BACKGROUND: Gallbladder cancer (GBC) is the most common malignant tumor of biliary tract. Isoliquiritigenin (ISL) is a natural compound with chalcone structure extracted from the roots of licorice and other plants. Relevant studies have shown that ISL has a strong anti-tumor ability in various types of tumors. However, the research of ISL against GBC has not been reported, which needs to be further investigated. METHODS: The effects of ISL against GBC cells in vitro and in vivo were characterized by cytotoxicity test, RNA-sequencing, quantitative real-time polymerase chain reaction, reactive oxygen species (ROS) detection, lipid peroxidation detection, ferrous ion detection, glutathione disulphide/glutathione (GSSG/GSH) detection, lentivirus transfection, nude mice tumorigenesis experiment and immunohistochemistry. RESULTS: ISL significantly inhibited the proliferation of GBC cells in vitro . The results of transcriptome sequencing and bioinformatics analysis showed that ferroptosis was the main pathway of ISL inhibiting the proliferation of GBC, and HMOX1 and GPX4 were the key molecules of ISL-induced ferroptosis. Knockdown of HMOX1 or overexpression of GPX4 can reduce the sensitivity of GBC cells to ISL-induced ferroptosis and significantly restore the viability of GBC cells. Moreover, ISL significantly reversed the iron content, ROS level, lipid peroxidation level and GSSG/GSH ratio of GBC cells. Finally, ISL significantly inhibited the growth of GBC in vivo and regulated the ferroptosis of GBC by mediating HMOX1 and GPX4 . CONCLUSION ISL induced ferroptosis in GBC mainly by activating p62-Keap1-Nrf2-HMOX1 signaling pathway and down-regulating GPX4 in vitro and in vivo . This evidence may provide a new direction for the treatment of GBC.
Animals ; Mice ; Carcinoma in Situ ; Chalcones/pharmacology* ; Ferroptosis ; Gallbladder Neoplasms/genetics* ; Glutathione Disulfide ; Kelch-Like ECH-Associated Protein 1 ; Mice, Nude ; NF-E2-Related Factor 2/genetics* ; Reactive Oxygen Species ; Humans

Gallbladder cancer(GBC)is one common type of bile tract cancers with poor prognosis. This review summarizes the recent development of studies about somatic mutation, molecular subtype, microenvironment heterogeneity, organoid, orthotopic model, patient-derived xenograft and clinical translation on GBC in aspects of genomic,transcriptome,single cell omics and clinical translation. We expect this review will provide new ideas on dissecting molecular mechanisms underlying the development and emerging chemoresistance of GBC following therapy and promote GBC precision medicine.
Humans ; Gallbladder Neoplasms/genetics* ; Prognosis ; Tumor Microenvironment

OBJECTIVETo examine the expression of tissue factor pathway inhibitor-2 (TFPI-2) in gallbladder cancer (GBC) and to investigate the anti-cancer activities of TFPI-2 against the growth of GBC.

METHODSTFPI-2 expression in gallbladder normal tissues, gallbladder polyp (GBP) tissues and GBC tissues were examined by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunohistochemical staining. Adenovirus carrying human TFPI-2 gene (Ad5-TFPI-2) were constructed and its anti-cancer effects were investigated in xenograft tumors. Xenograft tumors were constructed by injection of GBC-SD and SGC-996 cells into the flank of nude mice and the volume of xenograft tumors was measured every 3 days until the sacrifice of mice. The apoptosis index of xenograft tumors was examined by TUNEL assay. The status of Bax, Bcl-2 and caspase-3 was examined by Western blot assay.

RESULTSTFPI-2 expression was profoundly lower in GBC tissues (87.0%) when compared to normal tissues (23.3%) and GBP tissues (52.2%; χ(2) = 21.104, P = 0.000). Ad-TFPI-2 significantly inhibited the growth of xenograft tumors in nude mice. Ad-TFPI-2 inhibited GBC-SD cell growth through the induction of apoptosis. The means of total apoptotic cells per field were much higher in Ad5-TFPI-2 group than those in PBS and Ad5-GFP groups. Ad5-TFPI-2 elevated the expression of Bax and cleaved caspase-3, while it decreased the expression of Bcl-2.

CONCLUSIONSTFPI-2 gene and protein was down-regulated in GBC and the down-regulation of TFPI-2 may play a role in the tumorigenesis of GBC. Adenovirus-mediated TFPI-2 can inhibit GBC growth through the induction of apoptosis.

