Pulsatilla chinensis saponins improve SCFAs regulating GPR43-NLRP3 signaling pathway in the treatment of ulcerative colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulsatilla decoction has been extensively used to treat ulcerative colitis (UC) in recent years. Pulsatilla chinensis saponin (PRS), the active ingredient of its monarch medicine Pulsatilla chinensis (Bunge) Regel, plays a crucial role in the treatment of UC, but its specific mechanism of action has not been fully elucidated. AIM OF THE STUDY: This study aims to investigate the protective effect and possible mechanism of PRS on DSS-induced ulcerative colitis in rats. MATERIALS AND METHODS: In this study, the DSS-induced colitis model was used to explore the metabolism and absorption of PRS under UC, detect the content of short-chain fatty acids (SCFAs) in colon tissue, the expression of receptor G Protein-Coupled Receptor 43 (GPR43) protein and inflammasome NLRP3, and observe the expression level of IL-1ß, IL-6 and TNF-α in colon tissue. The protective effect of the PRS was also observed. RESULTS: It was found that in the UC group, the absorption rate and extent of drugs increased, and the elimination was accelerated. Compared with the control group, PRS increased the content of short-chain fatty acids (SCFAs) in colon tissue, promoted the expression of SCFAs receptor GPR43 protein, inhibited the activation of the NLRP3 inflammasome, and decreased the content of IL-1ß, IL-6 and TNF-α. PRS protects the colon in DSS-induced inflammatory bowel disease by increasing the content of SCFAs, promoting the expression of GPR43 protein, inhibiting the activation of the NLRP3 inflammasome, and reversing the increase in IL-1ß, IL-6 and TNF-α levels. CONCLUSIONS: PRS can increase the content of colonic SCFAs, activate the GPR43-NLRP3 signaling pathway, and reduce the levels of pro-inflammatory cytokines, thereby improving the symptoms of DSS-induced colitis.

Cyclophilin D: Guardian or Executioner for Tumor Cells?

Cyclophilin D (CypD) is a peptide-proline cis-trans isomerase (PPIase) distributed in the mitochondrial matrix. CypD regulates the opening of the mitochondrial permeability conversion pore (mPTP) and mitochondrial bioenergetics through PPIase activity or interaction with multiple binding partners in mitochondria. CypD initially attracted attention due to its regulation of mPTP overopening-mediated cell death. However, recent studies on the effects of CypD on tumors have shown conflicting results. Although CypD has been proven to promote the aerobic glycolysis in tumor cells, its regulation of malignant characteristics such as the survival, invasion and drug resistance of tumor cells remains controversial. Here, we elaborate the main biological functions of CypD and its relationships with tumor progression identified in recent years, focusing on the dual role of CypD in tumors.

Extrahepatic biliary tract visualization using near-infrared fluorescence imaging with indocyanine green: optimization of dose and dosing time.

BACKGROUND: The dose and dosing time of indocyanine green (ICG) vary among fluorescence cholangiography (FC) studies. The purpose of this prospective, randomized, exploratory clinical trial was to optimize the dose and dosing time of ICG. METHODS: PubMed was searched to determine the optimal dose. To optimize the dosing time of ICG, a clinical trial was designed with two parts. The first part included patients with T tubes for more than 1 month. After the patient was injected with ICG, bile was collected at 10 time points to explore the change and trends of bile fluorescence intensity (FI). In addition, the results of the first experiment were used to setup a randomized controlled trial (RCT) that aimed to find the optimal dosing timing for ICG injections for laparoscopic cholecystectomy (LC). During surgery, imaging data were collected for analysis. RESULTS: After performing a systematic review, the ICG injection dose for each patient in the clinical trial was 10 mg. Five patients were included in the first part of the study. Bile collected 8 h after ICG injection had a higher FI than bile collected at other time points (p < 0.05), and the FI of bile collected 20 h after ICG injection was nearly zero. In the second part of the experiment, 4 groups of patients (6 patients per group) were injected with 10 mg ICG at 8, 10, 12 and 14 h prior to surgery. The distribution of bile duct FI (p = 0.001), liver FI (p < 0.001), and common bile duct (CBD)-to-liver contrast (p = 0.001) were not the same in each group. Further analysis with the Bonferroni method revealed the following: (1) the FI of the CBD in the 8 h group was significantly different from that in the 14 h group (adjusted p < 0.001); (2) the liver FI of the 8 h group was higher than that of the 10 h group (adjusted p = 0.042) and the 14 h group (adjusted p < 0.001); and (3) the CBD-to-liver contrast of the 8 h group was lower than that of the 10 h group (adjusted p = 0.013) and the 14 h group (adjusted p = 0.001). CONCLUSION: ICG FC enables the real-time identification of extrahepatic bile ducts. The optimal effect of FC can be achieved by performing 10 mg ICG injections 10 to 12 h prior to surgery.

