CYP2E1 deficit mediates cholic acid-induced malignant growth in hepatocellular carcinoma cells

CYP2E1 deficit mediates cholic acid-induced malignant growth in hepatocellular carcinoma cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CYP2E1 deficit mediates cholic acid-induced malignant growth in hepatocellular carcinoma cells Zhiwei Hao, Xuemin Liu, Huanhuan He, Zhixuan Wei, Xiji Shu, Jianzhi Wang, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4070826/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background An increased serum cholic acid (CA) is concurrently appeared with a decreased CYP2E1 expression in hepatocellular carcinoma (HCC). However, whether and how CA may be involved in promoting hepatocarcinogenesis has not been elucidated. This study was aimed to investigate the role of CYP2E1 on CA-induced HCC cell growth and its underlying mechanism. Methods Our proteomic analysis of liver tumor tissues from DEN-induced male SD rats treated with CA administration revealed that CA downregulated CYP2E1 level. The proliferative ability of CA-treated HCC cells was examined by colony formation assays. Autophagic flux was detected by immunofluorescence and confocal microscopy. The protein levels of CYP2E1, mTOR, AKT, p62 and LC3Ⅱ were detected by Immunoblotting. The effect of CYP2E1 on CA-induced hepatocellular carcinogenesis was explored in vivo by establishing a xenograft tumor model in nude mice. We also investigated the clinical value of CYP2E1 in HCC patients. Results CA increased the clonogenicity of HCC cells and promoted the growth of xenograft tumors with a simultaneous reduction of CYP2E1 expression. Further studies revealed that both in vitro and in vivo, upregulating CYP2E1 could effectively inhibit cell growth of HCC with a blockage of autophagic flux, downregulation of AKT phosphorylation and upregulation of mTOR phosphorylation. CYP2E1 was involved in CA-activated autophagy through the AKT/mTOR signaling, which promoted CA-stimulated HCC cell growth. Finally, a decreased CYP2E1 expression was observed in the tumor tissues of HCC patients and the CYP2E1 level in tumor was negatively correlated with the serum level of total bile acids (TBA) and gamma-glutamyltransferase (GGT). Conclusions CYP2E1 deficit contributes to CA-induced HCC development with the mechanisms involving regulation of autophagy, thus CYP2E1 may serve as a potential target for HCC drug development. Cholic acid CYP2E1 Hepatocellular cancer Cell growth Autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Primary liver cancer is the fourth most commonly diagnosed cancer and the second leading cause of cancer-related death in China with approximately 367,700 new cases and 316,500 deaths in 2022(Han et al. 2024 ). Hepatocellular carcinoma (HCC) accounts for the majority (>80%) of liver cancers (Tamai et al. 2023 ). Risk factors for HCC include chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), aflatoxin-contained food, alcohol consumption, metabolic syndrome, etc (Toh et al. 2023 ). Recently, bile acids (BAs) were also found to be associated with HCC development (Conde et al. 2021 ; Shen et al. 2022 ). Bile acids are metabolites from cholesterol and mainly synthesized in the liver. BAs serve as endocrine signaling molecules sensed by nuclear or membrane-localized receptors to trigger specific signaling pathways and regulate many biological processes (Song et al. 2020 ; Cai et al. 2022 ). Studies from clinical patients and animal models have shown an association of BAs abnormalities with liver diseases such as HCC (Stepien et al. 2022 ). Clinically, increased levels of primary and secondary BAs were detected in HCC and cholangiocarcinoma carcinoma (CCA) patients (Changbumrung et al. 1990 ). Children with the progressive familial intrahepatic cholestasis type 2 (PFIC type 2) disease, characterized by a genetic deficiency of the canalicular bile salt export pump BSEP or ABCB11, and severe cholestasis with elevated serum and liver BAs levels, such as CA and chenodesoxycholic acid (CDCA), is predisposed to HCC (Knisely et al. 2006 ; Baptissart et al. 2013 ). The experiments in mice have showed that a CA-enriched diet strongly promoted N-nitrosodiethylamine-induced liver carcinogenesis (Yang et al. 2007 ). Our studies have also shown that CA administration promoted DEN-induced initiation and progression of HCC. However, the molecular mechanisms by which BAs promote HCC is still unknown. Autophagy, a “self-eating” process that clears intracellular waste, is crucial for development, differentiation, survival, and homeostasis (Tabibzadeh 2023 ). Accumulating evidence indicates that autophagy can be upregulated in response to a multitude of stresses including starvation, hypoxia and intracellular pathogens, which in most contexts promotes tumorigenesis(Li, He, and Ma 2020 ). Increased autophagy activity with high LC3B expression benefits tumor proliferation, invasion, or metastasis (Wu et al. 2014 ). Autophagy is involved in HCC progression, resulting in drug resistance and poor prognosis of HCC patients (Shi et al. 2011 ; Li, He, and Ma 2020 ; Xu et al. 2020 ). The protective role of autophagy in cholestasis-induced liver injury was also reported (Gao et al. 2014 ). High levels of total bile acids (TBA) in HCC patients were found to be strongly correlated with their autophagy level and poor survival (Gao et al. 2019 ). Autophagy triggered by glycochenodeoxycholate (GCDC) is a protective mechanism in invasion and metastasis of HCC(Gao et al. 2019 ). How dysregulated levels of BAs influence autophagy, however, is still poorly understood Cytochrome P450 2E1 (CYP2E1), a member enzyme of the cytochrome P450 superfamily, is an important hepatic metabolic enzyme that is responsible the metabolism of xenobiotics including ethanol, acetone, drugs and procarcinogens (Torres et al. 2019 ; Yang et al. 2022 ). Research effort has been focused on its role in drug metabolism and alcoholic liver diseases for a long time. Therefore, the role of CYP2E1 in HCC development is largely unknown. Recent studies have shown that the level of CYP2E1 in the tumor tissues of HCC patients was significantly lower than the adjacent nontumor tissues and the cirrhotic and/or normal liver tissues (Ho et al. 2004 ). The level of CYP2E1 expression is negatively associated with aggressive tumor type and poor prognosis of HCC patients (Ho et al. 2004 ), suggesting the inhibitory activity of CYP2E1 in the initiation and progression of HCC. HepG2 cells overexpressing CYP2E1 exhibited a slow rate of cell growth accompanied with increased CYP2E1-induced cytotoxicity (Cederbaum et al. 2001 ; Alwadei et al. 2023 ). In DEN-induced HCC rat model, CYP2E1 expression was declined along with the initiation, promotion and progression of HCC(Sanchez-Meza et al. 2023 ). These studies indicate that downregulation of CYP2E1 expression is involved in HCC tumorigenesis. In this study, we aimed to investigate the involvement of CYP2E1 in CA-induced autophagy and promotion of malignant growth of hepatocellular carcinoma cells. We found that CA induced autophagy and promoted malignant growth of human hepatocellular carcinoma HepG2 and Huh7 cells, which is regulated by CYP2E1. Clinically, decreased level of CYP2E1 expression was found in the tumor tissues of HCC patients with high TBA level. Materials and Methods Chemicals and antibodies CA was purchased form Sigma-Aldrich (St Louis, MO, USA). MK-2206 dihydrochloride was obtained from MedChemExpress (USA). Antibodies against p62 (18420-1-AP), CYP2E1 (19937-1-AP), AKT (10176-2-AP) and p-AKT (66444-1-lg) were from Proteintech Group (Chicago, IL, USA). The mTOR (380411) antibody was from Zen BioScience (Chengdu, Sichuan, China). p-mTOR (#5536), LC3A/B (#12741) and Alexa Fluor®594 Conjugate (#8889S) were from Cell Signaling Technology (Danvers, MA, USA). β-actin (AF0003) was purchased from Beyotime Biotechnology (Nanjing, China). Alexa Fluor 488 (A21206) was from Sigma-Aldrich Co. (St. Louis, MO, USA). Clinical tumor samples HCC cancerous and para-cancerous tissues were obtained from Union Hospital, Tongji Medical College, Huazhong University of Science and Technology between May 2019 and December 2023.The samples were from forty men and six women aged 42–78 years (mean 61.7 years), and none of them had received any anticancer treatment prior to surgery. Cell culture and stable cell lines Human HCC HepG2 and Huh7 cell lines were obtained from Shenogen Pharma Group (Beijing, China), and maintained in DMEM with 10% FBS at 37℃ in a humidified incubator with 5% CO 2 . Stable CYP2E1 expression cell lines and their control cell lines were generated by stable transfection of pcDNA-CYP2E1and pcDNA empty vector (Fenghui Biotechnology Co., LTD) respectively. Then, the CYP2E1 expression in the stable cell lines were verified by Western blot analysis. Western blot analysis Cells and liver tissues were lysed with the RIPA buffer. Proteins were separated through SDS-PAGE, transferred onto a PVDF membrane that was blocked and incubated with the indicated antibody. Target proteins were detected by ECL assay System. Animal experiments Male BALB/c-nu mice (5 weeks old) were purchased from Beijing SiPeiFu Animal Technology Co. LTD and housed in specific pathogen-free conditions with the constant temperature (24 ± 2℃) and relative humidity (60%). HepG2-CYP2E1 cells and HepG2-vector cells (5×10 6 cells/mice) were injected into the liver of BALB/c-nu mice (n = 4) to establish orthotopic liver xenograft tumor model with or without intragastric administration of CA (0.2%, every 2 days) and maintained for 28 days. Tumor volume was calculated according to the following formula: tumor volume = (major axis) × (minor axis) 2 /2. Male Sprague-Dawley (SD) rats, 6 weeks old (~ 230-250g), were obtained from the Hunan SJA Laboratory Animal Co., LTD, China. Rats were intraperitoneally injected with DEN (Sigma Chemical Co., LTD) of 75 mg/kg per body weight once a week for three weeks, then with DEN of 100 mg/kg per body weight once a week for three weeks (Matsuzaki et al. 1992 ) and (Shiota et al. 1999 ). All rats were subsequently fed with or without CA for 23 weeks. After euthanization, the livers were removed. Preparation and measurement of liver tumors were performed by Biotree Biotech Co., LTD., Shanghai, China as a custom service. Immunofluorescence staining After fixing with ice-cold 4% paraformaldehyde for 15 min, and permeabilizing with 0.1% Triton X-100 for 20 min, the samples were incubated with LC3 and p62 antibodies overnight at 4 ℃, washed and then with the Alexa Fluor conjugated secondary antibody for 1 h. The nuclei were stained with Hoechst 33342 (1 µg/mL) for 10 min. The images were obtained under a fluorescence microscope (Leica TCS SP8). pmCherry-EGFP-LC3 puncta assay Cells were seeded in a 24-well plate, detected with the pmCherryEGFP-LC3b