Tauroursodeoxycholic acid suppresses biliary epithelial cell apoptosis and endoplasmic reticulum stress by miR-107/NCK1 axis in a FXR-dependent manner.

Tauroursodeoxycholic acid (TUDCA) can activate farnesoid X receptor (FXR) to involve in the formation of gallstones. Here, this study aimed to probe the potential mechanism of TUDCA-FXR network in the formation of bile duct stone. The levels of TUDCA, FXR and NCK1 were decreased, while the level of miR-107 was increased in the serum of bile duct stone patients. FXR expression was positively correlated with TUDCA or NCK1 expression in patients, moreover, TUDCA pretreatment in biliary epithelial cells increased the levels of FXR and NCK1, and rescued the decrease of NCK1 caused by FXR knockdown in cells. Then functional analysis showed FXR knockdown caused apoptosis and endoplasmic reticulum stress (ERS) as well as suppressed proliferation in biliary epithelial cells in vitro, which were attenuated by TUDCA pretreatment or NCK1 overexpression Mechanistically, NCK1 was a target of miR-107, which was up-regulated by FXR silencing, and FXR knockdown-induced decrease of NCK1 was rescued by miR-107 inhibition. Additionally, miR-107 expression was negatively correlated with TUDCA expression in bile duct stone patients, and TUDCA pretreatment in biliary epithelial cells decreased miR-107 expression by FXR. Functionally, the pretreatment of TUDCA or FXR agonist suppressed miR-107-evoked apoptosis and ERS in biliary epithelial cells. In conclusion, TUDCA up-regulates FXR expression to activate NCK1 through absorbing miR-107, thus suppressing the apoptosis and ERS in biliary epithelial cells, these results provided a theoretical basis for elucidating the mechanism of bile duct stone formation.

WWP2 overexpression inhibits the antitumor effects of doxorubicin in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common liver cancer that accounts for 90% of cases. Doxorubicin exhibits a broad spectrum of antitumor activity and is one of the most active agents in HCC. WW domain-containing protein 2 (WWP2) is highly expressed in HCC tissues and activates protein kinase B (AKT) signaling pathway to enhance tumor metastasis. However, the role of WWP2 in the glycolysis and antitumor effects of doxorubicin and the epigenetic alterations of WWP2 in HCC remain to be elucidated. The levels of WWP2 and N6-methyladenosine methyltransferase-like 3 (METTL3) in clinical samples and cells were investigated. WWP2 were silenced or overexpressed to study the role of WWP2 in regulating cell proliferation, colony formation, and glycolysis. RNA immunoprecipitation was performed to test m6 A levels. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Western blot were used to measure mRNA and protein, respectively. WWP2 silencing inhibits cell proliferation, colony formation, and glycolysis, while WWP2 overexpression has the inverse effects via the AKT signaling pathway. Silencing WWP2 enhances doxorubicin's antitumor effect, while WWP2 overexpression suppresses doxorubicin's antitumor effect. Data also support that METTL3 mediates WWP2 m6A modification, and m6A reader, IGF2BP2, binds to the methylated WWP2 to promote the stability of WWP2, leading to upregulation of WWP2. METTL3 mediates WWP2 m6A modification, which can be recognized and bound by IGF2BP2 to increase the stability of WWP2, leading to WWP2 overexpression which inhibits the antitumor effects of doxorubicin through METTL3/WWP2/AKT/glycolysis axis.

G protein-coupled receptor 30 activation protects hepatic ischemia-reperfusion injury of liver tissue through inhibiting NLRP3 in the rat model.

One of the most prominent characteristics of hepatic ischemia-reperfusion injury (HI/R) is an intense inflammatory reaction, which plays a key role in inflammatory injury induced by ischemia-reperfusion. Nucleotide-binding oligomerization domain-containing protein (NOD-), leucine-rich repeat (LRR), and pyrin domains-containing protein 3 (NLRP3) are involved in the inflammatory injury of ischemia-reperfusion as an important pattern recognition receptor for innate immunity. G protein-coupled receptor 30 (GPR30) is a newly identified as 7-transmembrane G protein-coupled receptor and can be activated by many stimulations including estrogen. The current study aims to explore whether GPR30 agonist (G1) can alleviate hepatic ischemia-reperfusion injury HI/R by inhibiting NLRP3. An induced HI/R rat model was generated, blood and liver samples were gathered and subjected to histological examination, biochemical assays, Western blot assays, and qRT-PCR. Our results indicated GPR30 agonist (G1) pretreatment or NLRP3 silencing significantly decreased the serum levels of Interleukin 1ß (IL-1ß), alanine aminotransferase (ALT) and aspartate aminotransferase, improved histological alterations and hepatocyte apoptosis. Moreover, G1 pretreatment or NLRP3 silencing downregulated the protein level of Caspase-1 and pro-Interleukin 1ß (pro-IL-1ß) while G1 pretreatment upregulated the expression of GPR30 (p < 0.05). In conclusion, the salutary effects of GPR30 agonists on HI/R are mediated at least in part through downregulating NLRP3 expression. GPR30 may be used as a therapy target of HI/R.

