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supplemental Figure 1-12. Expression levels of 10 lncRNAs by qRT-PCR in 15 GBC tissues and paired non-tumor tissues (Figure 1). Expression levels of 8 mRNAs by qRT-PCR in 15 GBC tissues and paired non-tumor tissues (Figure 2). Molecular characteristics of GCASPC (Figure 3). Nucleotide sequence of the full-length human GCASPC gene (Figure 4). GCASPC has no coding capability (Figure 5). The expression level of GCASPC in gallbladder cancer cells (Figure 6). Manipulation of GCASPC expression level (Figure 7). Anti-proliferative activity of GCASPC (Figure 8). GCASPC acts in trans (Figure 9). The effect of GCASPC on the protein, activity and mRNA levels of PC (Figure 10). GCASPC destabilizes PC protein (Figure 11). GCASPC directly binds to miR-17-3p (Figure 12).

Long noncoding RNAs (lncRNA) are being implicated in the development of many cancers. Here, we report the discovery of a critical role for the lncRNA GCASPC in determining the progression of gallbladder cancer. Differentially expressed lncRNAs and mRNAs between gallbladder cancer specimens and paired adjacent nontumor tissues from five patients were identified and validated by an expression microarray analysis. Quantitative real-time PCR was used to measure GCASPC levels in tissues from 42 gallbladder cancer patients, and levels of GCASPC were confirmed further in a separate cohort of 89 gallbladder cancer patients. GCASPC was overexpressed or silenced in several gallbladder cancer cell lines where molecular and biological analyses were performed. GCASPC levels were significantly lower in gallbladder cancer than adjacent nontumor tissues and were associated with tumor size, American Joint Committee on Cancer tumor stage, and patient outcomes. GCASPC overexpression suppressed cell proliferation in vitro and in vivo, whereas GCASPC silencing had opposite effects. By RNA pull-down and mass spectrometry, we identified pyruvate carboxylase as an RNA-binding protein that associated with GCASPC. Because GCASPC is a target of miR-17-3p, we confirmed that both miR-17-3p and GCASPC downregulated pyruvate carboxylase level and activity by limiting protein stability. Taken together, our results defined a novel mechanism of lncRNA-regulated cell proliferation in gallbladder cancer, illuminating a new basis for understanding its pathogenicity. Cancer Res; 76(18); 5361–71. ©2016 AACR.

Supplementary Methods. Description of additional methods and procedures used in the study.

Supplemental Table 1-9. Clinical characteristics of 5 GBC patients used for lncRNA array (Table 1). Clinicopathological characterisitcs of 42 GBC patients (Cohort 1) (Table 2). Clinicopathological characterisitcs of 89 GBC patients (Cohort 2) (Table 3). Correlation between GCASPC expression and GBC clinicopathological characteristics in 42 patients (Cohort 1) (Table 4). Univariate and multivariate analyses of OS in 89 GBC patients by Cox regression analysis (Table 5). Proteins in the three bands specific to GCASPC identified by MALDI-TOF mass spectrometry (Table 6). miRNAs binding sites of GCASPC by MirTarget2 (Table 7). Information on antibodies used for correlation analysis (Table 8). Primer sequence used in this study (Table 9). supplemental Figure 1-12. Expression levels of 10 lncRNAs by qRT-PCR in 15 GBC tissues and paired non-tumor tissues (Figure 1). Expression levels of 8 mRNAs by qRT-PCR in 15 GBC tissues and paired non-tumor tissues (Figure 2). Molecular characteristics of GCASPC (Figure 3). Nucleotide sequence of the full-length human GCASPC gene (Figure 4). GCASPC has no coding capability (Figure 5). The expression level of GCASPC in gallbladder cancer cells (Figure 6). Manipulation of GCASPC expression level (Figure 7). Anti-proliferative activity of GCASPC (Figure 8). GCASPC acts in trans (Figure 9). The effect of GCASPC on the protein, activity and mRNA levels of PC (Figure 10). GCASPC destabilizes PC protein (Figure 11). GCASPC directly binds to miR-17-3p (Figure 12).

