Identification of HLA-A3 restricted cytotoxic T-lymphocyte epitopes from cancer-testis antigen MAGEC2

AIM:To predict and identify an HLA-A3 supertype-restricted cytotoxic T-lymphocyte (CTL) epitope derived from MAGEC2, which is utility in epitope design for the development of HLA-based vaccines and immunotherapeutics. METHODS:HLA-A3 epitopes from MAGEC2 protein were predicted by BIMAS, SYFPEITHI and IEDB. The binding affinity of the peptides to HLA-A*03 molecule was evaluated by T2A3 cell binding assay. ELISPOT assay was used to investigate the ability of the peptides inducing specific restricted CTLs to release interferon-γ (IFN-γ). The ability of the peptides to induce T-cell response was investigated by cytotoxicity assay in vitro. RESULTS:The candidate peptides P147, P167, P196, P229 and P251 showed moderate affinity toward HLA-A3 molecule. ELISPOT assay showed that P167, P196 and P251 were able to induce specific CTLs and higher levels of IFN-γ were released. The CTLs induced by P196 and P251 were able to lyse target cells. CONCLUSION:The peptides P196 and P251 have higher binding affinity with HLA-A3 and retain immunogenicity. They are excellent HLA-A3-restricted CTL epitopes from tumor antigen MAGEC2, which could serve as new candidates towards antitumor peptide vaccines.     

Identification and molecular modification of cytotoxic T-lymphocyte epitopes from osteosarcoma high-expressing antigen PBF

AIM: To observe whether modified epitopes from osteosarcoma high-expressing antigen papillomavirus-binding factor (PBF) have HLA-A2 restricted antitumor ability, and to develop peptide-based immunotherapy for osteosarcoma. METHODS: RT-PCR and Western blot were used to determine the expression of PBF in the osteosarcoma cell lines U2OS and Saos-2. HLA-A2 epitopes from PBF protein were predicted by NetCTL 1.2, SYFPEITHI and IEDB. The modified peptides from PBF containing HLA-A2 binding anchor motifs were designed by replacing the anchor residues. The peptides were synthesized by standard solid-phase methods, and the binding affinity of the peptides to HLA-A*0201 was evaluated by T2A2 cell binding assay. ELISPOT assay was used to investigate the seretion of interferon-γ (IFN-γ) from the peptide-induced specific cytotoxic T-lymphocytes (CTLs). The ability of inducing T-cell response was analyzed by lactate dehydrogenase (LDH) release assay and carboxyfluorescein succinimidyl ester (CFSE) cytotoxicity assay in vitro. RESULTS: The expression of PBF was observed in the U2OS and Saos-2 cells. The candidate peptides P75-1Y2L, P412-1Y, P416-1Y2L9V, P107-1Y and P435-1Y2L showed moderate affinity toward HLA-A2 molecule. The modified peptides showed significantly higher affinity with HLA-A2 than the native peptide. ELISPOT assay showed that P412, P412-1Y, P416, P416-1Y2L9V and P435-1Y2L induced specific CTLs to secrete IFN-γ, and P412-1Y and P416-1Y2L9V induced more secretion of IFN-γ than the native peptide. The CTLs induced by P412, P412-1Y, P416 and P416-1Y2L9V lysed U2OS cells. P412-1Y and P416-1Y2L9V peptide-specific CTLs showed higher cytotoxicity against U2OS cells than the native peptide-specific CTLs. CONCLUSION: Compared with the native peptide, modified epitopes P412-1Y and P416-1Y2L9V have higher binding affinity with HLA-A*0201 and retain immunogenecity. In addition, the anti-tumor immunity effects of modified epitopes P412-1Y and P416-1Y2L9V are stronger than the native peptide. The peptides P412-1Y and P416-1Y2L9V is excellent HLA-A*0201 restricted CTL epitopes from tumor antigen PBF, which could serve as new candidates towards antitumor peptide vaccines.     