Identification of potential functional genes in papillary thyroid cancer by co-expression network analysis.

Interactions between multiple genes are involved in the development of complex diseases. However, there are few analyses of gene interactions associated with papillary thyroid cancer (PTC). Weighted gene co-expression network analysis (WGCNA) is a novel and powerful method that detects gene interactions according to their co-expression similarities. In the present study, WGCNA was performed in order to identify functional genes associated with PTC using R package. First, differential gene expression analysis was conducted in order to identify the differentially expressed genes (DEGs) between PTC and normal samples. Subsequently, co-expression networks of the DEGs were constructed for the two sample groups, respectively. The two networks were compared in order to identify a poorly preserved module. Concentrating on the significant module, validation analysis was performed to confirm the identified genes and combined functional enrichment analysis was conducted in order to identify more functional associations of these genes with PTC. As a result, 1062 DEGs were identified for network construction. A brown module containing 118 highly related genes was selected as it exhibited the lowest module preservation. After validation analysis, 61 genes in the module were confirmed to be associated with PTC. Following the enrichment analysis, two PTC-related pathways were identified: Wnt signal pathway and transcriptional misregulation in cancer. LRP4, KLK7, PRICKLE1, ETV4 and ETV5 were predicted to be candidate genes regulating the pathogenesis of PTC. These results provide novel insights into the etiology of PTC and the identification of potential functional genes.

Bone marrow mesenchymal stromal cells attenuate liver allograft rejection may via upregulation PD-L1 expression through downregulation of miR-17-5p.

Studies have reported that bone marrow mesenchymal stem cells (BMSCs) play an important role in immune regulation after organ transplantation. Some of the BMSC-mediated regulatory mechanisms are related to PD-L1 expression. However, the regulatory mechanism of PD-L1 expression is not fully understood. In this experiment, we confirmed the regulatory role of BMSCs in the rejection of liver transplantation and explored the possible mechanism by which PD-L1 expression is regulated in BMSCs. An orthotopic liver transplantation model in rats was established based on the "double-cuff technique". Third-generation BMSCs were injected into the rejection model rats via portal vein. At the same time, sera were obtained from rats in the allograft, isograft and sham-operated groups. The sera from these groups were separately added to BMSCs complete medium to partially mimic the in vivo environment in which BMSCs are exposed. After predicting and analysing microRNAs that regulate PD-L1 expression, we examined the microRNA and PD-L1 expression in BMSCs of the three groups cultured in the conditioned media. Moreover, a luciferase reporter assay was used to confirm the interaction between PD-L1 and microRNA. The study found that the liver function and liver graft histopathology of the BMSC-treated group were better than those of the allograft group. The Kaplan-Meier survival curve analysis showed that the median survival time (MST) of the BMSC-treated group was longer than that of the allograft group. Bioinformatics prediction and analysis showed that miR-17-5p is highly likely to regulate PD-L1. Compared with the other two groups of cells, BMSCs cultured with serum from allograft models showed higher PD-L1 expression, but lower miR-17-5p expression. Pearson correlation analysis showed that miR-17-5p and PD-L1 expression was negatively correlated, and further luciferase reporter assays confirmed that miR-17-5p interacted directly with the 3´-untranslated region (UTR) of PD-L1 mRNA. The results of this study demonstrate that BMSCs can attenuate liver allograft rejection and provide us with the idea that BMSCs may further upregulate PD-L1 expression by downregulating miR-17-5p expression, thereby attenuating liver allograft rejection.

