Effect of Huaier granule on recurrence after curative resection of HCC: a multicentre, randomised clinical trial.

OBJECTIVE: There is little evidence that adjuvant therapy after radical surgical resection of hepatocellular carcinoma (HCC) improves recurrence-free survival (RFS) or overall survival (OS). We conducted a multicentre, randomised, controlled, phase IV trial evaluating the benefit of an aqueous extract of Trametes robinophila Murr (Huaier granule) to address this unmet need. DESIGN AND RESULTS: A total of 1044 patients were randomised in 2:1 ratio to receive either Huaier or no further treatment (controls) for a maximum of 96 weeks. The primary endpoint was RFS. Secondary endpoints included OS and tumour extrahepatic recurrence rate (ERR). The Huaier (n=686) and control groups (n=316) had a mean RFS of 75.5 weeks and 68.5 weeks, respectively (HR 0.67; 95% CI 0.55 to 0.81). The difference in the RFS rate between Huaier and control groups was 62.39% and 49.05% (95% CI 6.74 to 19.94; p=0.0001); this led to an OS rate in the Huaier and control groups of 95.19% and 91.46%, respectively (95% CI 0.26 to 7.21; p=0.0207). The tumour ERR between Huaier and control groups was 8.60% and 13.61% (95% CI -12.59 to -2.50; p=0.0018), respectively. CONCLUSIONS: This is the first nationwide multicentre study, involving 39 centres and 1044 patients, to prove the effectiveness of Huaier granule as adjuvant therapy for HCC after curative liver resection. It demonstrated a significant prolongation of RFS and reduced extrahepatic recurrence in Huaier group. TRIAL REGISTRATION: NCT01770431; Post-results.

Two immunogenic passenger dendritic cell subsets in the rat liver have distinct trafficking patterns and radiosensitivities.

UNLABELLED: The aim of this study was to investigate the trafficking patterns, radiation sensitivities, and functions of conventional dendritic cell (DC) subsets in the rat liver in an allotransplantation setting. We examined DCs in the liver, hepatic lymph, and graft tissues and recipient secondary lymphoid organs after liver transplantation from rats treated or untreated by sublethal irradiation. We identified two distinct immunogenic DC subsets. One was a previously reported population that underwent blood-borne migration to the recipient's secondary lymphoid organs, inducing systemic CD8(+) T-cell responses; these DCs are a radiosensitive class II major histocompatibility complex (MHCII)(+) CD103(+) CD172a(+) CD11b(-) CD86(+) subset. Another was a relatively radioresistant MHCII(+) CD103(+) CD172a(+) CD11b(+) CD86(+) subset that steadily appeared in the hepatic lymph. After transplantation, the second subset migrated to the parathymic lymph nodes (LNs), regional peritoneal cavity nodes, or persisted in the graft. Irradiation completely eliminated the migration and immunogenicity of the first subset, but only partly suppressed the migration of the second subset and the CD8(+) T-cell response in the parathymic LNs. The grafts were acutely rejected, and intragraft CD8(+) T-cell and FoxP3(+) regulatory T-cell responses were unchanged. The radioresistant second subset up-regulated CD25 and had high allostimulating activity in the mixed leukocyte reaction, suggesting that this subset induced CD8(+) T-cell responses in the parathymic LNs and in the graft by the direct allorecognition pathway, leading to the rejection. CONCLUSION: Conventional rat liver DCs contain at least two distinct immunogenic passenger subsets: a radiosensitive blood-borne migrant and a relatively radioresistant lymph-borne migrant. LNs draining the peritoneal cavity should be recognized as a major site of the intrahost T-cell response by the lymph-borne migrant. This study provides key insights into liver graft rejection and highlights the clinical implications of immunogenic DC subsets.

HBx induces HepG-2 cells autophagy through PI3K/Akt-mTOR pathway.