adenovirus (MiaoLingbio, China) for 6 h, incubated with CA for 24 h and examined with a confocal microscope (Leica TCS SP8). Clonogenic assay After seeding (4×10 3 cells/well), cells were treated with CA and cultured for 2 weeks. Then, the cell colonies were washed, fixed and stained with 0.1% crystal violet. The number of colonies were counted under a microscope. Immunohistochemistry staining After deparaffinizing, rehydrating gradually by gradient alcohol and incubating in a 3% hydrogen peroxide/methanol buffer, the slides were immersed in an ethylenediamine tetra acetic acid buffer (pH 8.0) and boiled for 5 min. Then, the slides were washed and incubated with the specific primary and secondary antibodies. Diaminobenzidine was used to generate signal, and images were captured. Gene and protein expression analysis GEPIA and UALCAN programs ( http://ualcan.path.uab.edu ) were used to analyze the relative mRNA expression of CYP2E1 in tumor and normal samples of HCC patients. The protein expression was analyzed using the data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC, http://ualcan.path.uab.edu/analysis-prot.html ). The GEPIA2.0 and Kaplan-Meier programs were then used for patient survival analysis. Statistical analysis Data were presented as the mean ± standard error of mean (SEM) using a GraphPad Prim 8.0 software with analysis of variance (ANOVA), followed by Bonferroni's post hoc tests. Statistical significance was set at p < 0.05. Spearman correlation coefficient was used to determine the correlations between abnormal distribution variables. Results CYP2E1 expression is downregulated in CA-treated hepatocellular carcinoma induced by DEN Our recent studies have found that CA administration promoted DEN-induced initiation and progression of HCC in rats. To explore the molecular mechanisms by which CA promotes hepatocellular carcinogenesis, liver tumor tissues from the DEN-treated, and both DEN and CA treated rats were used for whole-proteome label-free assessment (Fig. 1 A). Principal coordinates analysis (PCoA) showed a clear separation of proteins between these two groups (Fig. 1 B). One hundred and fifty-one proteins were expressed differentially between the tumor tissues from group DEN + CA and the group DEN (Fig. 1 C). The differentially expressed proteins between two groups were selected, according to the criteria of (log2 |fold-change| ≥ 1.2 and p < 0.05). The results showed that two hundred and seventy-three proteins were found upregulated, while one hundred and ninety-three were proteins downregulated in DEN, compared with DEN + CA (Fig. 1 D). Further analysis with the Protein-Protein Interaction Network Analysis using STRING database indicated involvement of three out of five enriched KEGG pathways (Fig S1 A). CYP2E1 expression was downregulated in the group DEN + CA rats (> 2-fold) compared to the DEN group. In Western blot analysis, we found that CYP2E1 expression was significantly downregulated in the liver tumors from the group DEN + CA compared to those in the group DEN (Fig. 1 E-F), consistent with the proteomics data. To examine whether CA regulates CYP2E1 expression, HepG2 and Huh7 cells were treated with different concentrations of CA for 24 h. Western blot analysis revealed that CA treatment reduced CYP2E1 expression in a dose-dependent manner (Fig. 2 A-B). In colony formation assays, CA significantly increased the colonogenicity of HCC cells at a concentration of 1 nM, compared to the vehicle control (Fig. 2 C-D). These results suggested that CA at the concentration of 1 nM was able to downregulate CYP2E1 expression and promote the malignant growth of HepG2 and Huh7 cells. CYP2E1 deficit contributes to CA-induced HCC growth with mechanism involving regulating autophagy. Bile acids such as glycochenodeoxycholate (GCDC) have been reported to induce autophagy, which promotes invasion and migration of HCC cells (Gao et al. 2019 ). To determine the role of CA in autophagic regulation, the levels of LC3Ⅱ (a marker of autophagosome) and p62 (a marker of autophagic flux) expression were examined in HepG2 and Huh7 cells treated with different concentrations of CA for 24 h. In Western blot analysis, we found that CA treatment upregulated the LC3Ⅱ expression while downregulated p62 expression (Fig. 2 E-F). We also found that the HCC cells treated with CA at the concentration of 1 nM exhibited more LC3-GFP puncta, compared to vehicle control treated cells (Fig. 2 G). These results thus indicate that CA is involved in activation of autophagy. To further confirm the role of CYP2E1 in CA-induced autophagy and growth of HCC cells, we established the HepG2 and Huh7 cells with forced expression of CYP2E1(Fig. 3 A-B). The colony formation assay showed that high level of CYP2E1 expression attenuated the growth of HCC cells (Fig. 3 G-H). Western blot analysis revealed the cells with forced expression of CYP2E1 exhibited increased level of LC3Ⅱ expression (Fig. 3 C-D), and immunofluorescence staining also showed increased LC3-GFP puncta (Fig. 3 E), suggesting that forced expression of CYP2E1 induces the accumulation of autophagosome in HCC cells. In addition, the cells with forced expression of CYP2E1 increased the expression level of p62 as shown by Western blot analysis (Fig. 3 C-D) and augmented the accumulation of p62 points by immunofluorescence staining (Fig. 3 F). Moreover, we infected HepG2 and Huh7 cells with the pmCherry-EGFP-LC3b adenovirus to monitor the synthesis of autophagosomes (yellow puncta) and autophagosome-lysosomal fusion (red puncta). Increased yellow puncta (autophagosomes) were detected in HepG2 and Huh7 cells with forced expression of CYP2E1 (Fig. 3 K). These results thus indicated that increased level of CYP2E1 in HCC cells attenuated cell growth and blocked autophagic flux which thus resulted in autophagosome accumulation. Next, HCC cells with or without forced CYP2E1 expression were treated with CA for 24h, and we found that CA failed to increase the accumulation of autophagosome, autophagic flux and cell growth in the HCC cells with forced CYP2E1 expression, which were obviously observed in the HCC cells without forced CYP2E1 expression (Fig. 3 I-J), indicating that CYP2E1 plays an important role in CA-induced growth of HCC cells and regulation of autophagy. AKT/mTOR signaling pathway is involved in CA-induced autophagy and cell growth of HCC regulated by CYP2E1. As the AKT/mTOR signaling plays a central role in regulation of tumor growth and autophagy, we measured the phosphorylated levels of AKT and mTOR in CA treatment of HCC cells using Western blot analysis with phosphorylation specific antibodies. Figure 4 A-B showed that CA upregulated the level of AKT phosphorylation, while downregulated the level of the mTOR phosphorylation. In addition, MK2206, an AKT inhibitor, significantly increased p62 expression (Fig S2A-B) and mTOR phosphorylation in the CA-treated cells, and inhibited the colony-forming ability of the HCC cells induced by CA treatment (Fig S2C-D), suggesting that AKT/mTOR pathway is involved in CA-induced autophagy and promotion of HCC cell growth (Fig. 4 C-D). Since the AKT phosphorylation was previously reported to be suppressed by CYP2E1 in chronic ethanol-induced fatty liver(Zeng et al. 2018 ). To determine whether CYP2E1 is involved in CA-activation of the AKT/mTOR signaling pathway, HCC cells with or without forced expression of CYP2E1 were treated with CA, and phosphorylation of AKT and mTOR was measured. We found that forced expression of CYP2E1 downregulated the AKT phosphorylation whereas upregulated mTOR phosphorylation, regardless CA treatment (Fig. 4 E-F). Consistently, as shown in (Fig. 3 K), CA failed to influence the accumulation of autophagosome, autophagic flux and cell growth in the HCC cells with forced CYP2E1 expression. Taken together, these results indicate that CYP2E1 plays an important role in CA-induced growth of HCC cells presumably through regulation of autophagy and the AKT/mTOR signaling pathway. CYP2E1 regulation of the AKT/mTOR pathway is involved in CA-promoted growth of HCC cells in vivo To further assess the role of CYP2E1 in CA-induced hepatocellular carcinogenesis, we performed xenograft experiment in nude mice. The nude mice were intrahepatically injected with the HepG2 cells with forced CYP2E1 expression (HepG2-CYP2E1) and a control cell line of the HepG2 cells transfected with an empty expression vector (HepG2-Vector), and maintained with or without intragastric supplement of CA for 28 days (Fig. 5 A). We found that tumors were formed in the livers of all mice, and the tumor volumes formed by the HepG2-Vector cells in the groups supplemented with CA were bigger compared with the group with the same volume of vehicle. The tumor volumes formed by the HepG2-CYP2E1 cells were much smaller than that from the HepG2-Vector cells. However, there was no changes between the HepG2-CYP2E1 groups with or without CA administration (Fig. 5 B-C), suggesting that upregulation of CYP2E1 is a critical mechanism by which CA influences HCC growth. Liver weights, body weight and the ratio of liver weight to body weight were not changed in all groups (Fig. 5 B-C). Western blot analysis revealed that CA increased the levels of LC3Ⅱ expression and AKT phosphorylation whereas decreased the levels of p62 expression and mTOR phosphorylation in the tumors formed by the HepG2-Vector cells (Fig. 5 D-E). Compared to the tumors formed by the HepG2-Vector cells, tumor tissues formed by the HepG2-CYP2E1 cells showed increased levels of LC3Ⅱ and p62 expression and mTOR phosphorylation, and decreased level of AKT phosphorylation, but the expression and phosphorylation of these proteins were without any changes after CA administration (Fig. 5 D-E). Similar results were observed by IHC staining (Fig. 5 F). These results thus indicated that CA administration accelerated tumor growth of HepG2 cells through the AKT/mTOR pathway regulated by CYP2E1. Low level of CYP2E1 expression correlates with poor progress and increased TBA as well as GGT in HCC patients To reveal the clinical relevance of CYP2E1 expression with clinical features of HCC patients, TCGA (The Cancer Genome Atlas) and GEPIA (Gene Expression Profiling Interactive Analysis) were used to evaluate CYP2E1 expression between the normal and tumor tissues of HCC patients. We found that CYP2E1 mRNA expression was lower in the tumor tissues than in the normal tissues (Fig. 6 A-B). The difference of the CYP2E1 protein between the tumor and normal tissues in HCC was also analyzed using the data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). We found decreased CYP2E1 protein expression in the tumor tissues compared with the normal tissues in HCC patients (Fig. 6 C). Then, we assessed the differences of the survival risk between the high CYP2E1 expression and low CYP2E1 expression