Adenoviridae ; genetics ; Aged ; Animals ; Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Female ; Gallbladder Neoplasms ; metabolism ; pathology ; therapy ; Genetic Therapy ; Glycoproteins ; genetics ; metabolism ; Humans ; Male ; Mice ; Mice, Nude ; Middle Aged ; Neoplasm Transplantation ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

BACKGROUNDThe precise molecular mechanisms underlying the gallbladder carcinoma (GBC) metastasis has not been fully elucidated.

METHODSIn the present study, metastasis-associated proteins were identified by comparative proteomic analysis. The functional study of the candidate protein vimentin was further investigated. First, a pair of higher and lower metastatic sublines (termed GBC-SD/M3 and GBC-SD, respectively), originated from the same parental cell line, was screened by spontaneous tumorigenicity and metastasis in vivo in animal study and further characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach comprised two-dimensional gel electrophoresis analysis and mass spectroscopy was used to identify and compare the protein expression patterns between higher metastatic GBC-SD/M3 and lower metastatic GBC-SD cell lines. Then twenty-six proteins were identified.

RESULTSAmong the 26 proteins identified, fourteen proteins were up-regulated and 12 proteins were down-regulated in GBC-SD/M3. Vimentin was identified and found to be overexpressed in GBC-SD/M3 as compared with GBC-SD. This result was further confirmed by quantitative PCR and Western blotting analysis. Furthermore, the cell migration and invasion potency of GBC-SD/M3 in vitro was remarkably suppressed after small interference RNA-mediated knockdown of vimentin. Moreover, immunoblot and immunohistochemical analysis on 12 human GBC specimens showed consistently increased vimentin expression in metastases compared with primary tumors.

CONCLUSIONTumor vimentin level may reflect the pathological progression in some GBC and may be a useful marker for predicting tumor metastasis and a therapeutic target for the treatment of GBC patients with metastases.

Animals ; Blotting, Western ; Cell Line, Tumor ; Cell Movement ; genetics ; physiology ; Electrophoresis, Gel, Two-Dimensional ; Gallbladder Neoplasms ; genetics ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Mice ; Mice, Nude ; Neoplasm Metastasis ; genetics ; pathology ; RNA Interference ; Reverse Transcriptase Polymerase Chain Reaction ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Vimentin ; genetics ; metabolism

CA-19-9 Antigen ; metabolism ; Carcinoembryonic Antigen ; metabolism ; Carcinoma ; pathology ; Carcinoma in Situ ; etiology ; genetics ; metabolism ; pathology ; Chromosomes, Human, Pair 17 ; Chromosomes, Human, Pair 5 ; Diagnosis, Differential ; Gallbladder Diseases ; pathology ; Gallbladder Neoplasms ; etiology ; genetics ; metabolism ; pathology ; Humans ; Loss of Heterozygosity ; Microsatellite Instability ; Polyps ; pathology ; Precancerous Conditions ; etiology ; genetics ; metabolism ; pathology

Carcinoma ; metabolism ; Cell Differentiation ; genetics ; Gallbladder Neoplasms ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Kangai-1 Protein ; genetics ; metabolism ; NM23 Nucleoside Diphosphate Kinases ; genetics ; metabolism

Antigens ; immunology ; Antigens, Neoplasm ; drug effects ; immunology ; metabolism ; Gallbladder Neoplasms ; immunology ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Neoplastic Stem Cells ; immunology ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Tumor Cells, Cultured

OBJECTIVE: To examine the expressive level of enhancer of zesle homolog 2 (EZH2) and phosphatase and tension homolog (PTEN), and to explore its clinicopathological significance in benign and malignant lesion of gallbladder. METHODS: EnVision immunohistochemical method was used to detect the expressive levels of EZH2 and PTEN in routinely paraffin-embedded sections in the resected specimens of gallbladder adenocarcinoma (n = 108), peritumoral tissues (n = 46), adenomatous polyp(n = 15), and chronic cholecystitis (n = 35). RESULTS: The positive rate of EZH2 was significantly higher in gallbladder adenocarcinoma than that in peritumoral tissues (chi(2) = 24.49, P < 0.01), adenomatous polyp(chi(2) = 11.68, P < 0.01), and chronic cholecystitis (chi(2) = 31.62, P < 0.01). The benign lesions in the positive cases of EZH2 and (or) the negative ones of PTEN showed the moderately- or severely- atypical hyperplasia of gallbladder epithelium. The positive rate of PTEN was significantly lower in gallbladder adenocarcinoma than that in peritumoral tissues(n = 20.20, P < 0.01), adenomatous polyp(chi(2)=10.81, P<0.01), and chronic cholecystitis (n = 29.83, P < 0.01).The positive rates of EZH2 were significantly lower in the highly-differentiated adenocarcinoma, the maximal diameter of mass < 2 cm, non-metastasis of lymphnodes, and non-infiltration of regional tissues than those in the moderately or low-differentiated adenocarcinoma, the maximal diameter > or = 2 cm, metastasis of lymphnode, and infiltration of regional tissues (P < 0.05 or P < 0.01). High inconsistency was found between the expression of EZH2 and PTEN in gallbladder adenocarcinoma (P < 0.05). CONCLUSION Expression of EZH2 and/or PTEN might be important biological markers in the carcinogenesis, progression, biological behaviors and prognosis of gallbladder adenocarcinoma.
Adenocarcinoma ; genetics ; pathology ; Adult ; Biomarkers, Tumor ; DNA-Binding Proteins ; biosynthesis ; Enhancer of Zeste Homolog 2 Protein ; Female ; Gallbladder Neoplasms ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; PTEN Phosphohydrolase ; biosynthesis ; Polycomb Repressive Complex 2 ; Prognosis ; Transcription Factors ; biosynthesis