Identification of HLA-A2 restricted cytotoxic T lymphocyte epitopes from tumor antigen PIWIL2

AIM: To identify the human leucocyte antigen A2 (HLA-A2) restricted cytotoxic T lymphocyte (CTL) epitopes from tumor antigen PIWIL2. METHODS: RT-PCR and Western blot was used to determine the expression of PIWIL2 in cancer cell lines MCF-7, SW480 and HT-29. HLA-A2 epitopes from PIWIL2 protein were predicted by the software of BIMAS, RankPep, NetMHC, NetCTL1.2 and IEDB. The peptides were synthesized by standard solid-phase methods. The binding affinity of the peptides to HLA-A2 molecules was evaluated by T2 cells binding assay. ELISPOT assay was used to investigate the levels of IFN-γ. The cytotoxicity assay in vitro was also used to determine the ability of inducing T cell response by the peptides. RESULTS: The expression of PIWIL2 was observed in MCF-7, SW480 and HT-29. The candidate peptide P485, P493 and P965 showed moderate affinity toward HLA-A2 molecule. ELISPOT assay showed P485 and P965 induced CTLs of IFN-γ release form CTLs. The CTLs induced by P485 and P965 lysed the MCF-7 cells. CONCLUSION: The peptides P485 and P965 are excellent HLA-A2 restricted cytotoxic T lymphocyte epitopes from the tumor antigen PIWIL2, which could serve as new candidates towards antitumor peptide vaccines.     

Expression of eEF1A2 in hepatocellular carcinoma

AIM: To study the expression of eukaryotic elongation factor 1A2 (eEF1A2) in the hepatocellular carcinoma (HCC) tissues and the effects of eEF1A2 over-expression on the biological behaviors of the HCC cells. ME-THODS: The expression of eEF1A2 at mRNA and protein levels in the HCC tissues and matched liver tissues from 62 HCC patients, and 20 normal liver tissues were detected by the methods of real-time PCR and immunohistochemical staining, respectively. The mRNA and protein expression of eEF1A2 in the HCC cells was also determined by real-time PCR and Western blot, respectively. The lentivirus containing eEF1A2 gene was constructed, and was used to infect the HCC cells with low eEF1A2 expression. The expression of eEF1A2 at mRNA and protein levels in the infected cells was detected by real-time PCR and Western blot, respectively. The cell activity, cell cycle and mRNA expression of albumin were measured by MTT assay, DNA ploid analysis and real-time PCR, respectively.RESULTS: The mRNA expression levels and protein expression positive rates of eEF1A2 in the 62 cases of HCC tissues, were significantly higher than those of 62 matched liver tissues and 20 normal liver tissues (P<0.01). eEF1A2 mRNA and protein were highly expressed in SMMC-7721 cells and BEL-7402 cells, and expressed in SK-HEP-1 cells at low level. The expression of eEF1A2 at mRNA and protein levels in the SK-HEP-1 cells was significantly enhanced by infection of GV287-eEF1A2 expression lentivirus.Compared with negative control group (transfected with negative control lentivirus), the cell activity in eEF1A2 over-expression group (transfected with GV287-eEF1A2 expression lentivirus) was significantly enhanced, the mRNA expression of albumin was remarkably reduced, and the cells in G₀/G₁ phase were significantly decreased with increased percentage of the cells in S and G₂/M phases.CONCLUSION: eEF1A2 is selectively over-expressed in human HCC cancer tissues. eEF1A2 might be a putative oncoprotein in HCC. eEF1A2 over-expression has noticeable effects on the HCC cell proliferation enhancement, differentiation inhibition, and cell cycle acceleration through the G₀/G₁ phase to S phase and G₂/M phases.     

