Effect of multi-target protein tyrosine kinase inhibitor Ponatinib on proliferation,adhesion and migration ability of human liver cancer cell line SK-Hep-1 / 中国生物制品学杂志

@#Objective To investigate the effect of a multi-target protein tyrosine kinase inhibitor,Ponatinib,on proliferation,homogeneity adhesion and migration ability of human liver cancer cell line SK-Hep-1.Methods SK-Hep-1 cells were cultured routinely and added with 24 tyrosine kinase inhibitors such as Ponatinib respectively,and the effect of Ponatinib on the survival and proliferation of SK-Hep-1 cells was detected by MTT assay.SK-Hep-1 cells were cultured routinely until the fusion degree reached 90%,then added with 0.1,0.5 and 1.0 μmol/L Ponatinib respectively,and the control group(without Ponatinib) was set up.The effect of Ponatinib on adhesion ability of SK-Hep-1 cells was detected by cell slow aggregation assay and dissociation assay,while the effect on migration ability by scratch test,and the effect on E-cadherin protein expression in SK-Hep-1 cells by Western blot.Results All 24 tyrosine kinase inhibitors inhibited SK-Hep-1 cells,among which Ponatinib showed the strongest inhibitory effect with a IC_(50) of(0.288±0.044) μmol/L.Compared with the control group,the number of cell mass(t=16.143,44.002 and 44.853 respectively,each P <0.001) and N_(TC)/N_(TE) [ratio of single cell number(N) after digestion by trypsin containing EDTA(TE) and CaCl_2(TC)](t=4.276,10.625 and 27.571 respectively,each P <0.05) decreased significantly and E-cadherin protein expression increased significantly(t=-3.757,-4.561and-6.922 respectively,each P <0.05) in 0.1,0.5 and 1.0 μmol/L Ponatinib groups;Scratch migration rate significantly decreased in 0.5 and 1.0 μmol/L Ponatinib groups(t=6.272～16.733 respectively,each P <0.01),while there was no significant difference in 0.1 μmol/L Ponatinib group(t=0.473 and 0.872 respectively,each P> 0.05) after 24 h and 48 h of scratch.Conclusion Ponatinib inhibited proliferation and migration of SK-Hep-1 cells and promoted cell adhesion.

Mechanism of Proteoglycan TPG-1 from Trametes robiniophila Inhibiting Growth of Human Hepatoma SK-HEP-1 Cells / 中国实验方剂学杂志

ObjectiveProteoglycan TPG-1 isolated from Trametes robiniophila（Huaier） has proved to have anti-hepatoma activity， and this paper aims to explore the molecular mechanism. MethodHuman hepatoma SK-HEP-1 cells were treated with TPG-1 （0， 0.05， 0.1， 0.25， 0.5， 1 g·L-1）. Then cell survival was detected by methyl thiazolyl tetrazolium （MTT） and apoptosis by flow cytometry. In addition， expression of genes in SK-HEP-1 cells treated with or without TPG-1 was examined by DNA microarray to preliminarily explore the anti-hepatoma molecular mechanism of TPG-1. ResultTPG-1 inhibited the proliferation of SK-HEP-1 cells as compared with the blank group （P<0.01）. After treatment with 1 g·L-1 TPG-1 for 48 h， the apoptosis rate of SK-HEP-1 cells increased （P<0.01）， and TPG-1 promoted the cleavage of cysteinyl aspartate specific proteinase （Caspase）-3 and Caspase-7， the key mediators of apoptosis （P<0.01）. Additionally， TPG-1 （1 g·L-1） suppressed the migration of SK-HEP-1 cells （P<0.05）. A total of 971 differentially expressed genes （DEGs） were identified in SK-HEP-1 cells after treatment with TPG-1， with 486 up-regulated and 485 down-regulated. These DEGs were mainly involved in the Gene Ontology （GO） terms of interleukin-6 （IL-6） biosynthesis， antigen processing and presentation， superoxide dismutase activity， positive regulation of mitogen-activated protein kinase kinase kinase （MAPKKK） cascade， nature killer （NK） cell chemotaxis， and chemokine biosynthesis， and the Kyoto Encyclopedia of Genes and Genomes （KEGG） pathways of nucleotide-binding oligomerization domain （NOD）-like receptor signaling pathway， apoptosis， Toll-like receptor signaling pathway， retinoic acid-inducible gene-Ⅰ （RIG-Ⅰ）-like receptor signaling pathway， T-cell receptor signaling pathway， and chemokine signaling pathway. Western blot results showed that TPG-1 （1 g·L-1） activated mitogen-activated protein kinase （MAPK） signaling pathway in SK-HEP-1 cells （P<0.01）. ConclusionProteoglycan TPG-1 inhibited the proliferation and migration， and induced apoptosis of human hepatoma SK-HEP-1 cells. Up-regulation of MAPK signaling pathway may be responsible for the growth inhibition of human hepatoma SK-HEP-1 cells by TPG-1.