Chronic hepatitis B virus infection is the dominant global cause of hepatocellular carcinoma (HCC), especially hepatitis B virus-X (HBx) plays a major role in this process. HBx protein promotes cell cycle progression, inactivates negative growth regulators, and binds to and inhibits the expression of p53 tumor suppressor gene and other tumor suppressor genes and senescence-related factors. However, the relationship between HBx and autophagy during the HCC development is poorly known. Previous studies found that autophagy functions as a survival mechanism in liver cancer cells. We suggest that autophagy plays a possible role in the pathogenesis of HBx-induced HCC. The present study showed that HBx transfection brought about an increase in the formation of autophagosomes and autolysosomes. Microtubule-associated protein light chain 3, Beclin 1, and lysosome-associated membrane protein 2a were up-regulated after HBx transfection. HBx-induced increase in the autophagic level was increased by mTOR inhibitor rapamycin and was blocked by treatment with the PI3K-Akt inhibitor LY294002. The same results can also be found in HepG2.2.15 cells. These results suggest that HBx activates the autophagic lysosome pathway in HepG-2 cells through the PI3K-Akt-mTOR pathway.

[Role of autophagy in HepG-2 cells induced by hepatitis B virus x protein].

OBJECTIVE: To investigate the role of autophagy in the injury of HepG-2 cells induced by hepatitis B virus x protein (HBx). METHODS: After HBx transfection, the cells were used to detect the formation of autophagosomes and observed by transmission electron microscopy, monodansylcadaverine (MDC) staining autophagic vacuole (AV), immunofluorescent ce staining microtubule-associated protein light chain 3 ( MAP1-LC3 ) protein, and Western blotting examining the ratio of LC3-II/LC3-I (gray level: 0.760 ± 0.078 vs 0.520 ± 0.086, P < 0.05), beclin 1 (gray level: 0.875 ± 0.093 vs 0.220 ± 0.087, P < 0.05)and lysosome associated membrane protein 2a ( lamp2a ) protein (gray level: 0.320 ± 0.061 vs 0.120 ± 0.064, P < 0.05) levels. RESULTS: (1) HBx transfected upregulated the expression of LC3-II, LC3-I, beclin 1 and lamp2a protein. (2) HBx transfected brought about an increase in the formation of autophagosomes and autolysosomes. CONCLUSION: HBx activates the autophagic lysosome pathway in HepG-2 cells through the LC3/beclin1 pathway.

[Application of psychological scales recommended by DC/TMD in patients with temporomandibular joint disorders].

PURPOSE: To observe psychological conditions such as anxiety, depression and somatic symptoms of temporomandibular disorders(TMD) patients using psychological scales recommended by DC/TMD and evaluate their clinical significance as the psychological axis for TMD diagnosis. METHODS: The experimental group included 100 TMD patients, and the control group comprised 100 normal prosthodontics outpatients without TMD symptoms. General information were collected including age, gender, education level, and personal income. The anxiety disorder scale (Generalized Anxiety Disorder-7, GAD-7), depression symptom scale (Patient Health Questionnaire-9, PHQ-9) and Patient Health Questionnaire-15 (PHQ-15) were used to evaluate patients' psychological conditions. SPSS 20.0 software package was used for data analysis. RESULTS: Patients less than 30 years old and between 30-50 years had similar TMD occurrence rates, both significantly higher than those older than 50 years old(P＜0.05). The proportion of highly educated patients in TMD group was significantly higher than that in the control group(P＜0.05), while the income level was not a risk factor for TMD (P=0.642). The incidence and average scores of anxiety, not the depression or somatic symptoms, in experimental group were significantly higher than the control group(P＜0.05). The level of anxiety and depression in painful TMD patients was significantly higher than patients with joint disease(P＞0.05). CONCLUSIONS: Gender(female), age (＜50 years old) and high education level (undergraduate and above) are risk factors of TMD, but the income level is irrelevant. The incidence and scores of anxiety in TMD patients are higher than normal prosthodontics outpatients, while there is no significant difference in the incidence of depression and somatic symptoms between two groups.

[Association of serum lipid profile with distant metastasis in breast cancer patients].

