal agent for drug-resistant tumor cells.

Analysis on the expression of the stem cells related genes in hepatocellular carcinoma cell lines

To investigate the expression of Oct4, Sox2, Nanog, SMO, beta-Catenin and Wnt5b mRNA in four hepatocellular carcinoma cell lines of SMMC-7721, Bel-7402, HepG2, MHCC-97 and normal hepatocellular cell line of L02, and to compare the response of these cell lines to all-trans retinoic acid. RT-PCR was used to detect expression of Oct4, Sox2, Nanog, SMO, beta-Catenin and Wnt5b mRNA in four hepatocellular carcinoma cell lines and normal hepatocellular cell line. Real time-PCR was used to quantify the expression of the genes. There are different levels of expression of the stem cell-related gene in hepatocellular carcinoma cell lines and control cell line (P less than 0.05). There are significant differences in HepG2 and L-02 for the response to all-trans retinoic acid (P less than 0.05). The stem cell-related genes are differentially expressed in different hepatocellular carcinoma cell lines.