Effect of lupeol on invasion and metastasis of human hepatoma HepG2 and SK-HEP-1 cells and its mechanism / 中国中药杂志

Epithelial-mesenchymal transformation(EMT) exists in embryonic development and is closely related to cell migration and invasion. The increased EMT level in tumors showed that E-cadherin was replaced by N-cadherin, and the expression of interstitial markers such as α-SMA and vimentin was up-regulated. It has been reported that lupeol can reduce the expression of matrix metalloproteinase-2(MMP-2), matrix metalloproteinase-9(MMP-9) and N-cadherin to inhibit the metastasis of osteoma cells. However lupeol has been less studied in liver cancer. Therefore, this paper investigated the effect of lupanol on invasion and metastasis of human hepatoma cell line HepG2 and SK-HEP-1 and its possible mechanism. MTT assay and Annexin V/PI double staining were used to investigate the effect of lupeol on activity and apoptosis of HepG2 cells and SK-HEP-1 cells. Moreover, the effect of lupeol on the invasion of HepG2 cells and SK-HEP-1 cells were evaluated by Transwell assay. The expressions of E-cadherin, N-cadherin, α-SMA, vimentin and MMP-9 were measured by Western blot. The model of subcutaneous transplantation of nude mice and the lung metastasis model of H22 hepatocellular carcinoma cells were established to evaluate the efficacy of lupeol in vivo on tumor growth and lung metastasis by HE staining combined with immunohistochemical assay. The results showed that lupeol inhibited the activity and invasion of HepG2 cells and SK-HEP-1 cells in a dose-dependent manner and induced apoptosis. Western blot showed that the expression of E-cadherin, a landmark protein for EMT, was induced by lupeol, and the expressions of N-cadherin, α-SMA, vimentin and MMP-9 were decreased. In vivo experiments showed that lupeol inhibited tumor growth in mice bearing xenograft. In addition, immunohistochemical experiments confirmed that lupeol could up-regulate the expression of E-cadherin in tumor tissues of nude mice, reduce the expression of N-cadherin, and inhibit the metastasis of liver cancer H22 cells in the lungs of mice. The above results indicated that the mechanism of lupeol inhibiting the invasion and metastasis of HCC cells may be related to the regulation of EMT process.

Screening of optimal siRNA interference sequence of CIT gene and its inhibition expression in hepatoma SK-Hep-1 / 中华肝脏病杂志

Objective@#To construct and screen optimal siRNA interference sequence of CIT gene and to detect its interference efficiency as well as proliferation effect in human hepatoma cell line SK-Hep-1.@*Methods@#Three siRNA target spots were designed and synthesized according to the CIT gene sequence. SK-Hep-1 HCC cells were transfected by liposome transfection. The knockdown efficiency of the target CIT gene was detected by real-time PCR and Western blot. Expressional change of CIT in SK-Hep-1 cells after 48 hours of siRNA interference were observed by immunohistochemistry and confocal microscopy. The proliferation of SK-Hep-1 cells after 48 hours of siRNA interference was detected by EdU cell proliferation assay. A t-test was used to compare the mean of two samples, and one-way ANOVA was used to compare the mean of multiple samples.@*Results@#Western blot results showed that the three interference sequences were targeted at different target spots. The expression level of CIT protein in KD-1,-2, and-3 groups were decreased (P < 0.01) than control, while the protein expression level of KD1 group was the lowest. Real-time PCR results showed that compared with the control group, the expression level of CIT mRNA in KD-1, -2, and -3 groups decreased (P < 0.01), while that in KD1 group was the lowest. Laser confocal microscopy also confirmed that the morphological expression of CIT attenuated significantly after transfection with siRNA. The results of EdU proliferation assay showed that siRNA transfected with CIT significantly attenuated the proliferation of SK-Hep-1 hepatoma cells (P < 0.05).@*Conclusion@#The successful construction and screening of siRNA fragments can effectively inhibit the expression and proliferation of CIT gene in hepatoma SK-Hep-1.