OBJECTIVE: In order to investigate whether the presence of distant metastases is associated with serum lipid abnormalities. METHODS: The fasting serum lipid profile and various clinicopathological data of 324 breast cancer patients with and without synchronous distant metastases were collected and analyzed. The serum lipid profile, including total cholesterol (TC), triglycerides (TG), low-density (LDL-C) and high-density lipoprotein cholesterol (HDL-C) was determined. The nutritional status, the serum albumin was measured and body mass index (BMI) was calculated. Univariate analysis and multiple logistic regression analysis were carried out to investigate the association of serum lipid profile with distant metastases. RESULTS: Univariate analysis showed that the distant metastasis rate was significantly higher in the breast cancer patients with an higher level of serum TC, TG, LDL-C, and LDL-C/HDL-C ratio (P < 0.05). Multiple logistic regression analysis showed that higher serum levels of TC, LDL-C and LDL-C/HDL-C ratio were independent risk factors for distant metastasis in breast cancer (OR = 2.324, 2.648 and 4.862, respectively). CONCLUSIONS: Hyperlipidemia is significantly associated with the distant metastasis in breast cancer patients. Monitoring of serum lipid profile may be helpful to predict the occurrence of distant metastasis in breast cancer patients.

Serum LDL-C and LDL-C/HDL-C ratio are positively correlated to lymph node stages in males with colorectal cancer.

BACKGROUND/AIMS: Although changes in serum lipid profile have been reported in CRC, the specific association between serum lipid profile and lymph node stages remains uncertain. METHODOLOGY: Fasting serum lipid profile, including TC, TG, HDL-C and LDL-C was retrospectively evaluated in 968 patients undergoing curative resection for primary CRC. To determine the nutritional status, the serum albumin levels were measured and BMI was calculated. Statistical analyses were performed to investigate the association of serum lipid profile with lymph node stages. RESULTS: Serum lipid levels correlated well with rate of lymph node metastasis and high LDL-C and low HDL-C levels tended to present more advanced lymph node stages. The observed elevation of the LDL-C/HDL-C ratio for patients with N2 stage was statistically significant when compared with patients with N1 stage. When separated by gender, multivariate logistic regression analysis showed that both LDL-C levels and LDL-C/HDL-C ratio had independent association with advanced N2 stage in males, but not in females. In addition, LDL-C/HDL-C ratio might be a more effective biomarker for identifying N2 stage than LDL-C levels alone (OR value: 2.85 vs. 1.63). CONCLUSIONS: Elevated serum LDL-C levels and an increased LDC-C/HDL-C ratio might favour development of lymph node metastasis and LDCC/HDL-C ratio might be a more effective biomarker for identifying advanced N2 stage than LDL-C levels alone, especially for male patients with CRC.

Comparison of immediate response to four conservative treatment modalities for management of masticatory myalgia with limited mandibular movement: a retrospective study.

OBJECTIVE: Conservative treatment modalities are recommended for managing masticatory myalgia in individuals with temporomandibular disorders. The aim of this study was to retrospectively review and compare the effectiveness of four conservative treatments: counseling and occlusal splint therapy, counseling and manipulation integrated with electrophysiotherapy, the combination of the two treatments, and counseling only. METHOD AND MATERIALS: One hundred and sixty-eight patients who had myalgia with limited jaw movement were retrospectively observed in this study. Between January 2015 and December 2017, 63 patients received counseling and stabilization occlusal splint therapy (Group 1), 35 patients received counseling and manipulation integrated with electrophysiotherapy (Group 2), 33 patients received the combination of counseling, splint therapy, and manipulation integrated with electrophysiotherapy (Group 3), and 37 patients received counseling only (Group 4). All subjects were followed up for 12 weeks. The intensity of spontaneous pain, palpation pain, chewing pain in the masticatory muscles, and range of pain-free maximal mouth opening were recorded in the clinical assessments. Intragroup and intergroup differences were examined by using analysis of variance (ANOVA) and the Kruskal-Wallis test. RESULTS: Spontaneous pain in the masticatory muscles was relieved significantly in all groups at the 6-week visit (P < .05), and no significant difference was found among the groups (P > .05). Palpation pain was relieved significantly at the 9-week visit in the counseling + occlusal splint therapy group, counseling + manipulation + electrophysiotherapy group, and counseling + occlusal splint + manipulation + electrophysiotherapy group (P < .05). In the treatment group with counseling alone, significant palpation pain relief occurred at 12 weeks. Chewing pain was relieved significantly at the 6-week visit in the counseling + occlusal splint therapy group, counseling + manipulation + electrophysiotherapy group, and counseling + occlusal splint + manipulation + electrophysiotherapy group (P < .05), yet no significant difference compared to the baseline was observed in the counseling-only group (P > .05). A significant increase in the maximal range of pain-free mouth opening was observed at the 9-week visit in the counseling + occlusal splint therapy group, and at the 3-week visit in the counseling + manipulation + electrophysiotherapy group and counseling + occlusal splint + manipulation + electrophysiotherapy group (P < .05). Nevertheless, no significant change in the range of mouth opening was found throughout the follow-up period in the counseling-only group (P > .05). CONCLUSION: Each of the included treatment modalities relieved spontaneous pain and tenderness to palpation of the masticatory muscles during the follow-up intervals. Counseling alone did not help patients with chewing pain and limited mouth opening in the short term. Treatment protocols including counseling, occlusal splint therapy, and manipulation, integrated with electrophysiotherapy showed the best short-term outcomes for symptomatic improvement.

