BACKGROUND: Glutamine synthetase (GS) and arginase 1 (Arg1) are widely used pathological markers that discriminate hepatocellular carcinoma (HCC) from intrahepatic cholangiocarcinoma; however, their clinical significance in HCC remains unclear. METHODS: We retrospectively analyzed 431 HCC patients: 251 received hepatectomy alone, and the other 180 received sorafenib as adjuvant treatment after hepatectomy. Expression of GS and Arg1 in tumor specimens was evaluated using immunostaining. mRNA sequencing and immunostaining to detect progenitor markers (cytokeratin 19 [CK19] and epithelial cell adhesion molecule [EpCAM]) and mutant TP53 were also conducted. RESULTS: Up to 72.4% (312/431) of HCC tumors were GS positive (GS+). Of the patients receiving hepatectomy alone, GS negative (GS-) patients had significantly better overall survival (OS) and recurrence-free survival (RFS) than GS+ patients; negative expression of Arg1, which is exclusively expressed in GS- hepatocytes in the healthy liver, had a negative effect on prognosis. Of the patients with a high risk of recurrence who received additional sorafenib treatment, GS- patients tended to have better RFS than GS+ patients, regardless of the expression status of Arg1. GS+ HCC tumors exhibit many features of the established proliferation molecular stratification subtype, including poor differentiation, high alpha-fetoprotein levels, increased progenitor tumor cells, TP53 mutation, and upregulation of multiple tumor-related signaling pathways. CONCLUSIONS GS- HCC patients have a better prognosis and are more likely to benefit from sorafenib treatment after hepatectomy. Immunostaining of GS may provide a simple and applicable approach for HCC molecular stratification to predict prognosis and guide targeted therapy.
Humans ; Carcinoma, Hepatocellular/metabolism* ; Sorafenib/therapeutic use* ; Liver Neoplasms/metabolism* ; Glutamate-Ammonia Ligase/metabolism* ; Hepatectomy ; Retrospective Studies ; Prognosis ; Neoplasm Recurrence, Local/surgery*

Objective: To investigate the effect and possible mechanism of Y-box-binding protein 1 (YB-1) on sorafenib resistance in hepatoma cells. Methods: Lentiviral vectors with YB-1 overexpression and knockdown were constructed, respectively, to stimulate human hepatoma cell lines (HepG2 and Huh7) alone or in combination with sorafenib.The overexpression part of the experiment was divided into four groups: overexpression control group (Lv-NC), YB-1 overexpression group (Lv-YB-1), overexpression control combined with sorafenib resistance group (Lv-NC+sorafenib), YB-1 overexpression combined with sorafenib resistance group (Lv-YB-1 + sorafenib). The knockdown part of the experiment was also divided into four groups: knockdown control group (Lv-shNC), YB-1 knockdown group (Lv-shYB-1), knockdown control combined with sorafenib resistance group (Lv-shNC + sorafenib), YB-1 knockdown combined with sorafenib resistance group (Lv-shYB-1 + sorafenib). The occurrence of cell apoptosis was detected by TUNEL. The protein expression levels of phosphorylated (p)-ERK and ERK, key proteins in the extracellular regulatory protein kinase (ERK) signaling pathway, were detected by Western blot and quantified by ImageJ software. Subcutaneous tumorigenesis experiments were performed in nude mice. The effect of YB-1 on the efficacy of sorafenib was verified in vivo. The comparison between the two sets of data was carried out by an independent sample t-test. One-way ANOVA was used for comparisons between the three groups of data above. Results: Sorafenib had accelerated the occurrence of apoptosis in hepatoma cells, while YB-1 overexpression had inhibited cell apoptosis, and at the same time also inhibited the apoptosis-accelerating impact of sorafenib. On the contrary, YB-1 knockdown accelerated cell apoptosis and amplified the induction effect of sorafenib on apoptosis. Furthermore, sorafenib resistance had down-regulated p-ERK levels (HepG2: Lv-NC 0.685 ± 0.143, Lv-NC + sorafenib 0.315 ± 0.168, P < 0.05; Huh7: Lv-NC 0.576 ± 0.078, Lv-NC + sorafenib 0.150 ± 0.131, P < 0.01), whereas YB-1 overexpression had inhibited sorafenib resistance p-ERK reduction (HepG2: Lv-NC + sorafenib 0.315 ± 0.168, Lv-YB-1 + sorafenib 0.688 ± 0.042, P < 0.05; Huh7: Lv-NC + sorafenib 0.150 ± 0.131, Lv-YB-1 + sorafenib 0.553 ± 0.041, P < 0.05). YB-1 knockdown further increased sorafenib-induced p-ERK downregulation (HepG2: Lv-shNC + sorafenib 0.911 ± 0.252, Lv-shYB-1 + sorafenib 0.500 ± 0.201, P < 0.05; Huh7: Lv-shNC + sorafenib 0.577 ± 0.082, Lv-shYB-1 + sorafenib 0.350 ± 0.143, P < 0.05), which was further verified in naked mice (Lv-shNC + sorafenib 0.812 ± 0.279, Lv-shYB-1 + sorafenib 0.352 ± 0.109, P < 0.05). Conclusion: YB-1 mediates the occurrence of sorafenib resistance via the ERK signaling pathway in hepatoma cells.
Humans ; Cell Line, Tumor ; Sorafenib/pharmacology* ; Drug Resistance, Neoplasm ; Y-Box-Binding Protein 1/metabolism* ; Carcinoma, Hepatocellular/metabolism* ; MAP Kinase Signaling System ; Animals ; Mice ; Mice, Nude