using the Kaplan-Meier (Fig. 6 D-E) and GEPIA (Fig. 6 F-G) survival analysis, and found that the HCC patients with low CYP2E1 expression exhibited worse disease-free survival and overall survival rates. Thus, our results suggested that downregulation of CYP2E1 contributes to HCC development, and may serve as an important indicator for poor prognosis in HCC patients. To confirm this, we examined CYP2E1 expression in forty-six cases of HCC specimens with Western blot analysis and IHC. Compared with the paired normal tissues, lower CYP2E1 expression was detected in 40 of 46 (86.96%) tumor specimens compared to the neighboring normal tissues (Fig. 6 H-I and Table 1 ). In addition, CYP2E1 expression was correlated with TBA and GGT ( p 0.05). Taken together, these findings indicate that downregulated CYP2E1 expression and increased serum TBA as well as GGT are involved in hepatocellular carcinogenesis. Table 1 Correlations between CYP2E1 level and liver function test in HCC patients. Factors CYP2E1 p -value Low High TBA 0.009** > 10 23 0 ≤ 10 17 6 ALT 0.171 > 40 10 0 5 ~ 40 30 6 AST 0.314 > 40 6 0 8 ~ 40 34 6 GGT 0.031* > 50 23 1 11 ~ 50 17 5 ALP 0.628 > 150 4 1 ≤ 150 36 5 AFP 0.462 > 20 18 2 ≤ 20 22 4 TBIL 0.756 > 19 9 2 5 ~ 19 31 4 * p < 0.05, ** p < 0.01was considered statistically significant. Discussion Our study demonstrated that CA treatment decreased CYP2E1 expression and promoted malignant growth of human HCC HepG2 and Huh7 cells both in vitro and in vivo. The promotion activity of CA in these cells was associated with CYP2E1-regulated induction of autophagy. We also found that high levels of TBA in HCC patients were strongly correlated with decreased CYP2E1 level. Hence, the CYP2E1-regulated autophagy is one of the underlying mechanisms of CA action in HCC. Studies have shown that BAs homeostasis was disturbed during HCC development. A retrospective cohort study identified persistently elevated TBA as a major independent risk factor for HCC development in chronic hepatitis B patients (Wang et al. 2016 ). Previously, bile acids are considered as tumor promoters and involved in the pathogenesis of HCC (Ma et al. 2018 ; Shen et al. 2022 ). Patients with cholestasis, a chronic and consistent exposure to BAs, often develop liver fibrosis and cirrhosis which eventually results in liver failure and increased risk of HCC or CCA (Eaton et al. 2013 ; Tomiyama et al. 2013 ). The increased levels of BAs, such as CA and CDCA in cholestatic liver diseases induce oxidative stress and apoptosis, thereby resulting in damage to the liver parenchyma and, eventually extrahepatic tissues (Monte et al. 2009 ). The unconjugated BAs including CA, CDCA, and DCA, are cytotoxic, and their accumulation could result in mitochondrial damage, disruption of cell membranes, production of reactive oxygen species (ROS) and induction of DNA damage and mutation in hepatocytes (Jang et al. 2012 ; Guicciardi et al. 2013 ). Toxic BAs-induced chronic inflammation and injury-repair response in the liver that likely contributes to tumor promotion (Li and Apte 2015 ). In this study, we also found that high levels of TBA were detected in 50% (23/46) cases of HCC patients, and increased level of TBA was correlated with decreased level of CYP2E1 expression. CYP2E1 is a key metabolic enzyme of the liver microsomal oxidase system and is involved in induction of oxidative stress(Cho et al. 2021 ). We choose CA for this study mainly because it is one of the major primary BAs produced in the liver and was increased in our DEN-induced HCC model. We used liver tumor tissues from DEN-induced liver tumor model with or without CA administration for whole-proteome label-free assessment, and found that CA administration increased the tumor volumes and decreased the level of CYP2E1 expression. HepG2 and Huh7 cells treated with CA and nude mice intrahepatically injected with human HCC HepG2 cells with or without CA administration for 28 days exhibited increased cell growth and decreased CYP2E1 expression. Thus, a possible relationship between CA and decreased CYP2E1 expression was suggested. Cytochrome P450 2E1 (CYP2E1) is mainly expressed in liver and plays a critical role in the metabolism of many environmental toxicants as well as cancer inducing agents, such as benzene, ethanol, carbon tetrachloride, and vinyl chloride (Gonzalez 2005 ; Kang et al. 2007 ; Hu et al. 2023 ; Yan et al. 2023 ). CYP2E1 is involved in an enhanced activation of procarcinogens to carcinogens (Harjumaki, Pridgeon, and Ingelman-Sundberg 2021 )as well as induction of oxidative stress (Cho et al. 2021 ), and it is suggested to be associated with the risk of liver cancer (Shun-Zhang Yu 2002 ). CYP2E1 expression is downregulated in tumor tissue of HCC patients, which is often associated with aggressive tumor type and poor prognosis of the patients (Zhu et al. 2022 ). By IHC, the mean CYP2E1 score was significantly lower in livers with cirrhosis and HCC compared to normal livers, and was considered that CYP2E1 might be directly induced by the factor(s) derived from component cells of tumors such as carcinoma cells themselves, whether primary or metastatic, stroma, and inflammatory cells (Hata et al. 2010 ). The results from DEN-induced HCC model also showed that, CYP2E1 expression in the liver tissues was slightly higher than that in normal rat liver in the first to third week, which may be explained by the fact that CYP2E1 is required in the metabolism of nitrosamines. With the aggravation of liver lesion and development of cirrhosis, CYP2E1 expression was gradually downregulated. Here, we found that, the level of CYP2E1 expression was decreased in forty of forty-six tumor specimens compare to the adjacent nontumor tissues as revealed by Western blot analysis, suggesting that the decreased expression of CYP2E1 may be involved in HCC tumorigenesis. A decrease in carcinogen metabolism (Liu et al. 2022 ) and an increase in procarcinogen activation have also been documented as HCC risk factors as well as changes in the metabolism of environmental toxins that arise from alterations in Cytochrome P450 (CYP) activity (Forrester et al. 1990 ; Guengerich 1992 ; Guengerich et al. 1996 ; Fontham et al. 2009 ; Cheng et al. 2022 ). CYPs function not only in the detoxification of internal and external xenobiotics, but also in the metabolic activation of carcinogens and may be further implicated in tumor initiation, promotion and progression (Singh et al. 2023 ). Previously, the functional characterization of CYP2E1 has been focused on its role in alcoholic liver diseases and drug metabolism (Nagappan et al. 2019 ), since it metabolizes and activates many toxicologically important compounds, such as aromatic hydrocarbons such as ethanol, carbon tetrachloride, acetaminophen, benzene, halothane, and many other halogenated substrates (Chen et al. 2019 ; Cui et al. 2019 ; Cho et al. 2023 ). However, the function of CYP2E1 in hepatocarcinogenesis is poorly known. Here, we found that CYP2E1 is involved in CA-induced promotion of malignant growth in HCC cells by activation of autophagy in vitro and in vivo. Forced CYP2E1 expression inhibited cell growth of HCC accompanied with autophagy inhibition. However, in the HCC cells with forced expression of CYP2E1, CA failed to influence CYP2E1 level and autophagy. Dysregulation of autophagy is involved in numerous diseases such as neurodegenerative disease, metabolic diseases, and cancer (Beckers, Tharkeshwar, and Van Damme 2021 ; Luo et al. 2021 ). Tumor cells upregulate autophagy to support their elevated metabolic demand for proliferation, survival and malignancy (Amaravadi, Kimmelman, and White 2016 ; Kimmelman and White 2017 ; Poillet-Perez and White 2019 ). Several signaling pathways are confirmed to regulate autophagy, including mTOR, PI3K-AKT and MAPK/ERK1/2, and mTOR is one of the main negative regulators of autophagy (Wang et al. 2017 ; Gao et al. 2019 ; Zhang et al. 2019 ). In this study, we found that autophagy is activated by CA, which is involved in CA’s role in promotion of HCC cell growth. CA upregulated the AKT phosphorylation but downregulated mTOR phosphorylation. Previously, it was reported that AKT phosphorylation was induced by CYP2E1-mediated oxidative stress (Zeng et al. 2018 ). The interplay between ROS and autophagy in tumor cells, function from tumor initiation to progression has been widely reported(Xing et al. 2022 ). During the initiation, progression and metastasis of tumor cells, the pro-tumoral role of autophagy was shown to eliminate ROS-induced metabolic stress and the production of nutrients required for tumor cell survival (Gao et al. 2019 ). CYP2E1, a key enzyme for generation of ROS, is a major contributor to the pathogenesis of many liver diseases such as alcoholic liver disease and HCC (Gao et al. 2019 ), but reports of its role in autophagy regulation are limited. Here, we found that forced CYP2E1 expression increased accumulation of autophagosomes as revealed by increased LC3 puncta, and blocked autophagic flux with increased p62 accumulation and blockage of autophagosome-lysosome fusion both in vitro and in vivo. Recent studies have shown that inhibition of autophagic flux resulted in accumulation of damaged organelles and dysfunctional proteins, which has the potential to turn autophagy into a serious and destructive process, leading to fatal toxic effects on tumor cells, providing an explanation of the inhibitory activity of CYP2E1 in HCC cell growth. We also showed that CYP2E1-induced autophagy and regulation of the AKT/mTOR signaling are involved in CA-mediated cell growth of HCC. Conclusions Our study revealed that CYP2E1 regulates CA-induced autophagy and promotion of HCC cell growth by regulating the AKT/mTOR signaling both in vitro and in vivo. Clinically, high TBA level in HCC tissue was strongly associated with decreased expression of CYP2E1. Targeting CYP2E1-mediated autophagy may be a novel and attractive therapeutic approach for amelioration of HCC with imbalance of BAs level. Abbreviations CYP2E1 Cytochrome P450 2E1 CA Cholic acid HCC Hepatocellular carcinoma DEN Diethylnitrosamine TBA Total bile acids CDCA Chenodesoxycholic acid HBV Hepatitis B virus HCV Hepatitis C virus BAs Bile acids GCDC Glycochenodeoxycholate GGT Gamma-glutamyltransferase CCA Cholangiocarcinoma carcinoma SD Sprague-Dawley Declarations Acknowledgments Not applicable. Author contributions FZQ, LYC and HSB conceived and designed the study; HZW, LXM, HHH, WZX, NY and WJC performed the experiments and acquired the data; HZW, LXM, ZHY and HZY analyzed and interpreted the data; SXJ, LYC and HSB reviewed the study; HZW drafted the manuscript; FZQ, WJZ, LYC and SBL contributed to revise manuscript and supervision. All the authors read and approved the final manuscript. Funding The present study was supported by the National Natural Science Foundation of China (Grant no. 81872040). Data availability All data generated or analyzed during this study are included in the submitted article and its supplementary files. Ethics approval and consent to participate All samples were used after written and informed consent of patients and approval of the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (2019 IEC(S646)). All experimental procedures were performed as previously described in compliance with the National Institutes of Health guidelines on the ethical use of animals and approved by the Ethics Committee of Jianghan University (JHDXLL2023-053). Conflict of interest The authors declare no conflicts of interest. Consent for publication Yes. References Alwadei N, Rashid M, Chandrashekar DV, Rahighi S, Totonchy J, Sharma A, Mehvar R. Generation and characterization of cyp2e1-overexpressing hepg2 cells to study the role of cyp2e1 in hepatic hypoxia-reoxygenation injury. Int J Mol Sci. 2023;24(9):8121. 