TBL1XR1 induces cell proliferation and inhibit cell apoptosis by the PI3K/AKT pathway in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest solid tumors. Identification of diagnostic and therapeutic biomarkers for PDAC is urgently needed. Transducin (ß)-like 1 X-linked receptor 1 (TBL1XR1) has been linked to the progression of various human cancers. Nevertheless, the function and role of TBL1XR1 in pancreatic cancers are unclear. AIM: To elucidate the function and potential mechanism of TBL1XR1 in the development of PDAC. METHODS: Ninety patients with histologically-confirmed PDAC were included in this study. PDAC tumor samples and cell lines were used to determine the expression of TBL1XR1. CCK-8 assays and colony formation assays were carried out to assess PDAC cell viability. Flow cytometry was performed to measure the changes in the cell cycle and cell apoptosis. Changes in related protein expression were measured by western blot analysis. Animal analysis was conducted to confirm the impact of TBL1XR1 in vivo. RESULTS: Patients with TBL1XR1-positive tumors had worse overall survival than those with TBL1XR1-negative tumors. Moreover, we found that TBL1XR1 strongly promoted PDAC cell proliferation and inhibited PDAC cell apoptosis. Moreover, knockdown of TBL1XR1 induced G0/G1 phase arrest. In vivo animal studies confirmed that TBL1XR1 accelerated tumor cell growth. The results of western blot analysis showed that TBL1XR1 might play a key role in regulating PDAC cell proliferation and apoptosis via the PI3K/AKT pathway. CONCLUSION: TBL1XR1 promoted PDAC cell progression and might be an effective diagnostic and therapeutic marker for pancreatic cancer.

RASSF1A expression inhibits the growth of hepatocellular carcinoma from Qidong County.

BACKGROUND AND AIM: The tumor-suppressing role of Ras-association domain family 1A (RASSF1A) has been described in several systems. In this study, we tested its tumor-suppressing ability and the potential molecular mechanisms in hepatocellular carcinoma (HCC) from Qidong County. METHODS: Reverse transcription polymerase chain reaction and Northern blotting were employed to detect the expression of RASSF1A in HCC. After establishing stable RASSF1A (wild type or mutant) expressing 'qi dong gan ai yan jiu suo' ([Qidong Institute of Liver Cancer] QGY)-7703 cell lines, we tested the effects of RASSF1A expression on cell growth by cell proliferation rate, cell colony formation, and cell cycle progression. We also tested the effects of RASSF1A expression on tumorigenesis in nude mice and on cellular sensitivity to mitomycin treatment. RESULTS: The RASSF1A transcript was not found in 75% (three of four) of HCC cell lines and 67% (32/48) of HCC primary biopsies. The stepwise regression analyses indicated that the loss of RASSF1A expression was more frequent in patients who were hepatitis B virus surface antigen positive (HBsAg+) compared to those who were HBsAg(-), both in tumor and corresponding non-cancerous tissues. The wild-type (wt)-RASSF1A expression in the QGY-7703 cell line resulted in fewer and smaller clones, decreased xenograft tumor volume and weight, and G(1)/S arrest in vitro and in vivo. The wt-RASSF1A expression also decreased the cyclin D1 protein expression, which appeared to be at the level of post-transcriptional control. In addition, the wt-RASSF1A expression increased cell growth inhibition and the percentage of cells with sub-G(1) DNA content when the cells were treated with mitomycin. CONCLUSION: RASSF1A is a tumor suppressor in HCC. The loss of RASSF1A expression may be related to HBsAg+ in hepatocarcinogenesis. Its inactivation may play an important role in the development of HCC.