Effects of avitinib on the pharmacokinetics of osimertinib in vitro and in vivo in rats.

BACKGROUND: Avitinib is one type of the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) for the treatment of non-small cell lung cancer (NSCLC) with EGFR mutations. The purpose of this study was to investigate the effect of avitinib on the pharmacokinetics of osimertinib, one FDA approved third-generation TIKI, both in vitro and in vivo. METHODS: The in vitro metabolic stability and inhibitory effect of avitinib on osimertinib were assessed with rat liver microsomes (RLM) to determine its IC50 values. For the in vivo study, 18 Sprague-Dawley rats were randomly divided into three groups: the avitinib multiple dose group (30 mg/kg avitinib once daily for seven days), the avitinib single dose group (PEG200 once daily for six days and a dose of 30 mg/kg avitinib in PEG200 on day 7) and the control group (equal amounts of PEG200 once daily for seven days). Next, all rats were given osimertinib at a dosage of 10 mg/kg. UPLC/MS-MS was used for the determination of the concentration of osimertinib in plasma. RESULTS: In vitro analysis revealed that the IC50 value of osimertinib in rat liver microsomes was 27.6 µM. When rats were pretreated with avitinib, the values of AUC and MRT of the osimertinib were increased, and its Cmax and Tmax were significantly extended, whereas the values of CLz/F were significantly decreased (P < 0.05). CONCLUSIONS: Both in vitro and in vivo results demonstrated that a drug-drug interaction between avitinib and osimertinib occurred and more attention should be paid when avitinib and osimertinib are synchronously administered in clinic. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: Osimertinib is the only market available third-generation EGFR-TKI and it has been reported that some drugs could have drug-drug interactions with it. WHAT THIS STUDY ADDS: For the first time, we systematically investigated the effect of avitinib, one newly developed third-generation EGFR-TKI, on the pharmacokinetics of osimertinib both in vitro and in vivo using a rat model.

Lamc1 promotes the Warburg effect in hepatocellular carcinoma cells by regulating PKM2 expression through AKT pathway.

Hepatocellular carcinoma (HCC), the most common aggressive malignancy of liver, is the third leading cause of cancer death across the world. Laminin gamma 1 (Lamc1), encodes laminin-γ1, an extracellular matrix protein involved in various progresses such as tumor cell proliferation and metabolism. In the present study, high expression of Lamc1 and PKM2 was observed in tumor tissues of HCC patients. In vitro, down-regulation of Lamc1 inhibited proliferation of HCC cells by promoting cell death, reduced glucose consumption and lactate production, accompanied by a decrease in the expression of glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA), and PTEN increased, as well as PTEN S380 and AKT S473/T308 phosphorylation decreased, while Lamc1 up-regulation had the opposite effect. The effects of PKM2 were similar to that of Lamc1 and markedly counteracted the effects of Lamc1 down-regulation. In addition, Lamc1-induced increase in PKM2 expression was strongly attenuated by a PI3K inhibitor, LY294002 or a si-p110 PI3K, with a significant decrease in GLUT1 and LDHA expression, as well as decreased AKT T308 phosphorylation. Thus, we speculated that Lamc1 was implicated in the progression of HCC probably by regulating PKM2 expression through PTEN/AKT pathway.

Inhibition of WWP2 suppresses proliferation, and induces G1 cell cycle arrest and apoptosis in liver cancer cells.

Primary liver cancer is one of the most common and aggressive human malignancies worldwide. As numerous studies have revealed that WW domain containing E3 Ub­protein ligase 2 (WWP2) exerts cancer­specific functions, the present study assessed the role of WWP2 in liver cancer. WWP2 was revealed to be significantly overexpressed in liver cancer tissues compared with paired normal tissues at the mRNA as well as at the protein level. Furthermore, small interfering RNA-mediated WWP2 knockdown in liver cancer cell lines was demonstrated to inhibit cell proliferation, cause cell cycle arrested in G1 phase and to induce apoptosis as revealed by a Cell Counting Kit-8 assay and flow cytometric analysis. In addition, western blot analysis revealed that WWP2 knockdown significantly increased the expression of apoptosis-associated markers caspase­7, caspase­8 and B-cell lymphoma 2 (Bcl-2)-associated X in liver cancer cell lines, while Bcl­2 was significantly decreased. In conclusion, the present study suggested that WWP2 may exert important functions in the over­proliferation and evasion of apoptosis of liver cancer, likely through regulating the expression of apoptosis-associated markers. Furthermore, WWP2 may represent a novel diagnostic marker and molecular therapeutic target for liver cancer.