10.3390/ijms24098121 . 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18:55:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":843036,"visible":true,"origin":"","legend":"\u003cp\u003eCA treatment decrease CYP2E1 level in HCC tumors induced by DEN. (A) Representative images of the livers from the male SD rats treated with DEN and then administrated with CA (group DEN+CA) and vehicle DMSO as a control (group DEN). (B-D) The general proteome information. (B) Principal Coordinates Analysis (PCoA) showed a clear separation of proteins between the group DEN and the group DEN+CA. (C) 151 differential proteins were identified in the tumor tissues of the group DEN+CA \u003cem\u003evs\u003c/em\u003e the group DEN, (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, increased proteins: red; decreased proteins: blue). (D) The increased (red) or decreased (blue) expression of proteins in the group DEN+CA \u003cem\u003evs\u003c/em\u003e the group DEN (*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05) were shown. (E-F) CYP2E1 protein expression from individual rats (n = 3) were validated by Western blot analysis (E) and then quantitatively analyzed (F). The data represent the mean ± SEM. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4070826/v1/90290d0d3a85756eae90bfa6.png"},{"id":52786903,"identity":"fff93d99-2129-43b3-aee6-678d15acfcae","added_by":"auto","created_at":"2024-03-15 18:55:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1786480,"visible":true,"origin":"","legend":"\u003cp\u003eCA treatment decreases CYP2E1 expression and activates autophagy in HepG2 and Huh7 cells. (A-B) HCC cells were treated with 0.1 and 1nM CA for 24 h and the same volume of DMSO was used as a vehicle control. CYP2E1 expression was assessed by Western blot analysis (A), and then quantitatively analyzed (B). The data represent the mean ± SEM (n = 3). (C-D) HCC cells were treated with CA (1nM) for 14 days, and the representative images of colony formation were shown (C) and then quantitatively analyzed (D). (E-F) HCC cells were treated with 0.1 and 1nM CA for 24 h and the same volume of DMSO was used as a vehicle control. The levels of LC3Ⅱ and p62 were assessed by Western blot analysis (E) and then quantitatively analyzed (F). The expression levels of p62 and LC3Ⅱ were normalized against that of β-actin. The data represent the mean ± SEM. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. (G) HCC cells were treated with CA (1nM) for 24 h and analyzed with immunofluorescence assay using the LC3Ⅱ antibody (488 green). Hoechst (blue) was used to stain the nuclei, and the stained HCC cells were then photographed under a fluorescence microscope. The scale bar is 20 μm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4070826/v1/3ee64803d90b032285b75fcb.png"},{"id":52786802,"identity":"b223966c-c18b-44d5-9bc5-e1c3cfa46b56","added_by":"auto","created_at":"2024-03-15 18:54:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2643657,"visible":true,"origin":"","legend":"\u003cp\u003eCYP2E1 deficit contributes to CA-induced HCC growth with mechanism involving regulating autophagy. (A-B) The level of CYP2E1 expression in the HepG2 and Huh7 cells with forced expression of CYP2E1 or vector control was measured by Western blot analysis (A) and quantitatively analyzed (B). (C-D) The levels of LC3II and p62 in the HCC cells with forced expression of CYP2E1 or vector control were measured with Western blot analysis (C) and quantitatively analyzed (D). (E-F) HCC cells with forced expression of CYP2E1 or vector control were analyzed with immunofluorescence assay using the LC3Ⅱ antibody (488 green) (E) and p62 antibody (594 red) (F). Hoechst (blue) was used to stain the nuclei, and the stained HCC cells were then photographed under a fluorescence microscope. The scale bar is 20 μm. The HCC cells with forced expression of CYP2E1 or vector control were treated with or without CA (1nM) for 14 days, and the representative images of colony formation were shown (G) and then quantitatively analyzed (H). (I-J) HCC cells with forced expression of CYP2E1 or vector control were treated with or without CA (1nM) for 24 h and the same volume of DMSO was used as a vehicle control. The levels of CYP2E1, LC3Ⅱ and p62 expression were assessed by Western blot analysis (I) and then quantitatively analyzed (J). The expression levels of CYP2E1, LC3Ⅱ and p62 were normalized against that of β-actin. The data represent the mean ± SEM. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. (K) The HCC cells with forced expression of CYP2E1 or vector control were infected with the mRFP-EGFP-LC3 adenovirus, and then treated with or without CA (1nM) for 24 h, and analyzed by confocal microscopy. The scale bar is 10 μm.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4070826/v1/bcb59a6106814316c1483e89.png"},{"id":52786812,"identity":"af2a1c7b-040c-4777-a215-7c50566d75f3","added_by":"auto","created_at":"2024-03-15 18:55:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1847729,"visible":true,"origin":"","legend":"\u003cp\u003eThe AKT/mTOR signaling pathway is involved in CA-induced autophagy and growth of HCC cells regulated by CYP2E1. (A-B) HCC cells were treated with 0.1 and 1nM cell CA for 24 h and the same volume of DMSO was used as a vehicle control. The levels of p-mTOR, mTOR, p-AKT and AKT were detected by Western blot analysis (A) and quantitatively analyzed (B). The levels of phosphorylated AKT and mTOR were normalized against the AKT and mTOR, respectively. (C-D) The HCC cells were exposed to MK2206 (20 μM) for 2 h, followed by CA (1nM) treatment for 24 h, and subsequently the expression levels of p-mTOR, mTOR, p-AKT and AKT were assessed by Western blot analysis (C) and then quantitatively analyzed (D). (E-F). The HCC cells with forced expression of CYP2E1 or vector were treated with or without CA (1nM) for 24 h and the same volume of DMSO was used as a vehicle control. The levels of p-mTOR, mTOR, p-AKT and AKT were assessed by Western blot analysis (E) and then quantitatively analyzed (F). The data represent the mean ± SEM. **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4070826/v1/9d09f85a2965164ce9720a9c.png"},{"id":52786867,"identity":"caa7c4aa-274c-49e7-916d-629a9086f6ac","added_by":"auto","created_at":"2024-03-15 18:55:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3083384,"visible":true,"origin":"","legend":"\u003cp\u003eCYP2E1 is involved in CA-induced promotion of HCC cell growth in nude mice. The nude mice were intrahepaticly injected with HepG2 cells with forced expression of CYP2E1 or vector control, and then intragastrically administrated with CA or the same volume of DMSO as a control for 28 days (A). Representative image of liver tumors in these nude mice (B) and quantitative analysis of liver tumor volumes, liver weight, body weight and the ratio of liver weight to body weight of the nude mice (C) were shown. (D-E) Expression levels of CYP2E1, LC3Ⅱ and p62 and the levels of the phosphorylated AKT and mTOR in the liver tumors from the nude mice with or without CA administration were assessed by Western blot analysis (D) and then quantitatively analyzed (E). β-actin was used as the internal loading control. The levels of phosphorylated AKT and mTOR were normalized against the AKT and mTOR, respectively. The data represent the mean ± SEM. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. The expression levels LC3Ⅱ and p62 in the liver tumors from the nude mice with or without CA administration were examined by immunohistochemical staining (F). The scale bar is 50 μm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4070826/v1/20c4d4b74cd35747a1a768f9.png"},{"id":52786874,"identity":"04d11c4d-dd7b-4ca8-b9c5-ffefc8939cea","added_by":"auto","created_at":"2024-03-15 18:55:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1566381,"visible":true,"origin":"","legend":"\u003cp\u003eLow expression of CYP2E1 correlates with poor progress and increased TBA as well as GGT in HCC patients. (A) Comparison of the CYP2E1 mRNA level across TCGA tumors, and the matched TCGA normal and GTEx data were included as controls. The box plot data were supplied. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. (B) Comparison of the CYP2E1 mRNA level between HCC and normal tissues in TCGA database. Statistical significance was assessed using two-tailed Student’s t-test. (C) CYP2E1 proteomic expression profile in HCC from CPTAC samples. Z-values represent standard deviations from the median across samples for HCC. N represents the number of samples. (D, E) Kaplan-Meier analysis of overall survival (D) and disease-free survival probability (E) of CYP2E1 levels in HCC patients. The statistical significance was assessed using two-sided log-rank test according to HCC patients with low or high expression of CYP2E1. (F, G) The overall survival (F) and disease-free survival probability (G) were compared between CYP2E1 high and low expression in the HCC patients from TCGA cohort. (H, I) The level of CYP2E1 expression in specimens of HCC tumor tissues (T) and adjacent non-tumor tissues (N) was assessed with Western blot analysis (H) and immunohistochemical staining (I).