cAMP responsive element binding protein-1 is a transcription factor of lysosomal-associated protein transmembrane-4 Beta in human breast cancer cells.

Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) is a potential proto-oncogene, whose overexpression is involved in cancer occurrence and progression. Its transcript is up-regulated in various types of solid tumors including breast cancer. However, its transcriptional regulation mechanism is still unclear. To investigate the mechanism of transcriptional regulation of LAPTM4B in human breast cancer cells, a series of luciferase reporter constructs and construct with mutated binding site for cAMP responsive element binding protein-1 (CREB1) were generated by PCR amplification and transiently transfected into breast cancer cells to determine the transcriptional activities of different promoter regions. The +10+292 promoter region was possessed the highest transcriptional activity. The ability of CREB1 to bind the LAPMT4B promoter was confirmed by electrophoretic mobility shift assay, super-shift and RNA interference experiments. Our study identified the core promoter region responsible for constitutive expression of LAPTM4B and clarified that CREB1 played an important role in LAPTM4B transcriptional regulation in human breast cancer cells.

Upregulation of LAPTM4B-35 promotes malignant transformation and tumorigenesis in L02 human liver cell line.

Hepatocellular carcinoma (HCC) is one of the most frequent malignant neoplasms worldwide and is the second leading cause of cancer death in China. We have previously demonstrated that LAPTM4B-35, encoded by lysosomal protein transmembrane 4 beta gene, is overexpressed in over 80% of HCCs and is a novel-independent prognostic factor for metastasis, recurrence, and postoperative survival in HCC. In this study, we investigated the role of LAPTM4B-35 in malignant transformation and tumorigenesis using L02 cells, a cell line originated from human normal liver cells. Our data show that replication-deficient adenovirus vector-mediated upregulation of LAPTM4B-35 promotes anchorage-independent proliferation and resistance to adriamycin-induced apoptosis. Study of the underlying mechanisms demonstrated alterations of molecular events involved in these processes, which included the activation of phosphoinositide 3-kinases (PI3K)/serine/threonine protein kinase B (PKB/AKT)/bcl-xL/bcl-2-associated death promoter homolog (Bad) signaling pathway, inhibition of caspase-3 activation, upregulation of Bcl-2, and downregulation of Bax. In addition, upregulation of LAPTM4B-35 in L02 cells resulted in tumorigenesis in 100% (6/6) of inoculated nude mice and accelerated the death of mice with xenografts in vivo. In conclusion, LAPTM4B-35 promotes malignant transformation and tumorigenesis in human liver L02 cell line through promotion of deregulated proliferation and inhibition of apoptosis. These findings suggest that overexpression of LAPTM4B-35 may play a critical role in hepatocarcinogenesis and therefore, may be a therapeutic target for HCC.

Overexpression of LAPTM4B-35 attenuates epirubucin-induced apoptosis of gallbladder carcinoma GBC-SD cells.

