Purpose:To study the difference in gene expression profile between HCC cell strains with high and low metastatic potential.Methods:The gene expression profile of high and low metastatic HCC cell strains were analyzed by cDNA microarray, and genes with differential expression were further verified by RT PCR.Results:384 differentially expressed genes were detected between high and low metastatic HCC strains, making up 32% of the total genes studied. RT PCR analysis on 4 genes chosen demonstrated the same results as those revealed by the array technique.Conclusions:Obvious difference in gene expression patterns was observed between high and low metastatic HCC cell strains, chromosome 8p may contain metastasis promote and inhibitory genes.

Objective:To identify the signature of tumor-infiltrating lymphocyte (TIL) subtypes that may affect cytokine expres-sion between different outcomes of hepatocellular carcinoma (HCC) patients by analyzing the CD molecular expression profiles of non-cancerous hepatic tissues. Methods:Surface markers of TIL in noncancerous hepatic tissues from 146 HCC patients were determined by using immunohistochemical method and flow cytometry. Univariate and multivariate Cox proportional hazards models and Kaplan-Meier method were used to analyze the association of their expression levels with tumor recurrence and survival. Results:More than 86.4%of TILs in patients were quiescent, as measured via CD4+or Foxp3 expression. Meanwhile, more than 90%of CD3+T cells ex-pressed CD8+. The proportion of T cells was low compared with CD8+T cells. The proportion of CD19 and CD20 in distant nontumor tissues almost was zero. The proportion of T cell subgroups isolated from HCC circulating whole blood did not show a significant shift compared with the normal control, as follows:CD4+T/CD8+T=1.167 ± 1.04, CD8+T/CD3+T=0.288 ± 0.116, and CD4+T/CD3+T=0.429 ± 0.178. The proportion of CD8+T cells in noncancerous hepatic tissues was higher than that in blood (P<0.001).Conclusion:TILs in HCC noncancerous hepatic tissues are increased and contain a subpopulation of CD3+CD8+T cells. CD8+T cells in cancerous tissues, rather than noncancerous tissues, show significant differences between different prognostic groups.

Objective To identify genes associated with hepatocellular carcinoma (HCC) as candidate diagnostic markers in a genome-wide scale. Methods The gene expression profiles of 40 pairs of HCC tumor tissue and peripheral non-tumorous liver tissue were analyzed by using gene chip technology.The gene chips were fabricated at the National Cancer Institute (NCI). Each gene chip contained 9 180 genes. The fluorescent targets were prepared by a direct labeling approach using two kinds of fluorescences as following: 100 ?g of total RNA from non-cancerous liver tissue was labeled with Cy3-dUTP and 200 ?g of total RNA from HCC was labeled with Cy5-dUTP. The targets were mixed together and hybridized with genes on the gene chips. Unsupervised hierarchical clustering analysis was done by CLUSTER and TREEVIEW software using median centered correlation and complete linkage. Results A total of 10 genes were found up-regulated in over 80% of primary tumors comparing with that of their corresponding non-tumorous liver tissues at a two-fold filter with an unsupervised hierarchical clustering algorithm, including protocadherin-alpha 9, ESTs, Homo sapiens cDNA FLJ, KPNA2, RPS20, SNRPE, CDKN2A, UBD, MDK and ANXA2. Conclusion These genes are supposed to be candidates for the diagnosis of HCC. Further investigation of these genes in a large scale of patients with HCC and patients with non-malignant hepatic diseases will be needed to disclose whether they could be used clinically as novel diagnostic tumor markers for HCC.

Objective:To explore the mechanism of the increased invasive and metastatic potential of hepatocellular carcinoma induced by sorafenib ,by studying the expression of stromal‐derived factor 1‐α(SDF 1‐α) .Methods :BALB/c nude mice models of hepatocellular carcinoma in situ were established with cell line HCCLM 3 .The mice were divided into sorafenib group and control group ,with 6 mice in each group .Sorafenib was administered intragastrically [30 mg/(kg · d)] in sorafenib group from 2 weeks after model had been established ,while 0 .9% sodium chloride solution was given in the control group .Mice were killed after treating for 4 weeks .The mRNA expressions of inflammation‐related factors in hepatocellular carcinoma tissues and pericarcinoma liver tissues of the two groups were detected by RT‐PCR .The protein expression of SDF1‐α in hepatocellular carcinoma tissues and pericarcinoma liver tissues was measured by immunohistochemistry .The protein expression of SDF1‐αin pericarcinoma liver tissues was also detected by Western blotting .ELISA was performed to detect the SDF1‐αconcentration in peripheral blood .Results:The mRNA expression of murine SDF1‐αin hepatocellular carcinoma tissues and pericarcinoma liver tissues was significantly increased by sorafenib therapy .The results of immunohistochemistry and Western blotting showed that protein expression level of SDF1‐α in hepatocellular carcinoma tissues and pericarcinoma liver tissues was significantly up‐regulated by sorafenib .The results of ELISA assay showed that the concentration of murine SDF1‐αin peripheral blood was increased by sorafenib .Conclusions :The expression of SDF1‐α in hepatocellular carcinoma tissues and pericarcinoma liver tissues can be up‐regulated by sorafenib therapy .