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4070826/v1/b33d49cd6f1cdc6349289db4.png"},{"id":52787512,"identity":"e54ddbab-258c-49e9-a7d2-f9f76c32541a","added_by":"auto","created_at":"2024-03-15 19:04:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4833375,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4070826/v1/a1230f4d-2fc4-45e0-8ea5-3085d481e163.pdf"},{"id":52786851,"identity":"142786a4-b277-4355-bd70-c657f7abf29d","added_by":"auto","created_at":"2024-03-15 18:55:12","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":20882,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-4070826/v1/49afbbba639fe65edd82a7bc.docx"}],"financialInterests":"","formattedTitle":"CYP2E1 deficit mediates cholic acid-induced malignant growth in hepatocellular carcinoma cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePrimary liver cancer is the fourth most commonly diagnosed cancer and the second leading cause of cancer-related death in China with approximately 367,700 new cases and 316,500 deaths in 2022(Han et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Hepatocellular carcinoma (HCC) accounts for the majority (\u0026gt;80%) of liver cancers (Tamai et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Risk factors for HCC include chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), aflatoxin-contained food, alcohol consumption, metabolic syndrome, etc (Toh et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Recently, bile acids (BAs) were also found to be associated with HCC development (Conde et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBile acids are metabolites from cholesterol and mainly synthesized in the liver. BAs serve as endocrine signaling molecules sensed by nuclear or membrane-localized receptors to trigger specific signaling pathways and regulate many biological processes (Song et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Cai et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Studies from clinical patients and animal models have shown an association of BAs abnormalities with liver diseases such as HCC (Stepien et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Clinically, increased levels of primary and secondary BAs were detected in HCC and cholangiocarcinoma carcinoma (CCA) patients (Changbumrung et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Children with the progressive familial intrahepatic cholestasis type 2 (PFIC type 2) disease, characterized by a genetic deficiency of the canalicular bile salt export pump BSEP or ABCB11, and severe cholestasis with elevated serum and liver BAs levels, such as CA and chenodesoxycholic acid (CDCA), is predisposed to HCC (Knisely et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Baptissart et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The experiments in mice have showed that a CA-enriched diet strongly promoted N-nitrosodiethylamine-induced liver carcinogenesis (Yang et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Our studies have also shown that CA administration promoted DEN-induced initiation and progression of HCC. However, the molecular mechanisms by which BAs promote HCC is still unknown.\u003c/p\u003e \u003cp\u003eAutophagy, a \u0026ldquo;self-eating\u0026rdquo; process that clears intracellular waste, is crucial for development, differentiation, survival, and homeostasis (Tabibzadeh \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Accumulating evidence indicates that autophagy can be upregulated in response to a multitude of stresses including starvation, hypoxia and intracellular pathogens, which in most contexts promotes tumorigenesis(Li, He, and Ma \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Increased autophagy activity with high LC3B expression benefits tumor proliferation, invasion, or metastasis (Wu et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Autophagy is involved in HCC progression, resulting in drug resistance and poor prognosis of HCC patients (Shi et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Li, He, and Ma \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The protective role of autophagy in cholestasis-induced liver injury was also reported (Gao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). High levels of total bile acids (TBA) in HCC patients were found to be strongly correlated with their autophagy level and poor survival (Gao et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Autophagy triggered by glycochenodeoxycholate (GCDC) is a protective mechanism in invasion and metastasis of HCC(Gao et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). How dysregulated levels of BAs influence autophagy, however, is still poorly understood\u003c/p\u003e \u003cp\u003eCytochrome P450 2E1 (CYP2E1), a member enzyme of the cytochrome P450 superfamily, is an important hepatic metabolic enzyme that is responsible the metabolism of xenobiotics including ethanol, acetone, drugs and procarcinogens (Torres et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Research effort has been focused on its role in drug metabolism and alcoholic liver diseases for a long time. Therefore, the role of CYP2E1 in HCC development is largely unknown. Recent studies have shown that the level of CYP2E1 in the tumor tissues of HCC patients was significantly lower than the adjacent nontumor tissues and the cirrhotic and/or normal liver tissues (Ho et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The level of CYP2E1 expression is negatively associated with aggressive tumor type and poor prognosis of HCC patients (Ho et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), suggesting the inhibitory activity of CYP2E1 in the initiation and progression of HCC. HepG2 cells overexpressing CYP2E1 exhibited a slow rate of cell growth accompanied with increased CYP2E1-induced cytotoxicity (Cederbaum et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Alwadei et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In DEN-induced HCC rat model, CYP2E1 expression was declined along with the initiation, promotion and progression of HCC(Sanchez-Meza et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These studies indicate that downregulation of CYP2E1 expression is involved in HCC tumorigenesis.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to investigate the involvement of CYP2E1 in CA-induced autophagy and promotion of malignant growth of hepatocellular carcinoma cells. We found that CA induced autophagy and promoted malignant growth of human hepatocellular carcinoma HepG2 and Huh7 cells, which is regulated by CYP2E1. Clinically, decreased level of CYP2E1 expression was found in the tumor tissues of HCC patients with high TBA level.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and antibodies\u003c/h2\u003e \u003cp\u003eCA was purchased form Sigma-Aldrich (St Louis, MO, USA). MK-2206 dihydrochloride was obtained from MedChemExpress (USA). Antibodies against p62 (18420-1-AP), CYP2E1 (19937-1-AP), AKT (10176-2-AP) and p-AKT (66444-1-lg) were from Proteintech Group (Chicago, IL, USA). The mTOR (380411) antibody was from Zen BioScience (Chengdu, Sichuan, China). p-mTOR (#5536), LC3A/B (#12741) and Alexa Fluor\u0026reg;594 Conjugate (#8889S) were from Cell Signaling Technology (Danvers, MA, USA). β-actin (AF0003) was purchased from Beyotime Biotechnology (Nanjing, China). Alexa Fluor 488 (A21206) was from Sigma-Aldrich Co. (St. Louis, MO, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eClinical tumor samples\u003c/h2\u003e \u003cp\u003eHCC cancerous and para-cancerous tissues were obtained from Union Hospital, Tongji Medical College, Huazhong University of Science and Technology between May 2019 and December 2023.The samples were from forty men and six women aged 42\u0026ndash;78 years (mean 61.7 years), and none of them had received any anticancer treatment prior to surgery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and stable cell lines\u003c/h2\u003e \u003cp\u003eHuman HCC HepG2 and Huh7 cell lines were obtained from Shenogen Pharma Group (Beijing, China), and maintained in DMEM with 10% FBS at 37℃ in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. Stable CYP2E1 expression cell lines and their control cell lines were generated by stable transfection of pcDNA-CYP2E1and pcDNA empty vector (Fenghui Biotechnology Co., LTD) respectively. Then, the CYP2E1 expression in the stable cell lines were verified by Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eCells and liver tissues were lysed with the RIPA buffer. Proteins were separated through SDS-PAGE, transferred onto a PVDF membrane that was blocked and incubated with the indicated antibody. Target proteins were detected by ECL assay System.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003eMale BALB/c-nu mice (5 weeks old) were purchased from Beijing SiPeiFu Animal Technology Co. LTD and housed in specific pathogen-free conditions with the constant temperature (24\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃) and relative humidity (60%). HepG2-CYP2E1 cells and HepG2-vector cells (5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mice) were injected into the liver of BALB/c-nu mice (n\u0026thinsp;=\u0026thinsp;4) to establish orthotopic liver xenograft tumor model with or without intragastric administration of CA (0.2%, every 2 days) and maintained for 28 days. Tumor volume was calculated according to the following formula: tumor volume = (major axis) \u0026times; (minor axis)\u003csup\u003e2\u003c/sup\u003e/2. Male Sprague-Dawley (SD) rats, 6 weeks old (~\u0026thinsp;230-250g), were obtained from the Hunan SJA Laboratory Animal Co., LTD, China. Rats were intraperitoneally injected with DEN (Sigma Chemical Co., LTD) of 75 mg/kg per body weight once a week for three weeks, then with DEN of 100 mg/kg per body weight once a week for three weeks (Matsuzaki et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) and (Shiota et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). All rats were subsequently fed with or without CA for 23 weeks. After euthanization, the livers were removed. Preparation and measurement of liver tumors were performed by Biotree Biotech Co., LTD., Shanghai, China as a custom service.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eAfter fixing with ice-cold 4% paraformaldehyde for 15 min, and permeabilizing with 0.1% Triton X-100 for 20 min, the samples were incubated with LC3 and p62 antibodies overnight at 4 ℃, washed and then with the Alexa Fluor conjugated secondary antibody for 1 h. The nuclei were stained with Hoechst 33342 (1 \u0026micro;g/mL) for 10 min. The images were obtained under a fluorescence microscope (Leica TCS SP8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003epmCherry-EGFP-LC3 puncta assay\u003c/h2\u003e \u003cp\u003eCells were seeded in a 24-well plate, detected with the pmCherryEGFP-LC3b adenovirus (MiaoLingbio, China) for 6 h, incubated with CA for 24 h and examined with a confocal microscope (Leica TCS SP8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eClonogenic assay\u003c/h2\u003e \u003cp\u003eAfter seeding (4\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well), cells were treated with CA and cultured for 2 weeks. Then, the cell colonies were washed, fixed and stained with 0.1% crystal violet. The number of colonies were counted under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry staining\u003c/h2\u003e \u003cp\u003eAfter deparaffinizing, rehydrating gradually by gradient alcohol and incubating in a 3% hydrogen peroxide/methanol buffer, the slides were immersed in an ethylenediamine tetra acetic acid buffer (pH 8.0) and boiled for 5 min. Then, the slides were washed and incubated with the specific primary and secondary antibodies. Diaminobenzidine was used to generate signal, and images were captured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGene and protein expression analysis\u003c/h2\u003e \u003cp\u003eGEPIA and UALCAN programs (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ualcan.path.uab.edu\u003c/span\u003e\u003cspan address=\"http://ualcan.path.uab.