BACKGROUND: It was shown previously that LAPTM4B promoted growth of gallbladder carcinoma (GBC) cells and predicted poor prognosis in GBC; however, its roles and relative mechanisms in apoptosis of GBC cells remain unknown. METHODS: The plasmids, pcDNA3-AE, containing the complete open reading frame of LAPTM4B and Mock (pcDNA3), were transfected transiently into GBC-SD cells, followed by induction of apoptosis by epirubicin. Cell apoptosis was determined by Hoechst 33258 staining, propidium iodide (PI) staining, and Annexin V/PI double staining flow cytometry. Protein expression was detected by immunoblotting. RESULTS: Overexpression of LAPTM4B-35 was observed in cells transfected with pcDNA3-AE. These cells possessed significantly less apoptosis ratios compared with cells transfected with the Mock plasmid, although the values were still greater than those in parent cells. Of the apoptosis-related molecules, expression of Bcl-2 and Bcl-xL was up-regulated in cells transfected with pcDNA3-AE, whereas expressions of Bax, Bid, and cleaved caspase-9 and -3 were down-regulated compared with their expression in other kinds of cells. CONCLUSION: Our data show that LAPTM4B-35 attenuated epirubicin-induced apoptosis of GBC-SD cells in vitro through a mitochondria-dependent pathway. Therefore, the protein LAPTM4B-35 might be associated with the chemoresistance of GBC.

LAPTM4B-35 overexpression is an independent prognostic marker in endometrial carcinoma.

BACKGROUND: Lysosomal protein transmembrane 4 ß-35 (LAPTM4B-35), a novel oncoprotein that belongs to the mammalian 4-tetratransmembrane spanning protein superfamily, has been implicated in oncogenesis and cancer progression in several solid malignances. However, the expression of LAPTM4B-35 and its role in endometrial cancer progression remain unknown. MATERIALS AND METHODS: We investigated the expression of the LAPTM4B-35 protein by immunohistochemistry in 30 normal endometrium specimens and 165 endometrial carcinomas and analyzed its correlation with various clinicopathologic features, including patient outcome. RESULTS: LAPTM4B-35 immunoreactivity was overexpressed in endometrial carcinoma cases compared with normal endometrium (P < 0.001). High LAPTM4B-35 expression was found in 117 (70.91%) of these 165 carcinomas and was positively correlated with the International Federation of Gynecology and Obstetrics stage, histological grade, depth of myometrial invasion, lymph node metastasis, lymph vascular space involvement, and recurrence, but not with age and histological type. Patients with high LAPTM4B-35 expression had significantly poorer overall survival and disease-free survival compared with patients with low expression of LAPTM4B-35 (P = 0.001 and P = 0.002, respectively). Multivariate analysis showed that high LAPTM4B-35 expression was an independent prognostic factor for both overall survival and disease-free survival of patients with endometrial carcinoma (both P = 0.005). CONCLUSIONS: These results showed that high LAPTM4B-35 expression was associated with progression and prognosis of endometrial carcinoma.

Expression of LAPTM4B in gallbladder carcinoma cells: the role in invasive potential.

BACKGROUND/AIMS: It was previously established that LAPTM4B-35 highly expressed in gallbladder carcinoma and being of clinicopathological and prognostic significances. However, expression of LAPTM4B gene in gallbladder carcinoma (GBC-SD), a gallbladder carcinoma cell line, and its role in invasive potential remain unclear. METHODOLOGY: Expression of LAPTM4B in GBC-SD cells was first detected. Plasmids, pcDNA3-AE (containing complete open reading frame of LAPTM4B) and Mock (pcDNA3), were transiently transfected into GBC-SD cells. Invasive phenotypes (migration and invasion) and relative molecules were then shown by transwell assay, crossing river test and Western blot analysis. RESULTS: Immunocytochemical staining revealed that LAPTM4B-35 positively expressed in cytoplasm of GBC-SD cells. But LAPTM4B-35 expression was obviously weaker in GBC-SD cells than that in BEL-7402 cells (positive control). Besides, cells transfected with pcDNA3-AE presented shorter crossing river time, less migrated and invaded cell numbers, compared with cells transfected with the Mock plasmid and parent cells. Finally, increased expressions of active uPA, MMP-9, pro MMP-2 and active MMP-2 were also observed in cells transfected with pcDNA3-AE. CONCLUSIONS: Our data suggested that LAPTM4B expressed in GBC-SD cells at a relatively low level. Forced overexpression of LAPTM4B increased invasive potential of GBC-SD cells, through modulating molecules associated with degradation of extracellular matrix.

