Baicalin protects against hepatocyte injury caused by aflatoxin B1 via the TP53-related ferroptosis Pathway.

OBJECTIVE: Baicalin has antioxidative, antiviral, and anti-inflammatory properties. However, its ability to alleviate oxidative stress (OS) and DNA damage in liver cells exposed to aflatoxin B1 (AFB1), a highly hepatotoxic compound, remains uncertain. In this study, the protective effects of baicalin on AFB1-induced hepatocyte injury and the mechanisms underlying those effects were investigated. METHODS: Stable cell lines expressing CYP3A4 were established using lentiviral vectors to assess oxidative stress levels by conducting assays to determine the content of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD). Additionally, DNA damage was evaluated by 8-hydroxy-2-deoxyguanosine (8-OHdG) and comet assays. Transcriptome sequencing, molecular docking, and in vitro experiments were conducted to determine the mechanisms underlying the effects of baicalin on AFB1-induced hepatocyte injury. In vivo, a rat model of hepatocyte injury induced by AFB1 was used to evaluate the effects of baicalin. RESULTS: In vitro, baicalin significantly attenuated AFB1-induced injury caused due to OS, as determined by a decrease in ROS, MDA, and SOD levels. Baicalin also considerably decreased AFB1-induced DNA damage in hepatocytes. This protective effect of baicalin was found to be closely associated with the TP53-mediated ferroptosis pathway. To elaborate, baicalin physically interacts with P53, leading to the suppression of the expression of GPX4 and SLC7A11, which in turn inhibits ferroptosis. In vivo findings showed that baicalin decreased DNA damage and ferroptosis in AFB1-treated rat liver tissues, as determined by a decrease in the expression of γ-H2AX and an increase in GPX4 and SLC7A11 levels. Overexpression of TP53 weakened the protective effects of baicalin. CONCLUSIONS: Baicalin can alleviate AFB1-induced OS and DNA damage in liver cells via the TP53-mediated ferroptosis pathway. In this study, a theoretical foundation was established for the use of baicalin in protecting the liver from the toxic effects of AFB1.

Clinical implications and molecular mechanisms of Cyclin-dependent kinases 4 for patients with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) was frequently considered as a kind of malignant tumor with a poor prognosis. Cyclin-dependent kinases (CDK) 4 was considered to be cell-cycle-related CDK gene. In this study, we explored the clinical significance of CDK4 in HCC patients. METHODS: Data of HCC patients were obtained from The Cancer Genome Atlas database (TCGA) and the Gene Expression Omnibus (GEO) database. Kaplan-Meier analysis and Cox regression model were performed to calculate median survival time (MST) and the hazard ration (HR), respectively. The joint-effect analysis and prognostic risk score model were constructed to demonstrate significance of prognosis-related genes. The differential expression of prognostic genes was further validated using reverse transcription-quantitative PCR (RT-qPCR) of 58 pairs of HCC samples. RESULTS: CDK1 and CDK4 were considered prognostic genes in TCGA and GSE14520 cohort. The result of joint-effect model indicated patients in CDK1 and CDK4 low expression groups had a better prognosis in TCGA (adjusted HR = 0.491; adjusted P = 0.003) and GSE14520 cohort (adjusted HR = 0.431; adjusted P = 0.002). Regarding Kaplan-Meier analysis, high expression of CDK1 and CDK4 was related to poor prognosis in both the TCGA (P < 0.001 and = 0.001 for CDK1 and CDK4, respectively) and the GSE14520 cohort (P = 0.006 and = 0.033 for CDK1 and CDK4, respectively). However, only CDK4 (P = 0.042) was validated in RT-qPCR experiment, while CDK1 (P = 0.075) was not. CONCLUSION: HCC patients with high CDK4 expression have poor prognosis, and CDK4 could be a potential candidate diagnostic biomarker for HCC.

High-Mobility Group AT-Hook 1 Served as a Prognosis Biomarker and Associated with Immune Infiltrate in Hepatocellular Carcinoma.

BACKGROUND: The protein high-mobility group AT-hook 1 (HMGA1) has been demonstrated that modulated cellular proliferation, invasion, and apoptosis with a poor prognosis in miscellaneous carcinomas. However, the mechanism of circumstantial carcinogenesis and association with the immune microenvironment of HMGA1 in hepatocellular carcinoma (HCC) had not been extensively explored. METHODS: The gene expression, clinicopathological correlation, and prognosis analysis were performed in the data obtained from TCGA. The results were further validated by ICGC and GEO database and external validation cohort from Guangxi. The HMGA1 protein expression was further examined in the HPA database. Biological function analyses were conducted by GSEA, STRING database, and Coexpedia online tool. Using TIMER and CIBERSORT method, the relationship between immune infiltrate and HMGA1 was investigated. RESULTS: In HCC, HMGA1 had much higher transcriptional and proteomic expression than in corresponding paraneoplastic tissue. Patients with high HMGA1 expression had a poor prognosis and unpromising clinicopathological features. High HMGA1 expression was closely related to the cell cycle, tumorigenesis, substance metabolism, and immune processes by regulating complex signaling pathways. Notably, HMGA1 may be associated with TP53 mutational carcinogenesis. Moreover, increased HMGA1 expression may lead to an increase in immune infiltration and a decrease in tumor purity in HCC. CIBERSORT analysis elucidated that the amount of B cell naive, B cell memory, T cells gamma delta, macrophages M2, and mast cell resting decreased when HMGA1 expression was high, whereas T cells follicular helper, macrophages M0, and Dendritic cells resting increased. CONCLUSION: In conclusions, HMGA1 is a potent prognostic biomarker and a sign of immune infiltration in HCC, which may be a potential immunotherapy target for HCC.