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used to analyze the relative mRNA expression of CYP2E1 in tumor and normal samples of HCC patients. The protein expression was analyzed using the data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ualcan.path.uab.edu/analysis-prot.html\u003c/span\u003e\u003cspan address=\"http://ualcan.path.uab.edu/analysis-prot.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The GEPIA2.0 and Kaplan-Meier programs were then used for patient survival analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM) using a GraphPad Prim 8.0 software with analysis of variance (ANOVA), followed by Bonferroni's post hoc tests. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Spearman correlation coefficient was used to determine the correlations between abnormal distribution variables.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCYP2E1 expression is downregulated in CA-treated hepatocellular carcinoma induced by DEN\u003c/h2\u003e \u003cp\u003eOur recent studies have found that CA administration promoted DEN-induced initiation and progression of HCC in rats. To explore the molecular mechanisms by which CA promotes hepatocellular carcinogenesis, liver tumor tissues from the DEN-treated, and both DEN and CA treated rats were used for whole-proteome label-free assessment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Principal coordinates analysis (PCoA) showed a clear separation of proteins between these two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). One hundred and fifty-one proteins were expressed differentially between the tumor tissues from group DEN\u0026thinsp;+\u0026thinsp;CA and the group DEN (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The differentially expressed proteins between two groups were selected, according to the criteria of (log2 |fold-change| \u0026ge; 1.2 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The results showed that two hundred and seventy-three proteins were found upregulated, while one hundred and ninety-three were proteins downregulated in DEN, compared with DEN\u0026thinsp;+\u0026thinsp;CA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Further analysis with the Protein-Protein Interaction Network Analysis using STRING database indicated involvement of three out of five enriched KEGG pathways (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). CYP2E1 expression was downregulated in the group DEN\u0026thinsp;+\u0026thinsp;CA rats (\u0026gt;\u0026thinsp;2-fold) compared to the DEN group. In Western blot analysis, we found that CYP2E1 expression was significantly downregulated in the liver tumors from the group DEN\u0026thinsp;+\u0026thinsp;CA compared to those in the group DEN (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F), consistent with the proteomics data.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine whether CA regulates CYP2E1 expression, HepG2 and Huh7 cells were treated with different concentrations of CA for 24 h. Western blot analysis revealed that CA treatment reduced CYP2E1 expression in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). In colony formation assays, CA significantly increased the colonogenicity of HCC cells at a concentration of 1 nM, compared to the vehicle control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). These results suggested that CA at the concentration of 1 nM was able to downregulate CYP2E1 expression and promote the malignant growth of HepG2 and Huh7 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCYP2E1 deficit contributes to CA-induced HCC growth with mechanism involving regulating autophagy.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBile acids such as glycochenodeoxycholate (GCDC) have been reported to induce autophagy, which promotes invasion and migration of HCC cells (Gao et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To determine the role of CA in autophagic regulation, the levels of LC3Ⅱ (a marker of autophagosome) and p62 (a marker of autophagic flux) expression were examined in HepG2 and Huh7 cells treated with different concentrations of CA for 24 h. In Western blot analysis, we found that CA treatment upregulated the LC3Ⅱ expression while downregulated p62 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F). We also found that the HCC cells treated with CA at the concentration of 1 nM exhibited more LC3-GFP puncta, compared to vehicle control treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). These results thus indicate that CA is involved in activation of autophagy.\u003c/p\u003e \u003cp\u003eTo further confirm the role of CYP2E1 in CA-induced autophagy and growth of HCC cells, we established the HepG2 and Huh7 cells with forced expression of CYP2E1(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). The colony formation assay showed that high level of CYP2E1 expression attenuated the growth of HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H). Western blot analysis revealed the cells with forced expression of CYP2E1 exhibited increased level of LC3Ⅱ expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D), and immunofluorescence staining also showed increased LC3-GFP puncta (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), suggesting that forced expression of CYP2E1 induces the accumulation of autophagosome in HCC cells. In addition, the cells with forced expression of CYP2E1 increased the expression level of p62 as shown by Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D) and augmented the accumulation of p62 points by immunofluorescence staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Moreover, we infected HepG2 and Huh7 cells with the pmCherry-EGFP-LC3b adenovirus to monitor the synthesis of autophagosomes (yellow puncta) and autophagosome-lysosomal fusion (red puncta). Increased yellow puncta (autophagosomes) were detected in HepG2 and Huh7 cells with forced expression of CYP2E1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). These results thus indicated that increased level of CYP2E1 in HCC cells attenuated cell growth and blocked autophagic flux which thus resulted in autophagosome accumulation. Next, HCC cells with or without forced CYP2E1 expression were treated with CA for 24h, and we found that CA failed to increase the accumulation of autophagosome, autophagic flux and cell growth in the HCC cells with forced CYP2E1 expression, which were obviously observed in the HCC cells without forced CYP2E1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-J), indicating that CYP2E1 plays an important role in CA-induced growth of HCC cells and regulation of autophagy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAKT/mTOR signaling pathway is involved in CA-induced autophagy and cell growth of HCC regulated by CYP2E1.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs the AKT/mTOR signaling plays a central role in regulation of tumor growth and autophagy, we measured the phosphorylated levels of AKT and mTOR in CA treatment of HCC cells using Western blot analysis with phosphorylation specific antibodies. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B showed that CA upregulated the level of AKT phosphorylation, while downregulated the level of the mTOR phosphorylation. In addition, MK2206, an AKT inhibitor, significantly increased p62 expression (Fig S2A-B) and mTOR phosphorylation in the CA-treated cells, and inhibited the colony-forming ability of the HCC cells induced by CA treatment (Fig S2C-D), suggesting that AKT/mTOR pathway is involved in CA-induced autophagy and promotion of HCC cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince the AKT phosphorylation was previously reported to be suppressed by CYP2E1 in chronic ethanol-induced fatty liver(Zeng et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To determine whether CYP2E1 is involved in CA-activation of the AKT/mTOR signaling pathway, HCC cells with or without forced expression of CYP2E1 were treated with CA, and phosphorylation of AKT and mTOR was measured. We found that forced expression of CYP2E1 downregulated the AKT phosphorylation whereas upregulated mTOR phosphorylation, regardless CA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F). Consistently, as shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK), CA failed to influence the accumulation of autophagosome, autophagic flux and cell growth in the HCC cells with forced CYP2E1 expression. Taken together, these results indicate that CYP2E1 plays an important role in CA-induced growth of HCC cells presumably through regulation of autophagy and the AKT/mTOR signaling pathway.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCYP2E1 regulation of the AKT/mTOR pathway is involved in CA-promoted growth of HCC cells in vivo\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further assess the role of CYP2E1 in CA-induced hepatocellular carcinogenesis, we performed xenograft experiment in nude mice. The nude mice were intrahepatically injected with the HepG2 cells with forced CYP2E1 expression (HepG2-CYP2E1) and a control cell line of the HepG2 cells transfected with an empty expression vector (HepG2-Vector), and maintained with or without intragastric supplement of CA for 28 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We found that tumors were formed in the livers of all mice, and the tumor volumes formed by the HepG2-Vector cells in the groups supplemented with CA were bigger compared with the group with the same volume of vehicle. The tumor volumes formed by the HepG2-CYP2E1 cells were much smaller than that from the HepG2-Vector cells. However, there was no changes between the HepG2-CYP2E1 groups with or without CA administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C), suggesting that upregulation of CYP2E1 is a critical mechanism by which CA influences HCC growth. Liver weights, body weight and the ratio of liver weight to body weight were not changed in all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). Western blot analysis revealed that CA increased the levels of LC3Ⅱ expression and AKT phosphorylation whereas decreased the levels of p62 expression and mTOR phosphorylation in the tumors formed by the HepG2-Vector cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E). Compared to the tumors