Objective:To investigate the effect of AMD3100 ,an inhibitor of receptor of stromal cell derived factor 1‐α (SDF1‐α) ,combined with sorafenib on nude mice with hepatocellular carcinoma (HCC) .Methods:Nude mice models of HCC in situ was established and the mice were divided into control group ,sorafenib group and sorafenib combined with AMD3100 group , with six mice in each group .In sorafenib group ,sorafenib 30 mg/(kg · d) was given by garage .In sorafenib combined with AMD3100 group ,AMD3100 2 .5 mg/(kg · d) was administrated by intraperitoneal injection based on the method of sorafenib group .In the control group ,0 .9% sodium chloride solution was given by garage . The intrahepatic invasion , the tumor volume ,the situation of pulmonary metastasis and the survival time of nude mice ,were compared among the three groups . Results:When sorafenib was combined with AMD3100 ,the intrahepatic invasion and metastasis of HCC caused by sorafenib could be significantly inhibited .Thus the tumor volume was decreased and the pulmonary metastasis could be reduced .The survival time was prolonged .Conclusions:AMD3100 ,a SDF1‐α receptor inhibitor ,can enhance the efficacy of sorafenib .

Many advances have been achieved in the clinical and basic studies on metastasis and recurrence of hepatocellular carcinoma (HCC) over the past 20 years.The achievements mainly include the following aspects:(1) a group of molecules related to metastasis and recurrence including osteopontin have been identified,and multi-molecular predictive models for metastasis have been established and optimized;(2) it has been found that the imbalance of immune response in tumor microenvironment is important in promoting metastasis and can be used to predict metastasis and recurrence;(3) it has been found and confirmed that interferon can prevent postoperative recurrence,and patients with a lower miR-26a expression level can achieve greater benefits;(4) the breakthroughs in liquid biopsy and immunotherapy bring a promising future for the prediction,prevention,and treatment of HCC metastasis and recurrence.However,these predictive models still need to be validated by multi-center studies,and the effects of adjuvant transarterial chemoembolization and targeted therapy with sorafenib still need further evaluation.