Overexpression of LAPTM4B promotes growth of gallbladder carcinoma cells in vitro.

BACKGROUND: The overexpression of LAPTM4B-35 in gallbladder carcinoma (GBC) and its clinicopathologic and prognostic significance have been previously shown. Thus, this gene may play a role in the growth of GBC cells. METHODS: The pcDNA3-AE containing the complete open reading frame of LAPTM4B (lysosome-associated protein transmembrane-4beta) and mock (pcDNA3) plasmids were transiently transfected into GBC-SD cells. Cell proliferation, cell cycle distribution, and protein expression were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium assay, flow cytometry, and Western blot, respectively. RESULTS: Cells transfected with pcDNA3-AE revealed accelerated proliferation, less serum dependence, and significant cell cycle progression compared with cells transfected with mock plasmid and parent cells. These phenotypes were accompanied by upregulated expression of C-myc, c-Fos, c-Jun, cyclin D1, and cyclin E and downregulated expression of P16 and P-27. CONCLUSIONS: LAPTM4B overexpression promotes the growth of GBC cells in vitro by regulating the expression levels of some proliferation-associated proteins. Therefore, the LAPTM4B gene might be used as a novel therapeutic target of GBC.

LAPTM4B-35 overexpression is a risk factor for tumor recurrence and poor prognosis in hepatocellular carcinoma.

PURPOSE: Lysosomal protein transmembrane 4 beta-35 (LAPTM4B-35) is a tetra-transmembrane glycoprotein that is abundantly localized on membrane-bound organelles including endosomes and lysosomes, and promotes cell proliferation and tumorigenesis through regulation of cell cycle and signaling pathways. The aim of the present study is to determine the potential clinical implications of LAPTM4B-35 expression in hepatocellular carcinoma (HCC). METHODS: Immunohistochemistry assay was used to determine the expression of LAPTM4B-35 protein in normal and HCC tissues from 71 patients. The correlations of LAPTM4B-35 expression with clinicopathological parameters, including gender, age, background liver, viral status, tumor size, portal vein invasion, histopathological differentiation, serum AFP level, TNM staging and recurrence of HCC were assessed by Chi-squared test. Patient survival and their differences were determined by Kaplan-Meier method and log-rank test. Cox regression (Proportional hazard model) was adopted for multivariate analysis of prognostic factors. RESULTS: LAPTM4B-35 immunoreactivity was negative or low in normal liver tissues, but high in HCC tissues (51/71, 71.8%). The overexpression of LAPTM4B-35 was significantly associated with recurrence, TNM staging and portal vein invasion of HCC. Patients with high LAPTM4B-35 expression had significantly poorer overall survival (OS) and disease-free survival (DFS) (both P < 0.001) when compared with patients with the low expression of LAPTM4B-35. On multivariate analysis, LAPTM4B-35 expression was found to be an independent prognostic factor for OS and DFS (P = 0.018 and P = 0.001, respectively). CONCLUSION: LAPTM4B-35 expression showed a strong association with the potencies of recurrence and metastasis and progression of HCC, and that may be applied as a novel marker for the prediction of recurrence and metastasis potency of HCC, and helpful for improving the diagnosis, prognosis and treatment of HCC.

Reduced expression of ASS is closely related to clinicopathological features and post-resectional survival of hepatocellular carcinoma.