RNA modifications affect many biological processes and physiological diseases. The 5-methylcytosine (m⁵C) modification regulates the progression of multiple tumors. However, its characteristics and functions in hepatocellular carcinoma (HCC) remain largely unknown. Here, we found that HCC tissues had a higher m⁵C methylation level than the adjacent normal tissues. Transcriptome analysis revealed that the hypermethylated genes mainly participated in the phosphokinase signaling pathways, such as the Ras and PI3K-Akt pathways. The m⁵C methyltransferase NSUN2 was highly expressed in HCC tissues. Interestingly, the expression of many genes was positively correlated with the expression of NSUN2, including GRB2, RNF115, AATF, ADAM15, RTN3, and HDGF. Real-time PCR assays further revealed that the expression of the mRNAs of GRB2, RNF115, and AATF decreased significantly with the down-regulation of NSUN2 expression in HCC cells. Furthermore, NSUN2 could regulate the cellular sensitivity of HCC cells to sorafenib via modulating the Ras signaling pathway. Moreover, knocking down NSUN2 caused cell cycle arrest. Taken together, our study demonstrates the vital role of NSUN2 in the progression of HCC.
Humans ; ADAM Proteins ; Apoptosis Regulatory Proteins ; Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/metabolism* ; Membrane Proteins ; Methyltransferases/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Repressor Proteins ; RNA, Messenger/metabolism* ; Sorafenib ; RNA Methylation/genetics*

OBJECTIVE: To conduct a pan-cancer analysis of the expression of long non-coding RNA (lncRNA) MIR22HG and explore its association with clinical characteristics. METHODS: We analyzed the expression of MIR22HG in different tumors and its association with clinical staging, lymph node metastasis, tumor mutation burden (TMB) and microsatellite instability (MSI) using R package based on the Cancer Genome Atlas (TCGA) datasets. The relationship between MIR22HG expression and infiltrating immune cells was analyzed using TIMER algorithm. The association of MIR22HG gene alteration frequency with the clinical outcomes was examined using cBioPortal online software. Data form Genomics of Drug Sensitivity in Cancer (GDSC) were used to analyze the relationship between MIR22HG and the sensitivity of chemotherapy drugs. We specifically analyzed MIR22HG expression in hepatocellular carcinoma (HCC) and its correlation with sorafenib treatment using GEO database and verified the results in 12 pairs of HCC specimens. Kaplan-Meier analysis was performed to analyze the correlation of MIR22HG with the outcomes of sorafenib treatment. We also tested the effects of MIR22HG overexpression and knockdown on IC₅₀ of sorafenib in HCC cells. RESULTS: MIR22HG was downregulated in most tumors (P < 0.05), where its deletion mutations were frequent, and associated with a poor prognosis (P < 0.05). In many tumors, MIR22HG expression level was correlated with clinical stage, lymph node metastasis, TMB, MSI, immune cell infiltration, immune checkpoint-related genes, and sensitivity to common chemotherapeutic drugs (P < 0.05). Among the 6 common infiltrating immune cells in cancers, neutrophil infiltration had the strongest correlation with MIR22HG expression level, especially in breast cancer, rectal cancer and kidney renal papillary cell carcinoma (P < 0.05). MIR22HG was downregulated in HCC in association with HCC progression (P < 0.05). In HCC patients, a low MIR22HG expression was associated with a favorable outcome after sorafenib treatment (HR=2.94, P=0.075) and was capable of predicting the response to sorafenib treatment (AUC=0.8095). Compared with the negative control, MIR22HG overexpression obviously reduced sorafenib sensitivity (with IC₅₀ of 7.731 vs 15.61) while MIR22HG knockdown increased sorafenib sensitivity of HCC cells (with IC₅₀ of 7.986 vs 5.085). CONCLUSION MIR22HG expression level is correlated with clinical stage, lymph node metastasis, TMB, MSI, immune cell infiltration, and chemosensitivity in most cancer, suggesting its potential as an immunotherapeutic target and also a prognostic biomarker for tumors.
Biomarkers, Tumor/genetics* ; Carcinoma, Hepatocellular/pathology* ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms/pathology* ; Lymphatic Metastasis ; Microsatellite Instability ; RNA, Long Noncoding/genetics* ; Sorafenib/pharmacology*