Silencing of WWP2 inhibits adhesion, invasion, and migration in liver cancer cells.

The role and clinical implication of the WWP2 E3 ubiquitin ligase in liver cancer are poorly understood. In the current study, we investigated the expression level of WWP2 and its functions in cell adhesion, invasion, and migration in liver cancer. We used real-time PCR to detect the expression of WWP2 in liver cancer and adjacent samples from the People's Hospital of Lishui and also analyzed The Cancer Genome Atlas (TCGA) RNA-seq data by bioinformatics. Migration and invasion were detected by transwell analysis. We detected a strong WWP2 expression in tumor tissues of the People's Hospital of Lishui, and the survival rate was significantly higher in patients with lower WWP2-expressing tumors. WWP2 small hairpin RNA (shRNA) lentivirus stably infected cells (shWWP2), Huh7, showed slower growth speed compared with scramble control-infected cells in a xenograft mouse model. Knockdown of WWP2 Huh7 and BEL-7404 cells demonstrated a reduction in adhesion, invasion, and migration. Gene set enrichment analysis (GSEA) showed that WWP2 is positively correlated to cancer-related pathways including the chemokine signaling pathway. WWP2 also regulated MMP-9, caspase-9, CXCR3, and CCR5 expression in liver cancer cells. In addition, knockdown of CXCR3 and CCR5 significantly inhibited cell proliferation, adhesion, invasion, and migration in Huh7 and BEL-7404 cells. Our data suggest that targeting of WWP2 may be a therapeutic strategy for liver cancer treatment.

Laparoscopic surgery is more advantageous than laparotomic surgery for treatment of hypersplenism with gallstones.

BACKGROUND: The aim of this study was to compare laparoscopic vs. laparotomic surgery for the treatment of hypersplenism with gallstones. METHODS: Forty patients were treated with splenectomy and cholecystectomy using either totally laparoscopic surgery (laparoscopic group) or laparotomy (laparotomy group). The outcomes were duration of the surgery, intraoperative bleeding volume, postoperative complications, and number of postoperative hospitalization days. RESULTS: There was no difference in the duration of the surgery between both groups. No patient experienced intraoperative complications. There were postoperative pleural effusions (N.=3) and bleeding at the puncture site (N.=1) in laparoscopic group, and postoperative pleural effusions (N.=2), incision infection (N.=2) and peritoneal effusion (N.=1) in laparotomy group. The length of postoperative hospitalization was markedly shorter in laparoscopic group. The follow-up for 3 to 15 months after the surgery demonstrated that hypersplenism was corrected in all patients. All blood markers (white blood cells, platelets, and hemoglobin) recovered to normal levels. The portal vein thrombosis occurred in 6 patients of laparoscopic group and 5 patients of laparotomy group; this was controlled by oral administration of Warfarin and enteric coated Aspirin capsules. CONCLUSIONS: Treatment efficacy is comparable between laparoscopic surgery and traditional laparotomy. Laparoscopic surgery is more advantageous because of smaller trauma, faster postoperative recovery, and minimal invasiveness.

Ezrin expression in the primary hepatocellular carcinoma patients and associated with clinical, pathological characteristics.

OBJECTIVE: The aim of this study was to assess Ezrin expression in the primary hepatic carcinoma patients and associated with clinical, pathological characteristics. MATERIALS AND METHODS: Fifty-one patients with primary hepatocellular carcinoma (PHC) with completed clinical data were retrospectively analyzed in this study. The Ezrin expression in PHC and normal control liver tissue was tested by immunohistochemical assay. The Ezrin expression and relationship with clinical characteristics were evaluated. RESULTS: The Ezrin positive rate were 66.7% and 15.7% with expression score of 3.21 ± 1.46 and 0.60 ± 1.10, respectively, in the cancer tissue and control tissue with statistical difference (P < 0.05). The Ezrin expression was associated with the metastasis status of the patients (P < 0.05) but not associated with the age (P > 0.05), gender (P > 0.05), differentiation (P > 0.05), and tumor diameter (P > 0.05). CONCLUSION: Ezrin protein is highly expressed in human PHC tissue which can be used for the prediction of metastasis disease.