Argininosuccinate synthetase (ASS) has previously been proven to be reductively expressed in hepatocellular carcinoma (HCC) and various types of HCC cell lines. Arginine, the product of ASS, has been used as a target in HCC by recombinant human arginase or arginine deiminase, which is now in the phase II clinical trial stage. This study aimed to present the levels of ASS expression in HCCs and its correlation with clinicopathological features and prognosis of HCC patients. Immunohistochemical detection of ASS was performed on samples from 71 patients with HCC. Positive staining was found in 21 HCCs, with a score of 2, as well as in normal liver tissues. Reduced ASS staining was found in 70.4% (50/71) of HCC tissues, including 21 with a score of 0 and 29 with a score of 1. The staining score in cancer tissues was significantly associated with gender, background liver, histopathological differentiation, recurrence, TNM staging and portal vein invasion (P<0.05), but not with age, viral status, tumor size and serum α-fetoprotein level. Patients with a high ASS expression had significantly poorer overall and disease-free survival (P<0.001 and P<0.001, respectively). These data showed that ASS was reductively or negatively expressed in a large portion of HCC, and that ASS levels in HCCs correlated inversely with prognosis. In conclusion, a high expression of ASS may be a novel marker of poor prognosis of patients presenting with HCC.

Expression of LAPTM4B-35: a novel marker of progression, invasiveness and poor prognosis of extrahepatic cholangiocarcinoma.

LAPTM4B was proven to overexpress in hepatocellular carcinoma and relate to differentiation. We immunohistochemically investigated the expression and potential clinicopathological and prognostic significance of LAPTM4B encoded protein, LAPTM4B-35, in extrahepatic cholangiocarcinoma (EHCC) for specimens from consecutive 81 patients. LAPTM4B-35 staining was positive in cancer tissues from 59 patients (72.8%), including 12 with score 1, 22 with score 2 and 25 with score 3. No positive staining was found in non-cancer epithelia. The staining score in cancer tissues was not only significantly associated with TNM staging, histological grade, perineural and lymph node invasion (P<0.05), but also of comprehensive prognostic implications, including integrated estimation with CA19-9. These data established that LAPTM4B-35 positively expressed in a great portion of EHCC and might be a novel molecular maker of progression, invasiveness and poor prognosis.

[Relationship between LAPTM4B gene polymorphism and susceptibility of lung cancer].

OBJECTIVE: To investigate the possible association between the allelic variation of LAPTM4B and the genetic susceptibility of lung cancer. METHODS: The genotype of LAPTM4B was analyzed in 134 unrelated healthy adult individuals and 166 patients with lung cancer by utilizing polymerase chain reaction based on special primers. The genotypical distribution of LAPTM4B was analyzed by chi2 test. RESULTS: The allelic frequencies of the *2 were 40.1% and 28.0% in the lung cancer group and the healthy control group respectively, which was significantly different between the two groups (P=0.002). There was a significant difference in the overall genotypical distribution between the patients and the controls (P=0.005). The risk of suffering from lung cancer was increased 1.91 times in the individuals of the *1/2 genotype (95%CI: 1.178-3.110) and 3.26 times in the individuals of the *2/2 genotype of LAPTM4B (95%CI: 1.338-7.929) compared with the *1/2 genotype. No association was observed between the genotypical distribution of LAPTM4B and the clinical information on patients of lung cancer such as gender, age, pathological type, differentiation classification of TNM and infection of HBV. CONCLUSION: This study suggests that the allele *2 of LAPTM4B might be the risk factor of lung cancer, which could be associated with genetic susceptibility of lung cancer.

Expression of lysosome-associated protein transmembrane 4B-35 in cancer and its correlation with the differentiation status of hepatocellular carcinoma.