Gallbladder cancer (GBC) is the most common type of biliary tract cancer worldwide. Long noncoding RNAs (lncRNAs) play essential roles in physiological and pathological development. LncRNA MEG3, a tumor suppressor, has been reported to play important roles in some cancers, but the role of MEG3 in GBC remains largely unknown. The purpose of the present study was to explore the role of MEG3 in proliferation and invasion and the potential molecular mechanism in GBC. We found that MEG3 was downregulated in GBC tissues and cells, and low expression of MEG3 was correlated with poor prognostic outcomes in patients. Overexpression of MEG3 inhibited GBC cell proliferation and invasion, induced cell apoptosis and decreased tumorigenicity in nude mice. Moreover, we found that MEG3 was associated with EZH2 and attenuated EZH2 by promoting its ubiquitination. Furthermore, MEG3 executed its functions via EZH2 to regulate the downstream target gene LATS2. Taken together, these findings suggest that MEG3 is an effective target for GBC therapy and may facilitate the development of lncRNA-directed diagnostics and therapeutics against GBC.

Gallbladder cancer (GBC) is one of the most lethal cancers with poor prognosis. In this study, we report that the long non-coding RNA LINC00152 is significantly upregulated in GBC tissues and cell lines. The high LINC00152 levels correlated positively with tumor status progression, lymph node invasion and TNM stage advancement. Functionally, we revealed that LINC00152 dramatically promoted cell proliferation, metastasis and inhibited apoptosis in vitro. In vivo, LINC00152 overexpression significantly promoted tumor growth. Mechanistic analyses indicated that LINC00152 could participate in the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, and transcription factor specificity protein 1 (SP1) induces its overexpression. In summary, our findings suggest that LINC00152 contributes to the oncogenic potential of GBC and SP1/LINC00152/PI3K/AKT may be a potential therapeutic target for GBC.

Background The use of livers from nonviable fetuses is particularly attractive for its potential to solve the current limitations of organ availability for the pediatric recipient. Therefore, it is essential to study the feasibility of orthotopic fetal liver transplantation. Method We measured the hepatic and extra-hepatic anatomical structures of fetal and neonatal lambs and established an orthotopic liver transplantation model of the fetal lamb. Results Mean weight of the liver of fetal lambs at 142 to 145 days gestation was 34.75 g and the mean diameter of the portal vein was 3.03 mm, the supra-hepatic vena cava was 5.88 mm, and the infra-hepatic vena cava was 4.00 mm, which matched the corresponding sizes in neonatal lambs aged up to 2 weeks. Using standard surgical procedures we completed the vascular inosculation of fetal liver. However, all the newborn lamb recipients survived less than 24 hours. Conclusions Orthotopic transplantation of the fetal liver is anatomically and technically feasible. However, perioperative issues need to be resolved prior to clinical application.

Background and Aim: Ischemia reperfusion injury (IRI) remains a major cause of graft injury, dysfunction and even failure post-transplantation. Heme oxygenase 1 (HO-1) has been found to be an attractive target for anti-inflammatory therapies and a potential candidate responsible for cell injury. The objective of this study was to investigate whether preconditioning the donor liver with Nodosin perfusion upregulates HO-1 and then lessens IRI in rat models. Methods: Wistar rats were divided into four groups: experimental group, control group, positive control group and negative control group in which the donor liver was preconditioned with Nodosin, lactated ringer's solution, cobalt protoporphyrin and zinc protoporphyrin perfusion, respectively. We measured HO-1 expression and enzyme activity in rat livers of each group ex vivo at 0, 1 and 2 h after perfusion. At 1 h after perfusion, donor livers of Wistar rats were transplanted into Sprague–Dawley rats orthotopically. Serum transaminase levels, degree of cell apoptosis and Suzuki's score were used to assess ischemia/reperfusion injury in recipients at 24 h after transplantation. Results: Ex vivo, donor liver preconditioning with Nodosin perfusion induced HO-1 expression and enzyme activity significantly, compared with the control group (P