formed by the HepG2-Vector cells, tumor tissues formed by the HepG2-CYP2E1 cells showed increased levels of LC3Ⅱ and p62 expression and mTOR phosphorylation, and decreased level of AKT phosphorylation, but the expression and phosphorylation of these proteins were without any changes after CA administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E). Similar results were observed by IHC staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). These results thus indicated that CA administration accelerated tumor growth of HepG2 cells through the AKT/mTOR pathway regulated by CYP2E1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLow level of CYP2E1 expression correlates with poor progress and increased TBA as well as GGT in HCC patients\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo reveal the clinical relevance of CYP2E1 expression with clinical features of HCC patients, TCGA (The Cancer Genome Atlas) and GEPIA (Gene Expression Profiling Interactive Analysis) were used to evaluate CYP2E1 expression between the normal and tumor tissues of HCC patients. We found that CYP2E1 mRNA expression was lower in the tumor tissues than in the normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). The difference of the CYP2E1 protein between the tumor and normal tissues in HCC was also analyzed using the data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). We found decreased CYP2E1 protein expression in the tumor tissues compared with the normal tissues in HCC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Then, we assessed the differences of the survival risk between the high CYP2E1 expression and low CYP2E1 expression using the Kaplan-Meier (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E) and GEPIA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-G) survival analysis, and found that the HCC patients with low CYP2E1 expression exhibited worse disease-free survival and overall survival rates. Thus, our results suggested that downregulation of CYP2E1 contributes to HCC development, and may serve as an important indicator for poor prognosis in HCC patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm this, we examined CYP2E1 expression in forty-six cases of HCC specimens with Western blot analysis and IHC. Compared with the paired normal tissues, lower CYP2E1 expression was detected in 40 of 46 (86.96%) tumor specimens compared to the neighboring normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-I and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition, CYP2E1 expression was correlated with TBA and GGT (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), but not with AST, ALT, ALP or AFP (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Taken together, these findings indicate that downregulated CYP2E1 expression and increased serum TBA as well as GGT are involved in hepatocellular carcinogenesis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelations between CYP2E1 level and liver function test in HCC patients.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFactors\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCYP2E1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTBA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.009**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eALT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.171\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u0026thinsp;~\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAST\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.314\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u0026thinsp;~\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGGT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.031*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u0026thinsp;~\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eALP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.628\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAFP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.462\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTBIL\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.756\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u0026thinsp;~\u0026thinsp;19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01was considered statistically significant.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study demonstrated that CA treatment decreased CYP2E1 expression and promoted malignant growth of human HCC HepG2 and Huh7 cells both in vitro and in vivo. The promotion activity of CA in these cells was associated with CYP2E1-regulated induction of autophagy. We also found that high levels of TBA in HCC patients were strongly correlated with decreased CYP2E1 level. Hence, the CYP2E1-regulated autophagy is one of the underlying mechanisms of CA action in HCC.\u003c/p\u003e \u003cp\u003eStudies have shown that BAs homeostasis was disturbed during HCC development. A retrospective cohort study identified persistently elevated TBA as a major independent risk factor for HCC development in chronic hepatitis B patients (Wang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Previously, bile acids are considered as tumor promoters and involved in the pathogenesis of HCC (Ma et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Patients with cholestasis, a chronic and consistent exposure to BAs, often develop liver fibrosis and cirrhosis which eventually results in liver failure and increased risk of HCC or CCA (Eaton et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tomiyama et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The increased levels of BAs, such as CA and CDCA in cholestatic liver diseases induce oxidative stress and apoptosis, thereby resulting in damage to the liver parenchyma and, eventually extrahepatic tissues (Monte et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The unconjugated BAs including CA, CDCA, and DCA, are cytotoxic, and their accumulation could result in mitochondrial damage, disruption of cell membranes, production of reactive oxygen species (ROS) and induction of DNA damage and mutation in hepatocytes (Jang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Guicciardi et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Toxic BAs-induced chronic inflammation and injury-repair response in the liver that likely contributes to tumor promotion (Li and Apte \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In this study, we also found that high levels of TBA were detected in 50% (23/46) cases of HCC patients, and increased level of TBA was correlated with decreased level of CYP2E1 expression. CYP2E1 is a key metabolic enzyme of the liver microsomal oxidase system and is involved in induction of oxidative stress(Cho et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We choose CA for this study mainly because it is one of the major primary BAs produced in the liver and was increased in our DEN-induced HCC model. We used liver tumor tissues from DEN-induced liver tumor model with or without CA administration for whole-proteome label-free assessment, and found that CA administration increased the tumor volumes and decreased the level of CYP2E1 expression. HepG2 and Huh7 cells treated with CA and nude mice intrahepatically injected with human HCC HepG2 cells with or without CA administration for 28 days exhibited increased cell growth and decreased CYP2E1 expression. Thus, a possible relationship between CA and decreased CYP2E1 expression was suggested.\u003c/p\u003e \u003cp\u003eCytochrome P450 2E1 (CYP2E1) is mainly expressed in liver and plays a critical role in the metabolism of many environmental toxicants as well as cancer inducing agents, such as benzene, ethanol, carbon tetrachloride, and vinyl chloride (Gonzalez \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Kang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). CYP2E1 is involved in an enhanced activation of procarcinogens to carcinogens (Harjumaki, Pridgeon, and Ingelman-Sundberg \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)as well as induction of oxidative stress (Cho et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and it is suggested to be associated with the risk of liver cancer (Shun-Zhang Yu \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). CYP2E1 expression is downregulated in tumor tissue of HCC patients, which is often associated with aggressive tumor type and poor prognosis of the patients (Zhu et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). By IHC, the mean CYP2E1 score was significantly lower in livers with cirrhosis and HCC compared to normal livers, and was considered that CYP2E1 might be directly induced by the factor(s) derived from component cells of tumors such as carcinoma cells themselves, whether primary or metastatic, stroma, and inflammatory cells (Hata et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The results from DEN-induced HCC model also showed that, CYP2E1 expression in the liver tissues was slightly higher than that in normal rat liver in the first to third week, which may be explained by the fact that CYP2E1 is required in the metabolism of nitrosamines. With the aggravation of liver lesion and development of cirrhosis, CYP2E1 expression was gradually downregulated. Here, we found that, the level of CYP2E1 expression was decreased in forty of forty-six tumor specimens compare to the adjacent nontumor tissues as revealed by Western blot analysis, suggesting that the decreased expression of CYP2E1 may be involved in HCC tumorigenesis. A decrease in carcinogen metabolism (Liu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and an increase in procarcinogen activation have also been documented as HCC risk factors as well as changes in the metabolism of environmental toxins that arise from alterations in Cytochrome P450 (CYP) activity (Forrester et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Guengerich \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Guengerich et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Fontham et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). CYPs function not only in the detoxification of internal and external xenobiotics, but also in the metabolic activation of carcinogens and may be further implicated in tumor initiation, promotion and progression (Singh et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Previously, the functional characterization of CYP2E1 has been focused on its role in alcoholic liver diseases and drug metabolism (Nagappan et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), since it metabolizes and activates many toxicologically important compounds, such as aromatic hydrocarbons such as ethanol, carbon tetrachloride, acetaminophen, benzene, halothane, and many other halogenated substrates (Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Cui et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Cho et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the function of CYP2E1 in hepatocarcinogenesis is poorly known. Here, we found that CYP2E1 is involved in CA-induced promotion of malignant growth in HCC cells by activation of autophagy in vitro and in vivo. Forced CYP2E1 expression inhibited cell growth of HCC accompanied with autophagy inhibition. However, in the HCC cells with forced expression of CYP2E1, CA failed to influence CYP2E1 level and autophagy.