Huaier aqueous extract inhibits proliferation of human hepatoma SK-HEP-1 cells through up-regulation of autophagy / 中国中药杂志

The purpose of this study was to investigate the effect of Huaier on autophagy of human hepatoma SK-HEP-1 cells and the effect of autophagy on the proliferation of SK-HEP-1 cells. CCK-8 assay was used to evaluate the effect of Huaier on the proliferation of SK-HEP-1 cells under different concentrations and different times. Acridine orange staining was used to measure the effect of Huaier on the autolysosome formation in SK-HEP-1 cells. Immunofluorescence assay was applied to examine the effect of Huaier on the expression and distribution of autophagy marker LC3 in SK-HEP-1 cells. In addition， LC3 expression was also checked by immunoblot analysis in the presence of Huaier. At last， the effects of Huaier in combination with autophagy inhibitor bafilomycin A1 on the proliferation of SK-HEP-1 cells was detected by CCK-8 assay. The results showed that Huaier aqueous extract significantly inhibited the proliferation of human hepatoma SK-HEP-1 cells in a dose- and time-dependent manner. Huaier aqueous extract dramatically promoted the formation of autolysosome in SK-HEP-1 cells. Moreover， Huaier markedly increased the number and intensity of intracellular LC3 fluorescent puncta and up-regulated LC3-Ⅱ expression. These data indicated that Huaier evidently activated autophagy of SK-HEP-1 cells. Additionally， autophagy inhibition significantly attenuated the sensitivity of SK-HEP-1 cells to Huaier treatment. Therefore， autophagy activation is involved in the inhibitory effects of Huaier on the proliferation of human hepatoma SK-HEP-1 cells.

Construction of recombinant plasmids Egr1-XPO4 and its synergic inhibition with 5-FU against hepatocarcinoma SK-Hep1 cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To construct recombinant plasmid Egr1-XPO4 and evaluate its synergic inhibition with 5-FU against hepatocarcinoma SK-Hep1 cells. Methods: The XPO4 gene was inserted into vector carrying promoter Egr1 to construct a new recombinant vector, Egr1-XPO4, which was then transfected into human hepatocarcinoma cell line SK-Hep1 and sensitized with chemotherapeutic drug 5-FU. Western blotting was adopted to examine the protein expression of XPO4; CCK assay was used to detect SK-Hep1 cell proliferation after transfection, and Flow Cytometry with Annexin V-FITC/PI staining was used to detect the apoptosis of SK-Hep1 cells. SKHep1 cell xenograft model was constructed on nude mice, and the effect of Egr1-XPO4 in combination with 5-FU on the growth of xenograft was observed. Results: The recombinant plasmid Egr1-XPO4 was successfully constructed.With the sensitization of 5-FU, the expression of XPO4 protein in SK-Hep1 cells was significantly elevated after Egr1-XPO4 transfection, and the evlevation was in a 5FU dose-depend manner.The combined treatment of Egr1-XPO4 and 5-FU produced a significantly stronger inhibition against SKHep1 cell proliferation and greatly promoted apoptosis of SK-Hep1cells compared with 5-FU or pEgr-XPO4 mono-treatment group (all P<0.05). And in vivo antitumor experiment showed that the tumor volume in Egr1-XPO4+5-FU treatment group was significantly smaller than that of Egr1-XPO4 or 5-FU mono-treatment group (P<0.05). Conclusion: The recombinant plasmid Egr1-XPO4 in combination with 5-FU could exertsynergic inhibitionagainst hepatocarcinomaSK-Hep1 cells.