AIM: To produce high-quality polyclonal antibody to lysosome-associated protein transmembrane 4B-35 and to identify LAPTM4B-35 expression in cancer tissues and its correlation with differentiation status of hepatocellular carcinoma (HCC). METHODS: The 297 bp 5' end of LAPTM4B cDNA was obtained by PCR and inserted into prokaryotic expression vector pGEX-KG. Then the recombinant pGEX-KG-N(1-99) was transformed into E.coli JM109 to express GST-fusion protein. The fusion protein was purified by glutathione sepharose(TM) 4B agarose. The purified GST-LAPTM4B-N(1-99) was characterized by SDS-PAGE, and used to immunize rabbits. The titer and specificity of antisera were detected by ELISA and Western blot, respectively. The correlation between the expression levels of LAPTM4B-35 and the differentiation status of HCC was analyzed via Western blot. The expression of LAPTM4B-35 in HCC and other six cancer tissues was investigated via tissue chip and immunohistochemical analysis. RESULTS: About 6.2 mg of pure GST-LAPTM4B-N(1-99) was isolated from 1 L of bacteria. The GST-LAPTM4B-N(1-99) produced high titer antisera in rabbits and showed good immunity. Western blot showed specific reactions for the antibody to the LAPTM4B-35 in the total proteins from HCC tissues and BEL-7402 cells, also to the fusion protein purified or in the transformed bacteria. LAPTM4B-35 was remarkably expressed in several cancers, such as HCC, breast cancer, gastric carcinoma, lung cancer, and colon carcinoma, but not commonly expressed in esophageal cancer and rectum carcinoma. Notably, the expression levels of LAPTM4B-35 were significantly and inversely correlated to the differentiation of HCCs in a 20 case analysis. CONCLUSION: Specific polyclonal antibody (LAPTM4B-N(1-99)-pAb) to LAPTM4B-35 was produced. It identified the expression of LAPTM4B-35 in some cancer tissues originated from single layer cuboidal and columnar epithelial cells and firmly demonstrated that the expression of LAPTM4B-35 in HCC was inversely correlated with the differentiation of HCC.

Liver injury after intermittent or continuous hepatic pedicle clamping and its protection by reduced glutathione.

BACKGROUND: The debate is still going on about selection of several clamping patterns during hepatectomy. The aim of this study was to assess the safety and preference of normothermic intermittent or continuous hepatic pedicle clamping and confirm the protective effect of reduced glutathione (GSH). METHODS: Thirty-two adult male healthy Sprague-Dawley (SD) rats were divided into groups of intermittent clamping and GSH absent (IA), continuous clamping and GSH absent (CA), intermittent clamping and GSH present (IP) and continuous clamping and GSH present (CP). The clamping manners were successively 40 minutes in continuous clamping groups and two cycles of 20 minutes with an interval of 5 minutes in intermittent clamping groups, and reperfusion periods were 60 minutes. Experimental parameters included levels of malonaldehyde (MDA) and Cu/Zn superoxide dismutase (SOD), pathological and ultrastructural changes in liver tissues, activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in sera. RESULTS: In the same group, the activities of ALT and AST were significantly higher in post-clamping rats than in pre-clamping rats (P<0.05), but no significant differences were noted in levels of MDA and Cu/Zn SOD (P>0.05). The differences of all values between post-reperfusion rats and pre-clamping rats were significant (P<0.05). Pathological and ultrastructural changes could be observed, but no irreversible injury was present. The comparison of the groups showed that the values at relevant time points between the intermittent and continuous groups were not significantly different (P>0.05). The values were significantly different between the GSH absent and present groups after reperfusion (P<0.05). The morphological damages were also obviously alleviated in the GSH present group. CONCLUSIONS: Normothermic intermittent or continuous hepatic pedicle clamping could cause reversible liver ischemia/reperfusion injury when the clamping time lasts 40 minutes. The injury extent seems to be similar. Continuous clamping should be regarded as a proper method in liver surgery. GSH has been confirmed as an effective agent in preventing post-clamping liver injury.

Structure analysis and expressions of a novel tetratransmembrane protein, lysosoma-associated protein transmembrane 4 beta associated with hepatocellular carcinoma.