Ginsenoside Rh4 induces apoptosis of human hepatocellular carcinoma HepG2 cells

AIM: To investigate the apoptosis and molecular mechanism of human hepatocellular carcinoma HepG2 cells induced by ginsenoside Rh4. METHODS: Human hepatocellular carcinoma HepG2 cells were treated with ginsenoside Rh4 at doses of 10, 20 and 40 μmol/L, and the inhibitory effect of ginsenoside Rh4 on HepG2 cell viability was measured by MTT assay. The apoptotic rate of HepG2 cells was analyzed by flow cytometry. The morphological changes of the HepG2 cells were observed by Hoechst 33258 and TUNEL staining. The expression of apoptosis-related proteins Bax, Bcl-2, caspase-3 and caspase-9 was determined by Western blot. RESULTS: Ginsenoside Rh4 promoted apoptosis of HepG2 cells in a dose-dependent manner. TUNEL and Hoechst 33258 staining showed that the cells appeared obvious shrinking, swelling and rupture after treated with ginsenoside Rh4 for 24 h. The results of Western blot showed that with the increasing concentrations of ginsenoside Rh4, the expression of pro-apoptotic proteins Bax, cleaved caspase-3 and caspase-9 increased, while anti-apoptotic protein Bcl-2 decreased gradually. CONCLUSION: Ginsenoside Rh4 induces apoptosis of human hepatocellular carcinoma HepG2 cells, and the main mechanism may be related to down-regulation of Bcl-2 and up-regulation of Bax, cleaved caspase-3, and caspase-9.     

Expression of human leukocyte antigen E in hepatocellular carcinoma cell lines

AIM: To investigate the human leukcyte antigen E (HLA-E) expression in hepatocellular carcinoma cell lines. METHODS: The techniques of real-time PCR and Western blotting were used to study the HLA-E expression in the 5 cell lines of hepatocellular carcinoma and a fetal liver cell line at mRNA and protein levels. RESULTS: The results of real-time PCR showed that no statistical difference of HLA-E mRNA level between fetal liver cell L02 and other 4 cell lines of hepatocellular carcinoma (HepG2, Bel7402, PLC and MHCC97) was observed, and almost absence of HLA-E mRNA expression in Hep3B2.1-7 cells was detected. However, the results of Western blotting showed that there was a significant statistical difference of HLA-E protein levels between L02 cells and the 5 cell lines of hepatocellular carcinoma (HepG2, Bel7402, PLC, MHCC97 and Hep3B2.1-7), and no HLA-E protein in Hep3B2.1-7 cells was detectable. CONCLUSION: Asynchronization of HLA-E expression between mRNA and protein levels was found in hepatocellular carcinoma cell lines.     

FERMT2 expression in hepatocellular carcinoma and its effect on cell growth

AIM: To identify the expression of fermitin family homolog 2 (FERMT2) in hepatocellular carcinoma (HCC) tissues and the effect of FERMT2 on the cell growth and related protein expression. METHODS: Real-time PCR and immunohistochemistry were used to detect FERMT2 expression in the HCC tissues. The technique of CRISPR/Cas9 was applied to construct stable FERMT2 knockout MHCC97H cell line. WST-1 assay and flow cytometry were used to measure the cell viability, cell-cycle distribution and cell apoptosis. Western blot was used to determine the expression of related proteins in the MHCC97H cells. RESULTS: In HCC tissues, the expression level of FERMT2 was higher than that in adjacent liver tissues (P<0.05). High expression of FERMT2 was significantly correlated with postoperative recurrence of tumor. Knockout of FERMT2 gene evidently inhibited MHCC97H cell viability and accelerated cell apoptosis. Meanwhile, the expression levels of proliferating cell nuclear antigen, cyclins, cyclin-dependent kinases 2 and anti-apoptotic factors were significantly downregulated in MHCC97H cells with FERMT2 knockout (P<0.05). CONCLUSION: FERMT2 may function as a promoter of hepatocarcinogenesis and progression via regulating the cell viability, cell-cycle distribution and cell apoptosis, which is related with the expression of cell cycle regulators and anti-apoptotic factors.     