Role of the duodenum in regulation of plasma ghrelin levels and body mass index after subtotal gastrectomy.

AIM: To investigate the role of the duodenum in the regulation of plasma ghrelin levels and body mass index (BMI), and the correlation between them after subtotal gastrectomy. METHODS: Forty-two patients with T0-1N0-1M0 gastric cancer were divided into two groups after gastrectomy according to digestive reconstruction pattern, Billroth I group (n = 23) and Billroth II group (n = 19). Ghrelin levels were determined with radioimmunoassay (RIA) before and on d 1, 7, 30 and 360 after gastrectomy, and BMI was also measured. RESULTS: The two groups had identical postoperative trends in ghrelin alterations during the early stage, both decreasing sharply to a nadir on d 1 (36.7% vs 35.7%), then markedly increasing on d 7 (51.0% vs 51.1%). On d 30, ghrelin levels in the Billroth I group were slightly higher than those in the Billroth II group. However, those of the Billroth I group recovered to 93.6% on d 360, which approached, although lower than, the preoperative levels, and no statistically significant difference was observed. Those of the Billroth II group recovered to only 81.6% and manifested significant discrepancy with preoperative levels (P = 0.033). Compared with preoperative levels, ghrelin levels of the two groups decreased by 6.9% and 18.4% and BMI fell by 3.3% and 6.4%, respectively. The linear regression correlations were revealed in both groups between decrease of ghrelin level and BMI (R1(2) = 0.297, P = 0.007; R2(2) = 0.559, P < 0.001). CONCLUSION: Anatomically and physiologically, the duodenum compensatively promotes ghrelin recovery and accordingly enhances BMI after gastrectomy. Regarding patients with insufficient ghrelin secretion, ghrelin is positively associated with BMI.

Screening for implicated genes in colorectal cancer using whole­genome gene expression profiling.

To identify biologically relevant genes associated with the pathogenesis of colorectal cancer (CRC), genome wide expression profiles of 17 pairs of CRC tumor and adjacent tissues, previously published in a DNA microarray study, were analyzed. Cytoscape, String tools and DAVID tools were used to investigate the biological pathways encoded by the genes identified as being either upregulated or downregulated in CRC, to determine protein­protein interactions and to identify potential hub genes associated with CRC. As a result, a total of 3,264 genes were identified as being differentially expressed in CRC and adjacent tissues, including 1,594 downregulated and 1,670 upregulated genes. Furthermore, 306 genes were revealed to be clustered in a complex interaction network, and the top 20 hub genes in this network were determined by application of the Matthews Correlation Coefficient algorithm. In addition, the patterns of the expression levels of the 20 hub genes were investigated using reverse transcription­quantitative polymerase chain reaction. Gene Ontology analysis revealed that four of the 20 hub genes encoded small subunit processome components (UTP3 small subunit processome component; UTP14 small subunit processome component; UTP 18 small subunit processome component; and UTP20 small subunit processome component) and a further four encoded WD repeat domains (WD repeat­containing protein 3, WD repeat domain 12, WD repeat­containing protein 43 and WD repeat­containing protein 75). In conclusion, the present DNA microarray study identified genes involved in the pathogenesis of CRC. Furthermore, it was revealed that hub genes identified from among the total identified upregulated and downregulated genes in CRC encoding subunit processome components and WD repeat domains may represent novel target molecules for future treatments of CRC.

Laparoscopic hepatectomy versus open hepatectomy for hepatocellular carcinoma in 157 patients: A case controlled study with propensity score matching at two Chinese centres.

BACKGROUND: The aim of this study was to investigate the long-term outcomes and perioperative outcomes of laparoscopic hepatectomy (LH) versus open hepatectomy (OH) for hepatocellular carcinoma (HCC) between well-matched patient groups. METHODS: We retrospectively reviewed data from 1535 hepatocellular carcinoma patients who underwent liver resection between January 2002 and December 2016 at two Chinese centres. Propensity score matching of patients in a ratio of 1:1 was conducted and 157 patients were matched. RESULTS: The median blood loss (150 vs 380 ml, P < 0.001) was significantly less with LH. The laparoscopic group had shorter hospital stay (6 vs 10 days, P < 0.001) and less complication rate (6.4% vs 24.2%,P < 0.001). There were no significant differences in overall survival and disease-free survival between LH and OH. There were no significant differences in perioperative and long-term outcomes. CONCLUSION: Laparoscopic hepatectomy is technically feasible and safe in selected patients. LH showed similar long-term outcomes, associated with less blood loss, shorter hospital stay, and fewer postoperative complications in selected patients with HCC compared with OH.