\u003c/p\u003e \u003cp\u003eDysregulation of autophagy is involved in numerous diseases such as neurodegenerative disease, metabolic diseases, and cancer (Beckers, Tharkeshwar, and Van Damme \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Luo et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Tumor cells upregulate autophagy to support their elevated metabolic demand for proliferation, survival and malignancy (Amaravadi, Kimmelman, and White \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kimmelman and White \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Poillet-Perez and White \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Several signaling pathways are confirmed to regulate autophagy, including mTOR, PI3K-AKT and MAPK/ERK1/2, and mTOR is one of the main negative regulators of autophagy (Wang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gao et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, we found that autophagy is activated by CA, which is involved in CA\u0026rsquo;s role in promotion of HCC cell growth. CA upregulated the AKT phosphorylation but downregulated mTOR phosphorylation. Previously, it was reported that AKT phosphorylation was induced by CYP2E1-mediated oxidative stress (Zeng et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The interplay between ROS and autophagy in tumor cells, function from tumor initiation to progression has been widely reported(Xing et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). During the initiation, progression and metastasis of tumor cells, the pro-tumoral role of autophagy was shown to eliminate ROS-induced metabolic stress and the production of nutrients required for tumor cell survival (Gao et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). CYP2E1, a key enzyme for generation of ROS, is a major contributor to the pathogenesis of many liver diseases such as alcoholic liver disease and HCC (Gao et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), but reports of its role in autophagy regulation are limited. Here, we found that forced CYP2E1 expression increased accumulation of autophagosomes as revealed by increased LC3 puncta, and blocked autophagic flux with increased p62 accumulation and blockage of autophagosome-lysosome fusion both in vitro and in vivo. Recent studies have shown that inhibition of autophagic flux resulted in accumulation of damaged organelles and dysfunctional proteins, which has the potential to turn autophagy into a serious and destructive process, leading to fatal toxic effects on tumor cells, providing an explanation of the inhibitory activity of CYP2E1 in HCC cell growth. We also showed that CYP2E1-induced autophagy and regulation of the AKT/mTOR signaling are involved in CA-mediated cell growth of HCC.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study revealed that CYP2E1 regulates CA-induced autophagy and promotion of HCC cell growth by regulating the AKT/mTOR signaling both in vitro and in vivo. Clinically, high TBA level in HCC tissue was strongly associated with decreased expression of CYP2E1. Targeting CYP2E1-mediated autophagy may be a novel and attractive therapeutic approach for amelioration of HCC with imbalance of BAs level.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eCYP2E1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eCytochrome P450 2E1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eCholic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eHCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eHepatocellular carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eDEN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eDiethylnitrosamine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eTBA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eTotal bile acids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eCDCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eChenodesoxycholic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eHBV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eHepatitis B virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eHCV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eHepatitis C virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eBAs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eBile acids\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eGCDC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eGlycochenodeoxycholate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eGGT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eGamma-glutamyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eCCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eCholangiocarcinoma carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eSprague-Dawley\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFZQ, LYC and HSB conceived and designed the study; HZW, LXM, HHH, WZX, NY and WJC performed the experiments and acquired the data; HZW, LXM, ZHY and HZY analyzed and interpreted the data; SXJ, LYC and HSB reviewed the study; HZW drafted the manuscript; FZQ, WJZ, LYC and SBL contributed to revise manuscript and supervision. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was supported by the National Natural Science Foundation of China (Grant no. 81872040).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in the submitted article and its supplementary files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll samples were used after written and informed consent of patients and approval of the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (2019 IEC(S646)). All experimental procedures were performed as previously described in compliance with the National Institutes of Health guidelines on the ethical use of animals and approved by the Ethics Committee of Jianghan University (JHDXLL2023-053).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYes.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlwadei N, Rashid M, Chandrashekar DV, Rahighi S, Totonchy J, Sharma A, Mehvar R. Generation and characterization of cyp2e1-overexpressing hepg2 cells to study the role of cyp2e1 in hepatic hypoxia-reoxygenation injury. 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J Translational Med. 2022;20(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12967-022-03396-6\u003c/span\u003e\u003cspan address=\"10.1186/s12967-022-03396-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mome","sideBox":"Learn more about [Molecular Medicine](https://molmed.biomedcentral.com)","snPcode":"10020","submissionUrl":"https://submission.springernature.com/new-submission/10020/3","title":"Molecular Medicine","twitterHandle":"@MolecularMedic1","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cholic acid, CYP2E1, Hepatocellular cancer, Cell growth, Autophagy","lastPublishedDoi":"10.21203/rs.3.rs-4070826/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4070826/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAn increased serum cholic acid (CA) is concurrently appeared with a decreased CYP2E1 expression in hepatocellular carcinoma (HCC). However, whether and how CA may be involved in promoting hepatocarcinogenesis has not been elucidated. This study was aimed to investigate the role of CYP2E1 on CA-induced HCC cell growth and its underlying mechanism.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eOur proteomic analysis of liver tumor tissues from DEN-induced male SD rats treated with CA administration revealed that CA downregulated CYP2E1 level. The proliferative ability of CA-treated HCC cells was examined by colony formation assays. Autophagic flux was detected by immunofluorescence and confocal microscopy. The protein levels of CYP2E1, mTOR, AKT, p62 and LC3Ⅱ were detected by Immunoblotting. The effect of CYP2E1 on CA-induced hepatocellular carcinogenesis was explored in vivo by establishing a xenograft tumor model in nude mice. We also investigated the clinical value of CYP2E1 in HCC patients.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCA increased the clonogenicity of HCC cells and promoted the growth of xenograft tumors with a simultaneous reduction of CYP2E1 expression. Further studies revealed that both in vitro and in vivo, upregulating CYP2E1 could effectively inhibit cell growth of HCC with a blockage of autophagic flux, downregulation of AKT phosphorylation and upregulation of mTOR phosphorylation. CYP2E1 was involved in CA-activated autophagy through the AKT/mTOR signaling, which promoted CA-stimulated HCC cell growth. Finally, a decreased CYP2E1 expression was observed in the tumor tissues of HCC patients and the CYP2E1 level in tumor was negatively correlated with the serum level of total bile acids (TBA) and gamma-glutamyltransferase (GGT).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCYP2E1 deficit contributes to CA-induced HCC development with the mechanisms involving regulation of autophagy, thus CYP2E1 may serve as a potential target for HCC drug development.\u003c/p\u003e","manuscriptTitle":"CYP2E1 deficit mediates cholic acid-induced malignant growth in hepatocellular carcinoma cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-15 18:45:43","doi":"10.21203/rs.3.rs-4070826/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-03-12T21:20:24+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-12T20:28:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Molecular Medicine","date":"2024-03-12T15:42:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-12T06:56:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Medicine","date":"2024-03-11T21:57:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mome","sideBox":"Learn more about [Molecular Medicine](https://molmed.biomedcentral.com)","snPcode":"10020","submissionUrl":"https://submission.springernature.com/new-submission/10020/3","title":"Molecular Medicine","twitterHandle":"@MolecularMedic1","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f4f38b9f-5512-4f13-9e33-2c1881356146","owner":[],"postedDate":"March 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-23T01:46:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-15 18:45:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4070826","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4070826","identity":"rs-4070826","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}