Gallbladder cancer is a rare disease and it is associated with a poor clinical outcome and survival. A standard therapy for it has not been established yet. The aim of this study is to evaluate efficacy and safety of two modified ECF regimens in advanced gallbladder cancer patients. Clinical data of 38 patients with advanced gallbladder cancer treated with modified ECF regimen were reviewed retrospectively. Of them, 21 patients received an epirubicin, cisplatin, and 5-FU/LV combination therapy. Seventeen patients received a chemotherapy of epirubicin, cisplatin, and capecitabine. Partial response was achieved in fourteen (36.84%) patients with a median duration of 5 months (range, 3-13 months), while stable disease was achieved in eight patients (21.05%). The median time to progression was 4.0 months (95% CI, 3.62-4.58 months). And the median overall survival was 9.8 months (95% CI, 7.26-12.34 months). Responders demonstrated better survival than non-responders (median survival time: 16 vs. 6.9 months, P = 0.008). The median survival time for epirubicin-, cisplatin- and capecitabine-treated patients was 9.2 versus 8.9 months for epirubicin-, cisplatin- and 5-FU/LV-treated patients. There was no statistical difference between both treatment groups in terms of survival time (P = 0.769). Regimen-related toxicity resulted in at least one treatment delay or dosage reduction in 63.2 and 34.2% patients, respectively. There were no chemotherapy-related deaths during the study. Modified ECF regimen with epirubicin, cisplatin and 5-FU/LV or substituting capecitabine for 5-FU/LV is still a potentially effective therapeutic chemotherapy for patients with advanced gallbladder cancer, and toxicity was manageable. There was no remarkable difference in efficacy between the two regimens.

Gallbladder carcinoma is known to be an aggressive malignancy and nonsensitive to routine chemotherapy. Its prognosis is quite poor. We have illustrated that somatostatin (SST) can enhance chemosensitivity of gallbladder cancer to Doxorubicin (DOX) in our precious studies. Here, we explored the possible mechanisms by which SST used to enhance the cytotoxicity of DOX on gallbladder carcinoma cell line.Human gallbladder cancer cells line (GBC-SD cell line) were divided into four groups: control group, SST group, DOX group, SST+DOX co-treated-group. Cell cycle was detected by flow cytometry (FCM). Cell apoptosis index was detected by using Annexin V/Propidium Iodide Binding on FCM. The expressions of certain key cell cycle-related factors, including retinoblastoma protein (Rb) and E2F-1 protein were investigated by western blotting. ICBP90 protein, which could be a new downstream effector of E2F-1, was also detected by western blotting. The expression of Topo IIα protein, target enzyme of DOX, was assessed in synchronized GBC-SD cells by western blotting.After 24h treatment with SST alone, cell cycle was arrested at S phase in GBC-SD cells line, followed by indistinctive increment of apoptosis index. After 24h treatment with SST and DOX, apoptosis index significantly increased than that of DOX alone (P0.05). Compared with control group, the expressions of Rb and E2F-1 protein were significantly up-regulated at 24h after treatment with SST. Similarly, the expressions of ICBP90 and Topo IIα protein were also enhanced at 24h after treatment with SST.These results suggested that SST could induce cell cycle block in S phase in GBC-SD cells line, the most sensitive phase of the cell cycle for DOX, through up-regulating Rb, E2F-1 and ICBP90 protein expression. Furthermore, ICBP90 induced the enhanced expression of Topo IIα protein which is the target enzyme of DOX and enhanced its cytotoxic effect on GBC-SD cells. We concluded that the mechanisms of SST enhanced chemosensitivity of GBC-SD cell line to DOX might be cell cycle arrest plus up-regulated target enzyme.

In order to explore the anti-inflammatory effects of Nodosin from Isodon serra, a traditional Chinese herb medicine, mouse T lymphocytes were incubated with Nodosin. In the current study, Nodosin suppressed the overproduction of the T lymphocytes; moreover, cell mitosis cycle was modulated by interfering with DNA replication in G1 stages via inhibition of IL-2 cytokine secretion at the mRNA level by Nodosin. Interestingly, Xylene-induced mouse tumescence model results suggested Nodosin depressed the murine ear-swelling extent and the level of IL-2 in the blood serum. Finally, Nodosin possessed significant anti-inflammatory effects and is a potential candidate for further clinical trial.

Objective: To explore the change of chemosensitivity of gallbladder cancer cells pre-treated with somatostatin. Methods: Twenty-four hours after somatostatin treatment, gradient concentrated Doxorubicin was added and growth curve of gallbladder cancer cells was investigated to measure IC50, i.e., concentration of Doxorubicin at 50% cell viability. Results: Somatostatin ccould induce gallbladder cancer cell growth arrest in S phase. Inhibition of growth of cancer cell line was detected by Doxorubicin concentration- dependently (P