Downregulation of mir-23b in plasma is associated with poor prognosis in patients with colorectal cancer.

MicroRNAs (miRNAs) are short, non-coding RNA molecules that act as regulators of gene expression. Circulating blood miRNAs have potential as cancer biomarkers. The main objective of the present study was to assess the effect of miRNA-23b (miR-23b) expression in plasma on the diagnosis and prognosis of colorectal cancer (CRC). Reverse transcription-quantitative polymerase chain reaction (PCR) was used to measure miR-23b expression levels, and methylation-specific PCR was used to test the promoter methylation status. Subsequently, the expression level of miR-23b in plasma samples was compared between CRC patients and healthy control individuals. The miR-23b expression levels were significantly lower in CRC cells and primary CRC tissues than in nonmalignant colorectal tissues (P<0.001). It was also shown that miR-23b expression is downregulated by promoter methylation and can be restored by demethylation agent treatment. miR-23b was significantly decreased in plasma samples from CRC patients compared with the healthy control individuals (P<0.001). The value of the area under the receiver operating characteristic curve was 0.842 (sensitivity, 84.38%; specificity, 77.08%; 95% confidence interval, 0.763-0.922). Low plasma miR-23b expression was significantly associated with clinical stage, tumor depth, distant metastasis and tumor recurrence. CRC patients with low miR-23b expression in plasma exhibited a shorter recurrence-free survival time and poorer overall survival rate. The present results suggested that the downregulation of miR-23b in the plasma has the potential to be a diagnostic and prognostic biomarker in CRC.

Protective effect of eNOS overexpression against ischemia/reperfusion injury in small-for-size liver transplantation.

Ischemia/reperfusion (I/R) injury can occur during small-for-size liver transplantation, resulting in delayed graft function and decreased long-term graft survival. The aim of the present study was to evaluate the effects of genetic overexpression of endothelial nitric oxide synthase (eNOS) in protecting hepatocytes against I/R injury in a rat model of small-for-size liver transplantation. L02 liver cells were transfected with the eNOS gene using an adenovirus (Ad-eNOS). eNOS expression was detected using quantitative polymerase chain reaction and western blot analysis. To evaluate the effect of eNOS overexpression, L02 cells were placed in a hypoxic environment for 12 h and immediately transferred to an oxygen-enriched atmosphere. For in vivo testing, rats pretreated with Ad-eNOS or control underwent small-for-size liver transplantation. At 6 h after reperfusion, the bile quantity, serum transaminase and nitric oxide (NO) levels, and histological outcomes were evaluated. Cell apoptosis was assessed by flow cytometry or TUNEL assay. In vitro, Ad-eNOS prevented apoptosis in L02 cells with an increase in the level of NO in culture supernatant. In vivo, Ad-eNOS pre-treatment significantly increased bile production, improved abnormal transaminase levels, diminished apoptosis among liver cells, and decreased hepatocellular damage at 6 h after I/R injury. The eNOS-mediated renal protective effects might be associated with the downregulation of tumor necrosis factor-α and a reduction in macrophage activation in the early stage of reperfusion in small-for-size liver allografts. eNOS-derived NO production significantly attenuates hepatic I/R injury. Thus, eNOS overexpression constitutes a promising therapeutic approach to prevent liver I/R injury following small-for-size liver transplantation.

Influence of adriamycin on changes in Nanog, Oct-4, Sox2, ARID1 and Wnt5b expression in liver cancer stem cells.