Targeted and immunotherapy drugs for hepatocellular carcinoma (HCC) have been rapidly developed. Atezolizumab in combination with bevacizumab has been recommended as the first-line standard of care for unresectable or advanced HCC in several national and international guidelines. The combination therapies with sindilizumab and bevacizumab biosimilar, apatinib and carrilizumab, dulvalizumab and tremelimumab are also recommended as first-line standard regimens for advanced HCC in the guideline of Chinese Society of Clinical Oncology. Local therapy combined with targeted drugs (such as sorafenib and lenvatinib) or immune checkpoint inhibitors can significantly improve outcomes. Therefore, some progress has also been made in the study of single-agent or combination regimens as perioperative neoadjuvant therapy.
Humans ; Carcinoma, Hepatocellular/pathology* ; Sorafenib/therapeutic use* ; Liver Neoplasms/pathology* ; Immune Checkpoint Inhibitors/therapeutic use* ; Bevacizumab/therapeutic use* ; Biosimilar Pharmaceuticals/therapeutic use*

Brachytherapy ; Carcinoma, Hepatocellular/drug therapy* ; Chemoembolization, Therapeutic ; Combined Modality Therapy ; Humans ; Iodine Radioisotopes ; Liver Neoplasms/drug therapy* ; Portal Vein ; Retrospective Studies ; Sorafenib/therapeutic use* ; Thrombosis ; Treatment Outcome

OBJECTIVE: To compare the effects of medical ozone oil and urea ointment for prevention and treatment of hand-foot skin reaction (HFSR) caused by sorafenib in patients with hepatocellular carcinoma (HCC). METHODS: A total of 99 patients diagnosed with advanced HCC according to National Comprehensive Cancer Network (NCCN) who were scheduled to receive sorafenib treatment for the first time were enrolled in this study between April, 2018 and January, 2020. The patients were randomized into medical ozone oil group ( RESULTS: Eight patients were excluded for poor compliance or protocol violations, leaving a total of 91 patients for analysis, including 44 in medical ozone oil group and 47 in urea ointment group. Sixteen (36.4%) of patients in ozone oil group developed HFSR, a rate significantly lower than that in urea ointment group (57.4%; CONCLUSIONS Medical ozone oil can significantly reduce the incidence and severity of HFSR to improve the quality of life of HCC patients receiving sorafenib treatment.
Antineoplastic Agents/therapeutic use* ; Carcinoma, Hepatocellular/drug therapy* ; Hand-Foot Syndrome/prevention & control* ; Humans ; Liver Neoplasms/drug therapy* ; Niacinamide/therapeutic use* ; Ozone/therapeutic use* ; Phenylurea Compounds/adverse effects* ; Quality of Life ; Sorafenib/therapeutic use*

OBJECTIVE: To establish the drug-resistant cell lines of hepatocellular carcinoma (HCC) induced by sorafenib, and to screen out the high expression genes in drug-resistant cell lines of HCC induced by sorafenib, then to explore the genes related to sorafenib resistance in hepatocellular carcinoma. METHODS: The human PLC and Huh7 cell lines were obtained, then the PLC and Huh7 drug-resistant cell lines were induced with sorafenib by using intermittent induction in vitro. CCK8 assay was used to detect the IC50 value of sorafenib for evaluation of drug sensitivity of hepatocellular carcinoma cell lines in PLC and Huh7. All the up regulated genes in PLC and Huh7 drug-resistant cell lines induced by sorafenib were screened out using high-throughput cDNA sequencing (RNA-Seq), Ualcan database was used to analyze the correlations between the up regulated genes in PLC and Huh7 drug-resistant cell lines induced and four clinical biological characteristics of hepatocellular carcinoma, including the gene expressions between normal samples and tumor samples, tumor stage, tumor grade, and patient overall survival, to find the genes that might be involved in the mechanism of sorafenib resistance of hepatocellular carcinoma. RESULTS: All the up regulated genes detected by the using high-throughput cDNA sequencing (RNA-Seq) in PLC and Huh7 drug-resistant cell lines were further screened out by following conditions:(1) genes co-expressed in PLC and Huh7 drug-resistant cells induced by sorafenib, (2) the fold change was more than 4 times and the difference was statistically significant (P <0.05), the top 12 up regulated genes in PLC and Huh7 drug-resistant cell lines were found, which were TPSG1, CBX4, CLC, CLEC18C, LGI4, F2RL1, S100A6, HABP2, C15ORF48, ZG16, FOLH1, and EPCAM. Compared with the correlations between the twelve genes and the clinical biological characteristics by Ualcan database, the potentially significant gene CBX4 was screened out. CONCLUSION The human PLC and Huh7 drug-resistant cell lines of hepatocellular carcinoma induced by sorafenib were successfully established. CBX4, the gene related to sorafenib resistance in hepatocellular carcinoma, was screened out by the high-throughput cDNA sequencing (RNA-Seq) and further analysis using Ualcan database, which is providing a powerful basis for further research on the mechanism of sorafenib resistance of hepatocellular carcinoma.
Antineoplastic Agents/therapeutic use* ; Carcinoma, Hepatocellular/drug therapy* ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; Humans ; Ligases ; Liver Neoplasms/drug therapy* ; Polycomb-Group Proteins ; Serine Endopeptidases ; Sorafenib/therapeutic use*