OBJECTIVETo study the effects of low-power electromagnetic fields of different frequencies on proliferation and DNA damage of gallbladder cancer cells.

METHODSThe cell growth curve was drawn and single cell gel electrophoresis performed to evaluate the proliferation and DNA damage of gallbladder cancer cells respectively after the cells were exposed to electromagnetic fields of different frequencies.

RESULTSAfter exposure to low-power electromagnetic fields of different frequencies (0.1-40 MHz), the cells displayed significant changes with obvious cell proliferation inhibition and DNA strand breakage.

CONCLUSIONLow-power electromagnetic fields within the range of 0.1-40 MHz may impair the DNA strand and cause inhibition of proliferation of the gallbladder cancer cells, and these effects are related to the frequency of the electromagnetic fields but not in a linear fashion.

Cell Line, Tumor ; Cell Proliferation ; radiation effects ; Comet Assay ; DNA Damage ; Dose-Response Relationship, Radiation ; Electromagnetic Fields ; Gallbladder Neoplasms ; genetics ; pathology ; Humans

OBJECTIVETo explore the anti-tumor mechanism of norcantharidin (NCTD) for the implanted tumors of human gallbladder carcinoma in nude mice in vivo.

METHODSAnimal model of implanted tumors of human gallbladder carcinoma in nude mice was established. Mice were randomly divided into control, 5-FU, NCTD and NCTD + 5-FU groups and were taken different treatment. The expressions of PCNA, Ki-67, cyclin D1, p27, Bcl-2, Bax, Survivin, nm23/nm23-H1, MMP2 and TIMP2 proteins or genes in each tissue section of every group were determined by immunohistochemistry and RT-PCR.

RESULTS(1) On proliferation-related gene proteins, the expression of PCNA, Ki-67, cyclin D1 was significantly decreased, with significantly increased expression of p27 protein, in paraffin sections of NCTD group when compared with control group (P < 0.05); The expression of PCNA mRNA, cyclin D1 mRNA was decreased, with significantly increased expression of p27 mRNA in NCTD group. (2) On apoptosis-related gene proteins, the expression of Bcl-2 was significantly decreased in paraffin sections of NCTD group when compared with control group (P < 0.05); The expression of Bcl-2 mRNA, Survivin mRNA was significantly decreased, with significantly increased expression of Bax mRNA in NCTD group. (3) There was significant difference on invasion around tumor and lung metastasis in NCTD group when compared with control group (P < 0.01). On metastasis-related gene proteins, the expression of nm23 and TIMP2 was significantly increased, with significantly decreased expression of MMP2 in paraffin sections of NCTD group when compared with control group (P < 0.05); The expression of nm23-H1 mRNA, TIMP2 mRNA was significantly increased, with significantly decreased expression of MMP2 mRNA in NCTD group.

CONCLUSIONSThe anti-tumor mechanism of NCTD for human gallbladder carcinoma in nude mice might correlated with inhibition of cell proliferation, blockage of cell cycle, induction of cell apoptosis, reducing of cell motility and invasive capability, alteration of the expression of proliferation-, apoptosis- and metastasis-related gene proteins such as PCNA, Ki-67, cyclin D1, p27, Bcl-2, Bax, Survivin, nm23, MMP2 and TIMP2.

Animals ; Apoptosis ; drug effects ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Proliferation ; drug effects ; Cyclin D1 ; biosynthesis ; genetics ; Gallbladder Neoplasms ; drug therapy ; metabolism ; pathology ; Humans ; Ki-67 Antigen ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; bcl-2-Associated X Protein ; biosynthesis ; genetics