AIM: To determine the influence of Adriamycin (ADM) on the changes in Nanog, Oct4, Sox2, as well as, in ARID1 and Wnt5b expression in liver cancer stem cells. METHODS: The MHCC97-L and HCCLM3 liver cancer cell lines were selected as the cell models in this study, and were routinely cultured. The 50% lethal dose (LD50) in the cell lines was detected by the MTT assay. Expression changes in liver cancer stem cell related genes (Nanog, Oct-4, Sox2, ARID1, and Wnt5b) were detected by western blot following treatment with ADM (LD50). RESULTS: The LD50 of ADM in MHCC97-L cells was lower than that in HCCLM3 cells (0.4123 ± 0.0236 µmol/L vs 0.5259 ± 0.0125 µmol/L, P < 0.05). Wnt5b and Nanog were expressed in both MHCC97-L and HCCLM3 cells, while only Sox2 was expressed in HCCLM3 cells. However, neither ARID1A nor Oct4 was detected in these two cell lines. Genes, related to the stem cells, showed different expression in liver cancer cells with different metastatic potential following treatment with ADM (LD50). Wnt5b protein increased gradually within 4 h of ADM (LD50) treatment, while Nanog decreased (P < 0.05). After 12 h, Wnt5b decreased gradually, while Nanog increased steadily (P < 0.05). In addition, only Sox2 was expressed in HCCLM3 cells with high metastatic potential following ADM (LD50) treatment. The expression of Sox2 increased gradually with ADM (LD50) in HCCLM3 cells (P < 0.05). CONCLUSION: ADM increased the death rate of MHCC97-L and HCCLM3 cells, while the growth suppressive effect of ADM was higher in MHCC97-L cells than in HCCLM3 cells.

SIRT3 expression in hepatocellular carcinoma and its impact on proliferation and invasion of hepatoma cells.

OBJECTIVE: To observe expression of SIRT3 in normal liver tissue, cirrhotic tissue and hepatocellular carcinoma (HCC) tissues, and to explore the significance of SIRT3 in primary HCC. METHODS: SIRT3 expression was detected in 10 normal cases, 30 cases with, 30 HCC cases by immunohistochemical and Western-blotting method. RESULTS: Immunohistochemical assay showed that the SIRT3 positive expression rates were 100.0% (10/10), 96.7% (29/30) and 60.0% (18/30), respectively in normal group, paracancer group and HCC group. And the SIRT3 expression in HCC group was significantly lower than in normal group and paracancer group (P<0.05). Western-blotting showed the SIRT3 expression in cancer tissue was 0.29±0.07, significantly lower than that in paracancer group and normal group (P<0.05). SIRT3 expression was related to the differentiation degree and portal vein tumor thrombus (P<0.05). CONCLUSIONS: Abnormal expression of SIRT3 is closely related to the biological behavior of primary HCC.

Association between C1019T polymorphism in the connexin 37 gene and Helicobacter pylori infection in patients with gastric cancer.

OBJECTIVE: To investigate the association between the connexin 37 C1019T polymorphism and Helicobacter pylori infection in patients with gastric cancer. METHODS: 388 patients with gastric cancer (GC), 204 with chronic superficial gastritis (CSG) were studied. H. pylori was detected by gastric mucosal biopsies biopsy dyeing method. Connexin 37 gene polymorphism 1019 site genotypes were determined by gene sequencing technology. Genotypes and alleles frequencies were compared. RESULTS: (1) Connexin37 gene 1019 site distribution frequency (CC type, TC type, TT type) in the CSG group was 18.1%, 45.1% and 36.8%; in the stomach cancer group it was 35.1%, 45.9% and 19.%, conforming to the Hardy-Weinberg euilibrium. (2) In comparison with CSG group, the frequency of Connexin37 C allele was higher in the gastric cancer group (58.0% vs 40.7%, OR=2.01, 95%CI=1.58-2.57, P<0.01). The prevalence of gastric cancer risk was significantly increased in the carriers of C allele (CC+TC) than in TT homozygote (OR=2.47, 5%CI=1.68-3.610. (3) Gastric cancer patients complicated with Hp infection 211 cases, gastric cancer group of the male patients with HP positive patients with 187 cases, 40 cases of female patients with negative patients, 24 cases were HP positive, negative in 137 cases, control group male patients, 28 cases were Hp positive, negative in 95 patients, female patients with Hp positive 6 cases, 75 cases were negative. On hierarchical analysis, the male group OR value was 15.9 (95%CI to 9.22-27.3), and the female OR was 2.19 (95%CI 0.88-5.59), indicating a greater contribution in males (P<0.01). After elimination of gender effects, positive HP and gastric cancer were closely related (OR 8.82, 95% CI: 5.45-14.3). (4) The distribution frequency of C allele in patients with Hp infection was much higher than that in Hp negative cases in the GC group (64.5% vs 47.0%, OR=2.05, 95%CI=1.54-2.74, P<0.01). Compared with TT homozygotes, (CC+TC) genotype prevalence of gastric cancer risk increased significantly (OR=2.96, 5%CI=1.76-2.99). CONCLUSION: The T allele in the connexin37 gene might not only be associated with gastric cancer but also with H. pylori infection.