The identification of cancer-associated long non-coding RNAs (lncRNAs) and the investigation of their molecular and biological functions are vital for understanding the molecular biology and progression of cancer. The lncRNA-LET, a newly identified lncRNA, was demonstrated to be down-regulated in hepatocellular cancer. However, little is known about its role in gallbladder cancer. In the present study, an obvious down-regulation of lncRNA-LET was observed in gallbladder cancer compared to their adjacent normal tissues. Meanwhile, patients with low expression of lncRNA-LET have significantly poorer prognosis than those with high expression. We confirmed that hypoxia decreased lncRNA-LET levels in gallbladder cancer cells. Moreover, lncRNA-LET overexpression was further validated to inhibit the invasion of gallbladder cancer cells under hypoxic or normoxic conditions in vitro. We demonstrated that lncRNA-LET overexpression conferred a proliferative advantage to tumor cells under hypoxic conditions. The ectopic expression of lncRNA-LET led to the promotion of cell cycle arrest at G0/G1 phase and to the induction of apoptosis under hypoxic conditions. Ectopic expression of LncRNA-LET also suppressed gallbladder tumor growth in vivo. Our findings indicate that lncRNA-LET may represent a prognostic marker and a potential therapeutic target for gallbladder cancer.

BACKGROUND: Tumor suppressor genes were studied in gallbladder disease including cancer for correlation. VEGF (vascular endothelial growth factor) expression was assessed against Nevin staging and metastasis of gallbladder carcinoma. The importance of p53, p16, and VEGF in gallbladder cancer was estimated. STUDY DESIGN: Twenty-four gallbladder carcinomas, 20 gallbladder adenomas, and 18 chronic cholecystitis specimens were immunohistochemically and histopathologically investigated for the relation of p53, p16, and VEGF to Nevin staging and pathologic grading. RESULTS: The expression rate of abnormal p53 in gallbladder carcinomas was significantly higher than that in gallbladder adenoma and chronic cholecystitis (p = 0.003, p=0.014). The expression rate of abnormal p53 in Nevin staging S 1 , S 2 , S 3 gallbladder carcinoma was significantly higher than that in S 4 , S 5 (p = 0.01). Abnormal p16 was highest in carcinoma, next in adenoma, and lowest in chronic cholecystitis (p = 0.031, p=0.017). Gallbladder carcinoma expressed VEGF far more often than adenoma or cholecystitis (p = 0.001); VEGF-positive rates were lower in S 1 , S 2 , S 3 than S 4 , S 5 by Nevin staging of gallbladder cancer (p = 0.044). CONCLUSIONS: Mutation of p53 and p16 genes might correlate with progression of of gallbladder carcinoma. Analysis of p53 and p16 can estimate the prognosis of gallbladder cancer. VEGF expression correlates with Nevin staging in gallbladder cancer.

To investigate the comprehensive measures for improving radical resection rate and safety of Bismuth-Corlette type III hilar cholangiocarcinoma.The clinical data of 15 patients with Bismuth-Corlette type III hilar cholangiocarcinoma who performed radical resection from June 2009 to December 2011 was analyzed retrospectively. There were 11 male and 4 female patients, aged from 45 to 74 years (mean 59 years). The preoperative evaluation were conducted by using magnetic resonance cholangiopancreatography (MRCP), dual source spiral CT combined with IQQA-Liver CT Imaging Analysis System providing three-dimensional reconstruction of tumor, bile duct, hepatic artery and portal vein, which could help to chose the appropriate treatment modality. All patients were treated with selective hemi-hepatic vascular control of removal liver, hemi-hepatectomy combined with whole caudate lobe resection and regional lymphadenectomy. The merits of each evaluation methods and measures of surgical treatment were analyzed thoroughly.The preoperative evaluation modalities including the dual source spiral CT combined with IQQA-Liver CT Imaging Analysis System could clearly show the involvement of bile duct, hepatic artery and portal vein invaded by the tumor. The satisfactory postoperative recovery could be achieved by the remnant liver volume of40% after the hemi-hepatectomy combined with whole caudate lobe resection through the selective preoperative biliary drainage and hemihepatic vascular control. The mean complication was transient aggravated liver dysfunction. There was no death reported during perioperative period in the group.The preoperative imaging evaluation modalities including MRCP, dual source spiral CT combined with IQQA-Liver CT Imaging Analysis System could do favor for the preoperative evaluation of invasion degree of hilar cholangiocarcinoma and the selection of appropriate surgical treatment. Hemi-hepatectomy combined with whole caudate lobe resection and regional lymphadenectomy could be an alternative management of Bismuth-Corlette type III hilar cholangiocarcinoma.