Effects of regenerated tissue extracts after liver injury on the proliferation, differentiation, migration and invasion of SK-HEP1 cells

Objective: To study the effects of regenerated tissue extracts after liver injury on the proliferation, differentiation, migration and invasion of SK-HEP1 cells. Methods: Regenerated tissue extracts after liver injury were used to induce SK-HEP1 cells after enrichment, their effects on the proliferation, differentiation, migration and invasion of SK-HEP1 cells were observed through in vitro cell culture, MTT, flow cytometry and transwell assays. Results: In response to the action of regenerated tissue extracts after liver injury, SK-HEP1 cells were blocked in G

Effects of regenerated tissue extracts after liver injury on the proliferation, differentiation, migration and invasion of SK-HEP1 cells

Objective:To study the effects of regenerated tissue extracts after liver injury on the proliferation, differentiation, migration and invasion of SK-HEP1 cells.Methods:Regenerated tissue extracts after liver injury were used to induce SK-HEP1 cells after enrichment, their effects on the proliferation, differentiation, migration and invasion of SK-HEP1 cells were observed through in vitro cell culture, MTT, flow cytometry and transwell assays.Results:In response to the action of regenerated tissue extracts after liver injury, SK-HEP1 cells were blocked in GConclusions:To a certain extent, regenerated tissue extracts after liver injury can inhibit the proliferation, differentiation, migration and invasion of hepatoma cells, showing an important potential of being a differentiating agent for the treatment of liver cancer.

Chemical constituents of Gleditsiae Spina and their antitumor activity / 中草药

Objective: To investigate the chemical constituents from Gleditsiae Spina (the thorns of Gleditsia sinensis) and their antitumor activitives. Methods: The chemical constituents were isolated and purified by the chromatography on repeated silica gel, Sephadex LH-20 column, semi-preparative HPLC, etc. Their structures were elucidated by NMR and MS spectroscopic data analyses; The cytotoxicity of compounds 1-4 and 6 was evaluated against human liver cancer SK-HEP-1 cells by MTT assay. Results: Twelve compounds were isolated from Gleditsiae Spina, and identified as 2-aminoimidazole (1), 2,3-dihydro-5-(2-formylvinyl)-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3-benzofuranmethanol (2), E-cinnamic acid (3), 3-O-trans-feruloylquinic acid (4), trans-caffeic acid (5), 4-hydroxy-3-methoxybenzamide (6), threo-guaiacylglycerol-β-coniferyl aldehyde ether (7), 5,7-dihydroxy-chromone (8), vanillic acid (9), protocatechuic acid (10), 3-O-caffeoylquinic acid methyl ester (11), and 3-O-caffeoylquinic acid ethyl ester (12); Compound 1 exhibited the potent cytotoxicity against SK-HEP-1 cells with IC50 value of 34.47 μg/mL. Conclusion: All the compounds except compound 5 are isolated from the plants of Gleditsia L. for the first time; Compound 1 shows significant cytotoxic activity against SK-HEP-1 cells.

The effects of plumbagin on proliferation and metastasis in human liver cancer SK-hep-1 cells / 中国癌症杂志

Background and purpose: Plumbagin is the main active components of traditional Chinese medicine of plumbago zeylanica. The present studies show that plumbagin has a killing effect on tumor cells. This study aimed to investigate the function and primary mechanism of plumbagin on invasion and metastasis of human liver cancer SK-hep-1 cells. Methods:With the treatment of plumbagin in vitro, cell proliferation and adhesion of SK-hep-1 cells were detected by MTS staining, cell cycle of SK-hep-1 cells were detected by lfow cytometry, the self-renewal and propagation abilities of SK-Hep-1 cells were conducted by colony formation assay , invasion in cells were performed using transwell invasion assay, and the p21 and MMP-2/9 mRNA levels were detected by real-time RT-PCR. Results:With the treatment of plumbagin, SK-Hep-1 cells proliferation was decreased with plumbagin concentration-dependency and the IC50 value of plumbagin in SK-Hep-1 cells was 22.04 mmol/L. The colony formation ability of SK-Hep-1 cells was decreased and the percentage of cells in G0/G1 phase was increased in a dose-dependent manner, as compared to control. The cell adhesion and invasion abilities were decreased. The real-time RT-PCR showed that p21 mRNA expression was increased and the MMP-2/9 mRNA was decreased. Conclusion:Plumbagin could suppress the proliferation and invasiveness of human liver cancer SK-hep-1 cells in vitro, and these effects may be by up-regulation of p21 and down-regulation of MMP-2 and MMP-9.