Clinical Significance and Potential Mechanisms of ATP Binding Cassette Subfamily C Genes in Hepatocellular Carcinoma.

The purpose of this investigation was to assess the diagnostic and prognostic significance of ATP binding cassette subfamily C (ABCC) genes in hepatocellular carcinoma (HCC). The Student t-test was used to compare the expression level of ABCCs between HCC and paraneoplastic tissues. Receiver operating characteristic curve (ROC) analysis was applied for diagnostic efficiency assessment. The Kaplan-Meier method and Cox proportional hazards model were respectively applied for survival analysis. Genes with prognostic significance were subsequently used to construct prognostic models. From the perspective of genome-wide enrichment analysis, the mechanisms of prognosis-related ABCC genes were attempted to be elaborated by gene set enrichment analysis (GSEA). It was observed in the TCGA database that ABCC1, ABCC4, ABCC5, and ABCC10 were significantly upregulated in tumor tissues, while ABCC6 and ABCC7 were downregulated in HCC tissues. Receiver operating characteristic analysis revealed that ABCC7 might be a potential diagnostic biomarker in HCC. ABCC1, ABCC4, ABCC5, and ABCC6 were significantly related to the prognosis of HCC in the TCGA database. The prognostic significance of ABCC1, ABCC4, ABCC5, and ABCC6 was also observed in the Guangxi cohort. In the Guangxi cohort, both polymerase chain reaction and IHC (immunohistochemical) assays demonstrated higher expression of ABCC1, ABCC4, and ABCC5 in HCC compared to liver tissues, while the opposite was true for ABCC6. GSEA analysis indicated that ABCC1 was associated with tumor differentiation, nod-like receptor signal pathway, and so forth. It also revealed that ABCC4 might play a role in HCC by regulating epithelial-mesenchymal transition, cytidine analog pathway, met pathway, and so forth. ABCC5 might be associated with the fatty acid metabolism and KRT19 in HCC. ABCC6 might impact the cell cycle in HCC by regulating E2F1 and myc. The relationship between ABCC genes and immune infiltration was explored, and ABCC1,4,5 were found to be positively associated with infiltration of multiple immune cells, while ABCC6 was found to be the opposite. In conclusion, ABCC1, ABCC4, ABCC5, and ABCC6 might be prognostic biomarkers in HCC. The prognostic models constructed with ABCC1, ABCC4, ABCC5, and ABCC6 had satisfactory efficacy.

CYP2C8 Suppress Proliferation, Migration, Invasion and Sorafenib Resistance of Hepatocellular Carcinoma via PI3K/Akt/p27kip1 Axis.

BACKGROUND: Cytochrome P450 2C8 (CYP2C8) gene is one of the members of the cytochrome P450 enzymes (CYPs) gene family. The aim of this study was to reveal the function of CYP2C8 in hepatocellular carcinoma (HCC) and its effect on the sorafenib resistance. METHODS: Differential expression analysis in multiple HCC datasets all suggested that CYP2C8 expression was significantly decreased in HCC tissues, compared with para-carcinoma liver tissues. The expression level of CYP2C8 was subsequently compared between HCC tissues and para-carcinoma liver tissues of 70 patients form Guangxi, China, with the result consistent with the above. Survival analysis and ROC analysis indicated that CYP2C8 was equipped with satisfactory diagnostic and prognostic value in HCC. To examine the effect of CYP2C8 on the malignant phenotype of HCC cells, stable transcriptional cell lines with CYP2C8 over-expression were established, and then Cell Counting Kit-8 (CCK8) assay, colony formation assay, cell cycle assay, cell invasion assay and wound healing assay were performed. RESULTS: The results of aforementioned assays suggested that CYP2C8 over-expression restricted the proliferation, clonality, migration, invasion and cell cycle of HCC cells but had no significant effect on cell apoptosis. The enrichment analysis in terms of sequencing data of HCC cell lines with stable CYP2C8 over-expression suggested that CYP2C8 might be related to PI3K/Akt/p27Kip1 axis. The inhibition of CYP2C8 over-expression on PI3K/Akt/p27Kip1 axis was subsequently demonstrated with Western blot assay. In the rescue experiment, it was observed that both P27 inhibitor and PI3K agonist counteracted the repressed malignant phenotype caused by CYP2C8 over-expression, which further demonstrated that CYP2C8 played a role in HCC cells via PI3K/Akt/p27Kip1 axis. DISCUSSION: The results demonstrated that CYP2C8 enhances the anticancer activity of sorafenib in vitro assays and in tumor xenograft model, with Ki-67 down-regulation and PI3K/Akt/p27Kip1 axis inhibition. In conclusion, these findings hinted that CYP2C8 restricted malignant phenotype and sorafenib resistance in HCC via PI3K/Akt/p27kip1 axis.