Effect of sirtuin 6 on proliferation of hepatocellular carcinoma cells

AIM: To illuminate the effect of sirtuin 6 (SIRT6) on the proliferation of hepatocellular carcinoma (HCC) cells. METHODS: The mRNA expression of SIRT6 in the peripheral blood from 200 cases of HCC patients and 50 cases of healthy people was detected by real-time quantitative PCR (RT-qPCR). The mRNA expression levels of SIRT6 in the peripheral blood from 200 cases of HCC patients were combined with multiple clinicopathologic parameters for statistical analysis. The protein expression of SIRT6 in primary hepatocytes, immortalized hepatocytes and 4 hepatoma cell lines were determined by Western blotting. The silencing of SIRT6 was conducted by transfection of vector expressing short hairpin RNA targeting on SIRT6, and the protein level of SIRT6 was measured by Western blotting. The viability of HCC cells was tested by MTS assay. DNA synthesis was analyzed by Cell-Light^TM EdU Apollo® 488 In Vitro Imaging Kit. The abilities of colony formation and anchorage-dependent growth were measured by colony formation assay and soft agar assay, respectively. RESULTS: The mRNA expression of SIRT6 in the peripheral blood of HCC patients was significantly higher than that in the healthy people, and its expression was highly associated with tumor size, tumor grade and vascular invasion. SIRT6 expression in 4 hepatoma cell lines was significantly higher than that in the others. SIRT6 silencing led to a significant decrease in the cell viability as tested by MTS assay. EdU staining revealed that SIRT6 silencing reduced DNA synthesis. SIRT6 silencing reduced the ability of colony formation and anchorage-dependent growth as determined by colony formation assay and soft agar assay, respectively. CONCLUSION: Sirtuin 6 promotes the proliferation and malignant transformation of HCC cells.     

In vivo immunogenic characteristics of cytotoxic T lymphocyte epitopes from pancreatic tumor antigen MUC4

AIM: Peptide vaccines have been conceived as promising therapies for tumor-inflicted patients due to their easy production and capability of inducing specific immune response required for defending the tumor. During our previous research, 4 HLA-A2-restricted peptides had been identified as immunogenic in vivo. In this study, we aimed to testify the in vivo immunogenicity of the 4 peptides. METHODS: BALB/c mice were vaccinated with HLA-A2 restricted peptides emulsified in incomplete Freund's adjuvant (IFA) subcutaneously in combination with the epitope at the adjacent location. After the 3rd peptide vaccination for 10 d, the peptide-specific immune response was evaluated by ELISPOT and ELISA. The ability to induce T cell response was investigated by using cytotoxicity assay in vivo and the presence of peptide-specific CD8⁺ T cells capable of recognizing the MHC-peptide was detected by flow cytometry. RESULTS: Among the 4 candidate HLA-A2 restricted peptides, the immune response elicited by P2004-1Y9V was superior to that of the other 3 peptides. The CTLs induced by P2004 and P2004-1Y9V lysed CAPAN-2 cells. P2004-1Y9V peptide-specific CTLs showed higher cytotoxicity against pancreatic tumor cell lines of CAPAN-2 than the native peptide-specific CTLs. Intracellular cytokine staining assay indicated the presence of P2004-1Y9V specific CD8⁺T cells in the P2004-1Y9V vaccinated mice. CONCLUSION: P2004-1Y9V is the most immunogenic peptide in vivo, and can be explored as potential tumor peptide vaccine in the future clinical study.     

Artesunate induces apoptosis of human hepatocellular carcinoma HepG2 cells by increasing generation of reactive oxygen species

AIM: To investigate the effect of reactive oxygen species (ROS) on the apoptosis of HepG2 cells induced by artesunate. METHODS: The effect of artesunate on the viability of HepG2 cells was measured by MTT assay. The morphological changes of the apoptotic cells were observed by the method of Hoechst 33258 fluorescence staining.The apoptosis of HepG2 cells was analyzed by flow cytometry. DCFH-DA was used to detect the changes of ROS generation during the process of apoptosis. The protein levels of Bax, Bcl-2, cleaved caspase-3 and cytochrome C (Cyt C) were determined by Western blot. HepG2 cells were pretreated with apocynin and then Western blot was used to detect the expression of p47^phox and p22^phox, and ROS changes were analyzed by flow cytometry. RESULTS: Compare with control group, the cell viability was obviously inhibited after treatment with artesunate for 24 h (P<0.05). The nuclei were densely stained, and the proportion of apoptotic cells was increased (P<0.05). ROS was increased significantly (P<0.05). The results of Western blot demonstrated that the expression level of Bax was increased, Bcl-2 was decreased, the ratio of Bax/Bcl-2 was increased, and the protein levels of cleaved caspase-3 and Cyt C were increased. Pretreatment with apocynin reduced the expression of p47^phox and p22^phox and the generation of ROS in the artesunate treatment group. CONCLUSION: Artesunate induces the apoptosis of HepG2 cells. The possible mechanism may be related to the increase in the generation of ROS.     