Construction of recombinant adenovirus vectors containing Akt gene and its effect on survival of hepatic cancer cells / 第二军医大学学报

Objective To construct a recombinant adenovirus encoding the Akt gene and to investigate its influence on the survival of hepatic cancer patients. Methods The Akt-wt and Akt-dn genes were subcloned into pacAd5 CMVK-NpA plasmid separately, and then the adenoviral recombinant adenovirus encoding the Akt gene was constructed with the RAPAd® CMV Adenoviral Expression System and was identified by PCR. The expression of Akt, GSK3β and p-GSK3β protein was detected by Western blotting analysis. SK-HEP-1 cells were infected with pacAd5 CMV-Akt-wt, pacAd5 CMV-Akt-dn and pacAd5 CMV-GFP, respectively. The cell survival was observed under fluorescence microscope after 24 h-starvation. Results pacAd5 CMV-Akt-wt and pacAd5 CMV-Akt-dn were enzymatically digested into two fragments(6 kb and 1. 44 kb). PCR result of the viral supernatant yielded a band of about 1. 44 kb. Western blotting analysis showed p-GSK3β expression in the Akt-wt group was stronger than that in the Akt-dn group. The survival rate of the SK-HEP-1 cells significantly decreased in the Akt-dn group than in the Akt-wt group. Conclusion We have successfully constructed the recombinant adenoviruses pacAd5-Akt-wt and pacAd5-Akt-dn, and they have pro-death effect in hepatic cancer cells, which paves a way for further studying Akt gene and the related signal pathway in hepatocellular carcinoma.

Construction of GPC3 green fluorescent protein eukaryotic expression vector and its influence on growth promoting effect of growth factors in SK-Hep-1 cells / 第二军医大学学报

Objective: To construct a GPC3 green fluorescent protein eukaryotic expression vector pEGFP-N2-G-PC3, and analyze its effects on the growth promoting effect of growth factors (fibroblast growth factor-2, FGF2; insulin-like growth factor-2, IGF2; transforming growth factor-β1, TGF-β1; and bone morphogenetic protein-4,BMP4) in human hepatoma cell line GPC3-SK-Hep-1. Methods: A eukaryotic expression vector for GPC3 genes (pEGFP-N2-GPC3) was constructed by recombinant DNA technique and was transfected into SK-Hep-1 cells by Lipofectamine™ 2000; the cells stably expressing GPC3 were screened out by G418 (600 μg/ml). The mRNA expression of GPC3 was detected by RT-PCR method and the protein expression of GPC3 by Western blotting and fluorescence microscope. Effects of GPC3 gene on the growth promoting effects of the above growth factors were examined by MTT. Results: The recombinant plasmid was verified to be correctly constructed by restriction endonuclease analysis and DNA sequencing. The green fluorescence was detected in the transfected SK-Hep-1 cells under fluorescence microscope. RT-PCR and Western blotting both confirmed that GPC3 was successfully expressed in SK-Hep-1 cells. FGF2-induced cell proliferation was significantly decreased by GPC3 gene, whereas the growth promoting effects of IGF2, TGF-β1 and BMP4 were not altered by GPC3 gene. Conclusion: We have successfully obtained the synthetic GPC3 protein, which has the same amino acid sequence as that of human GPC3 protein. The eukaryotic expression vector pEGFP-N2-GPC3 has been correctly constructed and GPC3 protein has been successfully expressed in SK-Hep-1 cells. GPC3 may negatively modulate FGF2 signaling pathway.