Blockade of 4-1BB/4-1BB ligand interactions prevents acute rejection in rat liver transplantation.

BACKGROUND: Blocking the 4-1BB/4-1BB ligand (4-1BBL) signal may modulate the secretion of Th1/Th2 cytokines and prolong the survival of the grafts, which play a key role in organ transplantation tolerance. The aim of this study was to investigate the role of blockade of the 4-1BB/4-1BBL co-stimulatory pathway with 4-1BBL monoclonal antibody (mAB) in acute rejection of rat orthotopic liver transplantation. METHODS: The orthotopic liver transplantation model was set up, while male Lewis rats were used as liver donors and Brown-Norway rats as recipients. The recipient rats were intravenously injected with anti 4-1BBL mAB or isotype control antibody. Groups were monitored for graft survival after transplantation. Plasma chemistry, including aspartate transaminase (AST), alanine aminotransferase (ALT), and bilirubin (BIL), was assayed. The concentrations of interleukin (IL)-2, IL-10 and interferon (IFN)-gamma in plasma were also measured by enzyme-linked immunosorbent assay. Allograft histology images were collected under light microscope and electron microscope. RESULTS: Isotype antibody treated recipients exhibited elevated plasma levels of liver injury markers including AST, ALT and BIL, progressive portal and venous inflammation and cellular infiltration of the liver allografts, and a mean graft survival time (MST) of 10.9 days. Administration of anti 4-1BBL mAB resulted in a decrease in plasma levels of liver injury markers and the concentrations of IL-2, IL-10 and IFN-gamma. The histological grade of rejection on day 7 decreased and MST (17.3 days) increased substantially. CONCLUSIONS: These results demonstrate that attenuation of acute rejection follows the blockade of the 4-1BB/4-1BBL co-stimulatory pathway with 4-1BBL monoclonal antibody and strongly suggest it is a promising strategy to prevent progression of graft rejection by suppressing T cell-mediated immunity.

RASSF1A expression inhibits cell growth and enhances cell chemosensitivity to mitomycin in BEL-7402 hepatocellular carcinoma cells.

BACKGROUND: The antitumor role of Ras association domain family 1A (RASSF1A) gene and its potential molecular mechanisms are not well understood. The objective of this study was to observe the antitumor ability of RASSF1A in hepatocellular carcinoma, and study the mechanisms of cell apoptosis induced by RASSF1A. METHODS: After stably transfecting a RASSF1A (wild-type or mutant) expression vector into the BEL-7402 hepatocellular carcinoma cell line, RT-PCR and Western blotting was used to detect the RASSF1A expression levels in recombinant cells. The effects of wild-type RASSF1A on cell growth were observed in vitro by analyzing cell proliferation rate, cell colony formation, and in vivo by analyzing tumorigenesis in nude mice. In addition, the effect of RASSF1A gene expression on the chemosensitivity of human hepatocellular carcinoma cells to antitumor drugs was examined by inhibition of cell proliferation and the percentage of apoptotic cells. RESULTS: Wild-type RASSF1A, not the mutant, suppressed cell growth in vitro and in vivo. Re-expression of wild-type RASSF1A could enhance the inhibition of cell proliferation and the percentage of apoptotic cells following cell treatment with mitomycin, but had no significant effect when combined with adriamycin, etoposide, 5-fluorouracil and cisplatin treatment. CONCLUSION: Wild-type RASSF1A inhibits cell growth and enhances cell chemosensitivity to mitomycin in hepatocellular carcinoma, suggesting that RASSF1A may serve as a new target for gene therapy in hepatocellular carcinoma patients.