Effect of ZNF281 on proliferation of hepatocellular carcinoma cells

AIM:To investigate the role of zinc finger protein 281 (ZNF281) in the proliferation of hepatocellular carcinoma (HCC) cells. METHODS:The mRNA expression levels of ZNF281 in peripheral blood mononuclear cells from 80 cases of healthy people and 206 cases of HCC patients were determined by real-time PCR. Statistical analysis were used to illustrate the relationship between the mRNA expression levels of ZNF281 in the peripheral blood mononuclear cells and the clinicopathologic parameters of HCC patients. Real-time PCR and Western blot were used to detect the mRNA and protein expression levels of ZNF281 in hepatoma cell lines and immortalized hepatocytes. The silencing of ZNF281 was conducted by transfection of small interfering RNA targeting ZNF281, and then the proliferation of HCC cells was analyzed by MTS assay. The DNA synthesis of HCC cells was tested by Cell-Light^TM EdU Apollo^®488 In Vitro Imaging Kit. The ability of colony formation of the HCC cells was measured by colony formation assay, and the ability of anchorage-indepen-dent growth was detected by soft agar test. RESULTS:The mRNA expression level of ZNF281 in the peripheral blood mononuclear cells from HCC patients was significantly increased compared with the healthy people, and the high expression level was positively correlated with tumor size, tumor stage and tumor vascular invasion. Concurrently, the expression level of ZNF281 in hepatoma cell lines was significantly higher than that in immortalized hepatocytes. More importantly, the silencing of ZNF281 inhibited the proliferation, DNA synthesis, colony formation and anchorage-independent growth of the HCC cells. CONCLUSION:ZNF281 promotes the proliferation of HCC cells.     

Effects of zinc on biological behavior of hepatocellular carcinoma cell line BEL-7404

AIM: To observe the effects of exogenous zinc on the biological behavior of hepatocellular carcinoma (HCC) cell line BEL-7404. METHODS: BEL-7404 cells were cultured with zinc sulfate at various concentrations. The intracellular concentration of zinc, cell viability, cell cycle, cell apoptosis and migration and invasion abilities were measured by TSQ fluorescent probe, MTT assay, DNA ploid analysis, acridine orange/ethidium bromide fluorescence staining and Transwell assay, respectively. The mRNA and protein expression levels of albumin in the BEL-7404 cells were determined by real-time PCR and Western blot, respectively. RESULTS: With the elevated concentration of zinc in culture condition, the concentration of zinc in the BEL-7404 cells was increased (P<0.05). The cell viability and migration and invasion abilities were decreased, while the apoptotic rate was increased (P<0.05). The cells in G₀/G₁ phase were decreased, while the cells in G₂/M phase were increased. Additionally, the mRNA and protein expression of albumin also increased (P<0.05). CONCLUSION: The zinc ion inhibits the cell viability as well as migration and invasion abilities, blocks the cells in G₂/M phase, and may reduce cell malignant phenotype.     

Progress in RNA-binding protein HuR and its roles in development of hepatocellular carcinoma

RNA-binding proteins （RBP） are molecules with a variety of biological functions discovered in recent years. Among them, HuR is an important RBP, widely expressed in various tissues of the body, and is a member of the Hu/embryonic lethal abnormal vision （ELAV） protein family. It mainly affects the expression levels of target genes in the cells by regulating the stability and/or translation efficiency of the mRNA of the genes, thus participating in the regulation of cell life activities. In recent years, more and more studies on HuR have revealed its important role in inflammation and cancer. This review summarizes the functions of HuR and probes into the regulation of HuR functions. In addition, the roles of HuR in the occurrence and development of hepatocellular carcinoma （HCC） are also discussed, which provides important information for exploring the pathogenesis, biomarkers and therapeutic targets of HCC.     