Role of Caveolin-1 in Indomethacin-induced Death of Human Hepatoadenocarcinoma SK-Hep1 Cells

Caveolin-1 (CAV1) is an integral membrane protein that may function as a scaffold for plasma membrane proteins and acts as a tumor suppressor protein. One causative factor of chemotherapy- resistant cancers is P-plycoprotein (P-gp), the product of the multidrug resistance-1 gene (MDR1), which is localized in the caveolar structure. Currently, the interactive roles of CAV1 and MDR1 expression in the death of cancer cells remain controversial. In this study, we investigated the effects of indomethacin on the cell viability and the expression levels of MDR1 mRNA and protein in a CAV1- siRNA-mediated gene knockdown hepatoma cell line (SK-Hep1). Cell viability was significantly decreased in CAV1-siRNA-transfected cells compared with that of control-siRNA-transfected cells. Furthermore, the viability of cells pretreated with CAV1 siRNA was markedly decreased by treatment with indomethacin (400micrometer for 24 h). However, the protein and mRNA levels of MDR1 were unchanged in CAV1-siRNA-transfected cells. These results suggest that CAV1 plays an important role as a major survival enzyme in cancer cells, and indomethacin can sensitively induce cell death under conditions of reduced CAV1 expression, independent of MDR1 expression.

Influence of glypican-3 gene on proliferation,adhesion and invasion of hepatoma carcinoma cancer cell line SK-Hep-1 / 中国肿瘤生物治疗杂志

Objective:To study the influence of GPC3 gene on the proliferation,adhesion and invasion of hepatoma cell line SK-Hep-1.Methods:SK-Hep-1 cells were transfected with pEGFP-N2-GPC3 using Lipofectamine2000.RT-PCR was used to examine the GPC3mRNA expression in GPC3-SK-Hep-1 cells.MTT assay was used to examine the proliferation and calculate the adhesion rate of SK-Hep-1 cells.Transwell system was used to assess the migration and invasion of the cells.Results:SK-Hep-1 cells were successfully transfected with pEGFP-N2-GPC palsmid.GPC3 mRNA was detected in SK-Hep-1 cells.Transfection with CPC3 significantly suppressed the growth of SK-Hep-1 cells(P

Development of Dual Reporter System of Mutant Dopamine 2 Receptor (D2R) and Sodium Iodide Symporter (NIS) Transgenes / 대한핵의학회잡지

PURPOSE: Both human NIS and mutant D2R transgenes are proposed as reporting system in transplanted cell tracking. Using hepatoma cell lines, we constructed a dual reporter system containing human sodium-iodide symporter (hNIS) and dopamine 2 receptor (D2R) and compared its characteristics. MATERIALS AND METHODS: The recombinant plasmid (pIRES-hNIS/D2R) was constructed with IRES (internal ribosome entry site) under control of the CMV promoter. pIRES-hNIS/D2R was transfected to human hepatoma SK-Hep1 cell line with lipofectamine. HEP-ND (SK-Hep1-hNIS/D2R) cells stably expressing hNIS and D2R was established by selection with G418 for two weeks. RT-PCR was performed to investigate the expression of both hNIS and D2R genes. The expressions of hNIS and D2R were measured by 125I uptake assays and receptor binding assays. Specific binding of D2R to [3H]spiperone was verified by Scatchard plot with (+) butaclamol as a specific inhibitor. K (d) and B (max) values were estimated. The correlation between hNIS and D2R expression was compared by using each clone. RESULTS: Similar quantities of hNIS and D2R genes were expressed on HEP-ND as RT-PCR assays. HEP-ND cells showed 30 to 40 fold higher radioiodine uptakes than those of parental SK-Hep1 cells. 125I uptake in HEP-ND cells was completely inhibited by KClO4, a NIS inhibitor. Specific binding to HEP-ND cells was saturable and the K (d) and B (max) values for HEP-ND cells were 2.92 nM, 745.25 fmol/mg protein and 2.91nM, 1323 fmole/mg protein in two clones, respectively. The radioiodine uptake by hNIS activity and D2R binding was highly correlated. CONCLUSION: We developed a dual positron and gamma imaging reporter system of hNIS and D2R in a stably transfected cell line. We expect that D2R and hNIS genes can complement mutually as a nuclear reporting system or that D2R can be used as reporter gene when hNIS gene were used as a treatment gene.