OBJECTIVE: To explore the molecular mechanism of sorafenib resistance in hepatoma cells and identify for new targets to reverse drug resistance. METHODS: THP-1 cells were induced into M2 tumor-associated macrophages (M2-TAMs) in vitro and identified by immunofluorescence. SMMC-7721 cells were co-cultured with M2-TAMs with or without sorafenib treatment. CCK-8 assay was used to observe the inhibitory effect of sorafenib on the cell proliferation. Annexin V/PI double staining and protein immunoblotting were used to assess the effect of sorafenib on the proliferation, apoptosis and the expressions of apoptosis-related proteins and autophagy-related protein in SMMC-7721 cells co-cultured with M2-TAMs in the presence or absence of the autophagy inhibitor chloroquine (CQ). RESULTS: The IC of sorafenib at 48 h was 2.25 μmol/L in SMMC-7721 cells cultured alone, and increased to 4.72 μmol/L in the cells co-cultured with M2-TAMs. Compared with the cells cultured alone, the co-cultured SMMC-7721 cells showed significantly reduced apoptosis rate in response to sorafenib ( < 0.01) and significantly increased expression of Bcl-2 and Bcl-2/Bax ratio ( < 0.05) with also increased LC3-II/LC3-I ratio ( < 0.001) and lowered expression of p62 ( < 0.05), suggesting a significantly enhanced level of autophagy. CQ treatment significantly inhibited the proliferation of the co-cultured SMMC-7721 cells ( < 0.05), increased the cell apoptosis ( < 0.05) and reduced the Bcl-2/Bax ratio ( < 0.01). CONCLUSIONS M2-TAMs can attenuate the inhibitory effect of sorafenib on the proliferation of hepatoma cells by increasing the level of autophagy, suggesting a new strategy for reversing sorafenib resistance induced by the tumor microenvironment by inhibiting autophagy.
Apoptosis ; Autophagy ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Humans ; Liver Neoplasms ; Macrophages ; Sorafenib

OBJECTIVE: To investigate the role of E26 transformation-specific variant 4 (ETV4) in sorafenib and cisplatin resistance in hepatocellular carcinoma (HCC). METHODS: HCC cell lines SMMC-7721 and HCC-LM3 were transfected with an ETV4- overexpressing plasmid or small interfering RNAs (siRNAs) targeting ETV4. The cells with ETV4 overexpression or ETV4 interference were treated with DMSO, sorafenib (5 μmol/L) or cisplatin (5 μmol/L) for 48 h, and the total protein and total RNA were collected. Western blotting, flow cytometry, EdU proliferation assay were used to analyze the apoptosis and proliferation of the cells. We also obtained clinical specimens of HCC tissues and paired adjacent tissues from 11 patients for detecting ETV4 mRNA expression levels using real-time fluorescence quantitative PCR (q-PCR). The effect of ETV4 interference on the mRNA expression levels of immediate early response gene 3 (IER3) was examined in HCC cells that were treated with DMSO, sorafenib or cisplatin for 48 h. RESULTS: The expression of ETV4 mRNA was significantly higher in HCC tissues than in the paired adjacent tissues. Overexpression of ETV4 in the HCC cell lines obviously inhibited cell apoptosis induced by sorafenib or cisplatin. Conversely, ETV4 interference significantly enhanced the apoptosis and inhibited the proliferation of the HCC cells following treatments with sorafenib or cisplatin. In addition, ETV4 regulated the mRNA expression levels of IER3 in the cells treatmed with sorafenib and cisplatin. CONCLUSIONS ETV4 promotes resistance of HCC cells to sorafenib or cisplatin .
Apoptosis ; Apoptosis Regulatory Proteins ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Cell Proliferation ; Cisplatin ; Drug Resistance, Neoplasm ; Humans ; Liver Neoplasms ; Membrane Proteins ; Niacinamide ; Phenylurea Compounds ; Sorafenib