Effect of gold nanoparticles on reversing adriamycin resistance of human hepatocellular carcinoma HepG2/ADM cells

AIM: To investigate whether gold nanoparticles (GNPs) reverses adriamycin (ADM), resistance of human hepatocellular carcinoma drug-resistant cell line HepG2/ADM and to explore the potential mechanism. METHODS: The sensitivities of HepG2 cells and HepG2/ADM cells to ADM were tested by MTT assay before and after GNPs pretreatment. The apoptotic rate was examined by flow cytometry. The concentration of ADM in HepG2/ADM or HepG2 cells was determined by ultraviolet-visible spectrophotometer. The content of glutathione (GSH) in HepG2/ADM or HepG2 cells by DTNB method. RESULTS: The half maximal inhibitory concentrations (IC₅₀) of ADM for HepG2/ADM cells were(29.46±1.73) mg/L and (15.18±0.85) mg/L before and after GNPs pretreatment,respectively. The IC₅₀ of ADM for HepG2 cells was (9.16±2.03) mg/L before pretreatment. The apoptotic rate in GNPs+ADM group was higher than that in ADM group (P<0.05). The concentration of ADM in HepG2/ADM group was lower than that in HepG2 group (P<0.01). After GNPs pretreatment, the concentration of ADM in HepG2/ADM cells was higher than that before pretreatment. The content of GSH in HepG2/ADM group was higher than that in HepG2 group (P<0.01). After GNPs pretreatment, the content of GSH in the HepG2/ADM cells was lower than that before pretreatment. CONCLUSION: Gold nanoparticles can reverse ADM resistance of human hepatocellular carcinoma drug-resistant cell line HepG2/ADM, reduce the content of GSH and increase the concentration of ADM in HepG2/ADM cells.     

Effect of antisense locked nucleic acid blocking translation initiation region of c-myc exon 2 on apoptosis of hepatocellular carcinoma cells

AIM: To observe the effect of antisense locked nucleic acid (anti-LNA) blocking the translation initiation region of c-myc exon 2 on the viability and apoptosis of hepatocellular carcinoma cells.METHODS: The anti-LNA that was complementary to the translation initiation region of c-myc exon 2 was designed, synthesized, and introduced into the HepG2 cells by cationic liposome-mediated transfection. The mRNA and protein levels of c-Myc in the cells were determined by RT-PCR and Western blot. The change of cell apoptosis was analyzed by flow cytometry, and the toxicity of anti-LNA to the cells was detected by MTT assay.RESULTS: Five days after transfection, the mRNA level of c-Myc in anti-LNA group was 0.335±0.016, and the protein level was 0.448±0.037, significantly lower than those in control group (both P<0.05). The ratio of apoptotic cells in anti-LNA group was 32%±6%, which was higher than that in control group (P<0.05).CONCLUSION: Antisense locked nucleic acid targeting at the translation initiation region of c-myc exon 2 shows strong inhibitory effects on the apoptosis of hepatocellular carcinoma cells.     

Simultaneous ex vivo generation of cytomegalovirus pp65 and Epstein-Barr virus specific cytotoxic T-lymphocyte from human umbilical cord blood

AIM: To investigate the possibility of simultaneously ex vivo generating cytomegalovirus (CMV) pp65 and Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) from human umbilical cord blood (CB). METHODS: Mononuclear cell derived from CB (CBMC) was used to construct EBV-transformed B-lymphoblastoid cell lines (BLCL). Then BLCL were transduced with a recombinant retrovirus encoding pp65, the immunodominant CMV polypeptide. CBMC from the same CB donor were stimulated with pp65-expressing BLCL (BLCLpp65) weekly for 5~6 weeks. Chromium release assays (CRA) were performed to detect the specific cytotoxicity of the CTL against EBV and CMV. RESULTS: Western blot analysis and immunocytochemistry confirmed that BLCLpp65 could simultaneously express CMVpp65 and EBV antigen. CRA results showed that the generated CTL possessed specific cytotoxic against EBV and CMV, and the cytotoxicity was mediated by CD₈⁺ CTL. CONCLUSION: BLCLpp65 can be used as antigen-presenting cells to stimulate expansion of EBV and CMV specific CTL simultaneously from the predominantly native T cell population in CB.     