Objective To explore the effect and mechanism of liver X receptor agonist T0901317 on angiogenesis phenotype of liver cancer.Methods The experimental study was conducted.Hepatocellular carcinoma MHCC97-H and Huh7 cells and human umbilical vein endothelial cells (HUVEC) were cultured in vitro.Each cell line was divided into 3 groups:control group (non-treated),low concentration group (treated using 1 μmot/L T0901317) and high concentration group (treated using 3 μmol/L T0901317).Cell proliferation was counted with a CCK-8 assay.Quantitative real-time polymerase chain reaction (PCR) was applied to confirm the relative mRNA expression of fatty acid synthetase (FAS) of liver X receptor target genes in 3 groups.Subcutaneous xenograft tumor volume and body mass were measured in MHCC97-H nude mice model.Then mice were sacrificed and tumor tissues were analyzed for CD31 relative expression by immunohistochemistry (IHC) staining.Migration and vessel angiogenesis of HUVEC were determined by Transwell method.Observation indicators:(1) effects of T0901317 on MHCC97-H,Huh7 and HUVEC cells proliferation,(2) effects of T0901317 on liver X receptor with MHCC97-H,Huh7 and HUVEC cells,(3) effects of T0901317 on subcutaneous xenograft tumor growth in MHCC97-H nude mice model,(4) effects of T0901317 on CD31 relative expression in subcutaneous xenograft tumor tissues of MHCC97-H nude mice model,(5) effects of T0901317 on migration of HUVEC,(6) effects of T0901317 on vessel angiogenesis of HUVEC.Measurement data with normal distribution were represented as x±s,and comparisons between groups were analyzed by the t test.Results (1)Effects of T0901317 on MHCC97-H,Huh7 and HUVEC cells proliferation:results of CCK-8 assay showed that percentage of living cells was respectively 100.0％± 1.7％,101.0％±0.7％ and 104.6％± 1.9％ in MHCC97-H control,low concentration and high concentration groups,with no statistically significant difference (F =2.632,P＞0.05).Percentage of living cells was respectively 100.0％ ± 2.7％,97.6％ ± 2.4％ and 103.7％ ± 2.8％ in Huh7 control,low concentration and high concentration groups,with no statistically significant difference (F =1.404,P＞0.05).Percentage of living cells was respectively 100.0％ ±0.7％,100.7％± 1.2％ and 101.3％ ±0.8％ in HUVEC control,low concentration and high concentration groups,with no statistically significant difference (F=0.471,P＞0.05).(2) Effects of T0901317 on liver X receptor with MHCC97-H,Huh7 and HUVEC cells:results of quantitative real-time PCR showed that relative mRNA expressions of FAS in MHCC97-H control,low concentration and high concentration groups were respectively 100.0 ％±2.2％,658.5％±7.7％ and 1 241.0％± 106.8％,with a statistically significant difference among groups (F=46.227,P＜0.05),and with a statistically significant difference between MHCC97-H control group and MHCC97-H low concentration and high concentration groups (t =70.025,8.274,P ＜ 0.05) and between MHCC97-H low concentration and high concentration groups (t =4.222,P ＜ 0.05).Relative mRNA expressions of FAS in Huh7 control,low concentration and high concentration groups were respectively 100.0％ ± 15.8％,1 225.0％ ± 26.7 ％ and 2 015.0％ ± 215.1％,with a statistically significant difference among groups (F =49.402,P＜ 0.05),and with a statistically significant difference between Huh7 control group and Huh7 low concentration and high concentration groups (t=39.460,8.879,P＜0.05) and between Huh7 low concentration and high concentration groups (t =2.836,P ＜ 0.05).Relative mRNA expressions of FAS in HUVEC control,low concentration and high concentration groups were respectively 100.0％ ± 19.6％,790.8％ ± 116.5％ and 1 756.0％ ± 55.0％,with a statistically significant difference among groups (F=185.395,P＜0.05),and with a statistically significant difference between HUVEC control group and HUVEC low concentration and high concentration groups (t =7.639,34.375,P＜0.05) and between HUVEC low concentration and high concentration groups (t =7.488,P＜0.05).(3) Effects of T0901317 on subcutaneous xenograft tumor growth in MHCC97-H nude mice model:results of assay showed that subcutaneous xenograft tumor volume in MHCC97-H control group and MHCC97-H T0901317 group were respectively (935±72)mm3 and (552 ± 47)mm3,with a statistically significant difference between groups (t=4.449,P＜0.05).Body masses of nude mice model in MHCC97-H control group and MHCC97-H T0901317 group were respectively (23.8±0.8) g and (21.7± 1.7) g,with no statistically significant difference between groups (t =1.059,P＞0.05).(4) Effects of T0901317 on CD31 relative expression in subcutaneous xenograft tumor tissues of MHCC97-H nude mice model:results of IHC staining showed that CD31 relative expression in subcutaneous xenograft tumor tissues of MHCC97-H nude mice model was 100％±11％ and 35％±7％ in MHCC97-H control group and MHCC97-H T0901317 group,with a statistically significant difference between groups (t =4.919,P＜0.05).(5) Effects of T0901317 on migration of HUVEC:results of Transwell method showed that percentages of membrane cells in HUVEC control,low concentration and high concentration groups were respectively 100.0％±4.0％,57.3％±1.5％ and 32.7％± 1.7％,with a statistically significant difference among groups (F=163.944,P＜0.05),and with statistically significant differences between HUVEC control group and HUVEC low concentration and high concentration groups (t =9.998,15.434,P＜0.05) and between HUVEC low concentration and high concentration groups (t =10.801,P ＜ 0.05).(6) Effects of T0901317 on vessel angiogenesis of HUVEC:results of vessel angiogenesis assay showed that length of vessel angiogenesis in HUVEC control,low concentration and high concentration groups were respectively 100.0％±3.4％,68.4％ ±3.5％ and 44.7％± 0.5％,with a statistically significant difference among groups (F =38.964,P ＜ 0.05),and with statistically significant differences between HUVEC control group and HUVEC low concentration and high concentration groups (t=6.268,9.831,P＜0.05) and between HUVEC low concentration and high concentration groups (t =3.460,P＜0.05).Conclusion Liver X receptor agonist T0901317 can inhibit vessel angiogenesis of liver cancer.

Cabozantinib, mainly targeting cMet and vascular endothelial growth factor receptor 2, is the second-line treatment for patients with advanced hepatocellular carcinoma (HCC). However, the lower response rate and resistance limit its enduring clinical benefit. In this study, we found that cMet-low HCC cells showed primary resistance to cMet inhibitors, and the combination of cabozantinib and mammalian target of rapamycin (mTOR) inhibitor, rapamycin, exhibited a synergistic inhibitory effect on the in vitro cell proliferation and in vivo tumor growth of these cells. Mechanically, the combination of rapamycin with cabozantinib resulted in the remarkable inhibition of AKT, extracellular signal-regulated protein kinases, mTOR, and common downstream signal molecules of receptor tyrosine kinases; decreased cyclin D1 expression; and induced cell cycle arrest. Meanwhile, rapamycin enhanced the inhibitory effects of cabozantinib on the migration and tubule formation of human umbilical vascular endothelial cells and human growth factor-induced invasion of cMet inhibitor-resistant HCC cells under hypoxia condition. These effects were further validated in xenograft models. In conclusion, our findings uncover a potential combination therapy of cabozantinib and rapamycin to combat cabozantinib-resistant HCC.
Anilides/pharmacology* ; Animals ; Carcinoma, Hepatocellular/drug therapy* ; Cell Line, Tumor ; Cell Proliferation ; Endothelial Cells/metabolism* ; Humans ; Liver Neoplasms/drug therapy* ; Pyridines/pharmacology* ; Sirolimus/pharmacology* ; Xenograft Model Antitumor Assays