Comparison of transumbilical single-port laparoscopic cholecystectomy and fourth-port laparoscopic cholecystectomy.

This work aims to compare the curative effect of transumbilical single-port laparoscopic cholecystectomy (TUSPLC) and four-port laparoscopic cholecystectomy (FPLC). 200 patients with cholecystolithiasis were enrolled in this study. They were randomly divided into TUSPLC group and FPLC group, 100 cases in each group, and the TUSPLC and FPLC was performed, respectively. The surgical time, intraoperative complication, conversions rate, postoperative pain, postoperative analgesic drug use, incision infection, postoperative hospitalization time and postoperative cosmetic results in two groups were compared. The total conversion rate, conversion rate with Nassar grade II, and conversion rate with Nassar grade III in TUSPLC group were significantly higher than FPLC group (P < 0.01), and the incision cosmetic result after 1 month in TUSPLC group was obviously better than FPLC group (P < 0.01), but the surgical time in TUSPLC group was significantly longer than FPLC group (P < 0.01). There was no significant difference of incision infection, intraoperative complication, and postoperative hospitalization time, incision pain in postoperative first and second day, postoperative use of analgesia drug and incision cosmetic result on discharge day between two groups (P > 0.05). TUSPLC has obvious advantage in treatment of Nassar grade I patients with cholecystolithiasis. It can be used as a supplement for standard laparoscopic gallbladder surgery. It is safe and feasible, without abdominal scar, thus achieving to excellent cosmetic result and high satisfaction in patients.

The association of renin-angiotensin system genes with the progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Angiogenesis is reported to play a pivotal role in the occurrence, development and metastasis of HCC. The renin-angiotensin system (RAS) is involved in the regulation of angiogenesis. Here, based on the analysis of HCC datasets from Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA), we found that there was a negative correlation between the mRNA levels of angiotensin converting enzyme 2 (ACE2) and CD34. To explore the association of RAS with the progression from fibrosis to cirrhosis to HCC, liver specimens and serum samples were collected from patients with hepatic fibrosis, cirrhosis and HCC. Relative hepatic mRNA levels of CD34 and ACE2 were determined by real-time PCR, and the serum concentrations of Angiotensin II (Ang II), Ang (1-7) and vascular endothelial growth factor (VEGF) were detected by ELISA. We found that ACE2 mRNA was gradually decreased, while CD34 mRNA was progressively increased with the increasing grade of disease severity. Concentrations of Ang II, Ang (1-7) and VEGF were higher in the sera of patients than in that of healthy volunteers. These proteins' concentrations were also progressively increased with the increasing grade of disease severity. Moreover, a positive correlation was found between VEGF and Ang II or Ang (1-7), while negative correlation was observed between mRNA levels of CD34 and ACE2. More importantly, patients with higher level of ACE2 expression had longer survival time than those with lower level of ACE2 expression. Taken together, our data suggests that the low expression of ACE2 may be a useful indicator of poor prognosis in HCC. The RAS may have a role in the progression of HCC.

Effect of DAPK1 gene on proliferation, migration, and invasion of carcinoma of pancreas BxPC-3 cell line.

DAPK1 can induce apoptosis in several cells; to determine the effect of DAPK1 would provide a new potential therapeutic strategy for treating pancreatic cancer. The aim of the present study was to investigate the effect of DAPK1 gene on proliferation, migration, and invasion of carcinoma of pancreas BxPC-3 cell line and explore the possible mechanisms. In our study, DAPK1 over-expressed cells were established by using the lentiviral transfection method, and DAPK1 obviously increased in BxPC-3 cells after transient transfection. Cell Counting Kit-8 (CCK-8) assay was used to determine the BxPC-3 cells proliferation after transfection. Apoptosis of the BxPC-3 cells was determined by using flow cytometry analysis. In addition, cell adhesion assay and in vitro invasion assay were performed. Western blotting was used to determine the protein expressions of caspase-3, DAPK1, VEGF, PEDF, MMP2, AKT, P-AKT, P-ERK, Bcl2, and Bax. Our results demonstrated that DAPK1 gene over-expression can suppress the proliferation, migration, and invasion of carcinoma of pancreas BxPC-3 cell line, and the possible mechanisms may be correlated to induction of mitochondria-mediated apoptosis, down-regulations of MMP-2 and VEGF, up-regulations of PEDF, through the PI3K/Akt and ERK pathways.