Effects of luteolin on invasion,migration and adhesion of human hepatocellular carcinoma HepG2 cells

AIM: To investigate the effects of luteolin on invasion, migration and adhesion of human hepatocelluar carcinoma HepG2 cells.METHODS: HepG2 cells were cultured and treated with luteolin at 10, 20 and 40 μmol/L respectively. The invasion capability was examined by cell invasion assay. The migration ability was examined by wound healing assay. The adhesion capability was measured by adhesion assay. The protein levels of E-cadherin, N-cadherin, vimentin and Snai1 were determined by Western blot analysis.RESULTS: Luteolin inhibited the invasion, migration and adhesion ability of HepG2 cells in vitro in a dose-dependent manner. After treatment with luteolin, the expression of E-cadherin was increased significantly and the expression of N-cadherin, vimentin and Snai1 were decreased significantly.CONCLUSION: Luteolin inhibits the invasion, migration and adhesion ability of human hepatocelluar carcinoma HepG2 cells. The mechanism may be related to the regulatory effects of luteolin on epithelial-mesenchymal transition.     

Effects of ZIP14 on biological behaviors of hepatocellular carcinoma cell line BEL-7404

AIM: To study the expression of zinc transporter ZRT/IRT-like protein 14 (ZIP14) in the hepatocellular carcinoma (HCC) tissues, and to investigate the effects of ZIP14 over-expression on the biological behaviors of HCC cells. METHODS: The expression of ZIP14 at mRNA and protein levels in the HCC tissues and adjacent non-tumor tissues were detected by real-time PCR and immunohistochemical staining, respectively. The lentivirus expression system containing GV365-ZIP14 was constructed, and was used to infect the HCC cell line BEL-7404, which had relatively poor expression of ZIP14. The expression of ZIP14 at mRNA and protein levels in the transfected cells were detected by real-time PCR and Western blot, respectively. Under the conditions of zinc sulfate stimulation at different concentrations, the cell viability, the cell cycle, and the cell migration and invasion abilities were detected by MTT assay, DNA ploid detection, and Transwell assay, respectively. RESULTS: The mRNA expression level and the strong-positive rate of protein expression of ZIP14 in the HCC tissues were significantly lower than those in the adjacent non-tumor liver tissues (P<0.01). The expression of ZIP14 at mRNA and protein levels in the BEL7404 cells was significantly enhanced by infection of GV365-ZIP14 expression lentivirus. Compared with negative control group (transfected with negative control lentivirus), the cell viability, migration and invasion in ZIP14 over-expression group (transfected with GV365-ZIP14 expression lentivirus) were significantly reduced, and the percentage of the cells in G₂/M phase was significantly increased, all of which were more obvious with the elevation of zinc concentration in the culture medium. CONCLUSION: ZIP14 is low expressed in the HCC tissues. The ZIP14 over-expression has inhibitory effects on the viability, migration and invasion of HCC cells, and blocks the cell cycle in G₂/M phase, which might be closely related to the elevation of zinc concentration in cytoplasma of HCC cells due to enchanced zinc transport by ZIP14.     

Treatment of hepatocellular carcinoma with CIK cell therapy combined with mini-invasive TACE

Transcatheter arterial chemoembolization (TACE) has become an important way to treat hepatocellular carcinoma (HCC). TACE can kill HCC cells directly. Cytokine-induced killer (CIK) cell therapy improves immunity. CIK sequential therapy combined with TACE improves treatment safety, efficacy, the quality of life and long-term outcome for the HCC patients. This review summarizes the status and latest progress of CIK cell therapy combined with TACE in the treatment of HCC.