AIM: To analyze the structure and expressions of the protein encoded by an HCC-associated novel gene, lysosome-associated protein transmembrane 4 beta (LAPTM4B). METHODS: Primary structure and fundamental characteristics of LAPTM4B protein were analysed with bioinformatics. Expressions of LAPTM4B in HCC tissues and various cell lines were detected using polyclonal antibodies and Western blot. RESULTS: LAPTM4B encoded two isoforms of proteins with molecular masses 35-ku and 24-ku, respectively. The expression level of LAPTM4B-35 protein in HCC tissues was dramatically upregulated and related to the differentiation status of HCC tissues, and it was also high in some cancer cell lines. Computer analysis showed LAPTM4B was an integral membrane protein with four transmembrane domains. LAPTM4B showed relatively high homology to LAPTM4A and LAPTM5 in various species. CONCLUSION: LAPTM4B gene encoded two isoforms of tetratransmembrane proteins, LAPTM4B-35 and LAPTM4B-24. The expression of LAPTM4B-35 protein is upregulated and associated with poor differentiation in human HCC tissues, and also at high levels in some cancer cell lines. LAPTM4B is an original and conserved protein.

Molecular cloning and characterization of LAPTM4B, a novel gene upregulated in hepatocellular carcinoma.

Lysosomal-associated protein transmembrane-4 beta (LAPTM4B), a novel gene upregulated in hepatocellular carcinoma (HCC), was cloned using fluorescence differential display, RACE, and RT-PCR. It contains seven exons and encodes a 35-kDa protein with four putative transmembrane regions. Both the N- and C-termini of the protein are proline-rich, and may serve as potential ligands for the SH3 domain. Immunohistochemical analysis localized the protein predominantly to intracellular membranes. Northern blot showed that the LAPTM4B mRNAs were remarkably upregulated in HCC (87.3%) and correlated inversely with differentiation status. LAPTM4B was also overexpressed in many HCC-derived cell lines. It was also highly expressed in fetal livers and certain adult normal tissues including the heart, skeletal muscle, testis, and ovary. Promoter function assays showed a distinct difference in the gene's activities between BEL7402 and HLE cell lines, suggesting that the transcription factors responsible for regulation of the gene in the two cell lines are different, and that possible negative regulatory cis-elements may exist upstream of the promoter region. It was demonstrated that the N-terminus of LAPTM4B was essential for survival of the cells. Cells harboring the full-length LAPTM4B cDNA expression clone displayed a slightly increased efficiency in colony formation. These results suggest that LAPTM4B is a potential protooncogene, whose overexpression is involved in carcinogenesis and progression of HCC. In normal cells, it may also play important roles such as regulation of cell proliferation and survival.

[Laminin and its alpha 6 integrin receptor in the regulation of human hepatocellular carcinoma cell phenotypes].

OBJECTIVE: To study the role of integrin alpha 6 in cell attachment, spread, survival/proliferation and differentiation of human hepatocellular carcinoma (HCC) BEL-7402 cells on various substrates by monoclonal antibody against the extracellular domain of alpha 6 subunit (IA6ED McAb). METHODS: The effect of McAb on attachment and spread of BEL-7402 cells on LN or FN substrate was examined. MTT analysis was used to examine the cell survival/proliferation, gelatin zymography to the matrix metalloproteinases (MMPs) secreted by BEL-7402 cells, and microparticle immunoabsorbent kit to AFP secretion while the cells were cultured on LN-, FN- or Matrigel-coated substrates. RESULTS: The cell attachment, spread and survival/proliferation were inhibited. Moreover importantly, the malignant cell dedifferentiation and abnormal differentiation on LN-coated substrate were also strongly inhibited by IA6ED McAb. CONCLUSION: LN and integrin alpha 6 regulate human HCC cell phenotypes of survival/proliferation and differentiation. The phenotypes of dedifferentiation and abnormal differentiation can be reversed by blocking the interaction between integrin alpha 6 and LN using IA6ED McAb, which may lower metastatic potency of tumor cells.