Objective As Helicobacter spp. have been successfully isolated from the biliary system, a hypothetical question was raised about the role of these organisms in the development of biliary tract cancer. This meta-analysis has been carried out to explore the association between Helicobacter spp. infection and biliary tract cancer. Methods A systematic literature search was carried out to identify all eligible articles. Meta-analysis used odds ratio and a random-effect model, and 95% confidence intervals for odds ratios were calculated. Heterogeneity was quantitatively assessed using the χ-test, with significance set at a P-value of 0.01, and was measured using the I-statistic. Results Ten studies published between 2002 and 2011 were finally included for meta-analysis. Helicobacter pylori, Helicobacter bilis, Helicobacter hepaticus, and Helicobacter ganmani were studied. With heterogeneity (I=0%, P=0.685), a significantly higher pooled infection rate of Helicobacter spp. was observed in the biliary tract cancer group compared with the normal group (P=0.0001) and the benign biliary disease group, respectively (P=0.0001). Studies from East Asia and South Asia showed a higher prevalence of Helicobacter spp. in the malignant group. Evidence supporting the higher presence of Helicobacter spp. in the cancer group was obtained using PCR and immunohistochemical analysis of specimens from bile and biliary tissues. Conclusion Our meta-analysis suggests a trend of a higher presence of Helicobacter spp. in patients with biliary tract cancers compared with normal controls or those with benign biliary diseases.

To 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.TFPI-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.TFPI-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.TFPI-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.

To determine the distribution of macrophages (MΦ) in both xanthogranulomatous cholecystitis (XGC) and gallbladder carcinoma (GBC) and to analyze the association between XGC and GBC.From January 2009 to June 2011, 110 patients with gallbladder diseases, including 35 with GBC, 45 with XGC and 30 with chronic cholecystitis (CC), were enrolled. Immunohistochemistry stain and real-time polymerase chain reaction using oncogenes (BCL-2, c-Myc) and anti-oncogene genes (p53, p21) were performed, serum expressions of tumor marker (CA19-9, CA724 and CA242) were also conducted. MΦ were used to determine their potential role in the carcinogenesis of GBC.BCL-2 and c-Myc expressions gradually increased among CC, XGC and GBC (P = 0.032 and P = 0.020, respectively); while p53 and p21 were similar in the three groups (P = 0.167 and P = 0.122, respectively). Serum BCL-2 and c-Myc were significantly correlated with their tissue levels; in terms of serum tumor markers, which gradually increased among CC, XGC and GBC, however, CA242 and CA724 were both negative in XGC but positive in GBC. Furthermore, gradually increasing MΦ counts were observed among CC, XGC and GBC groups; c-Myc and CA724 were independent predictors for the differentiation of XGC and GBC.XGC is an uncommon inflammatory condition distinct from CC and may be associated with the precancerous nature of GBC for its upregulated oncogenes and MΦ biology. c-Myc and CA724 were independent predictors for the differentiation of XGC and GBC.

Ischemia reperfusion injury (IRI) remains a major cause of graft injury, dysfunction and even failure post-transplantation. Heme oxygenase 1 (HO-1) has been found to be an attractive target for anti-inflammatory therapies and a potential candidate responsible for cell injury. The objective of this study was to investigate whether preconditioning the donor liver with Nodosin perfusion upregulates HO-1 and then lessens IRI in rat models.Wistar rats were divided into four groups: experimental group, control group, positive control group and negative control group in which the donor liver was preconditioned with Nodosin, lactated ringer's solution, cobalt protoporphyrin and zinc protoporphyrin perfusion, respectively. We measured HO-1 expression and enzyme activity in rat livers of each group ex vivo at 0, 1 and 2 h after perfusion. At 1 h after perfusion, donor livers of Wistar rats were transplanted into Sprague-Dawley rats orthotopically. Serum transaminase levels, degree of cell apoptosis and Suzuki's score were used to assess ischemia/reperfusion injury in recipients at 24 h after transplantation.Ex vivo, donor liver preconditioning with Nodosin perfusion induced HO-1 expression and enzyme activity significantly, compared with the control group (P0.05). In vivo, serum transaminase levels, cell apoptosis degree and Suzuki's score of representative recipients in the Nodosin group were lower than that in the control group (P0.05). Preconditioning with Nodosin perfusion induced HO-1 protein mainly in Kupffer cells.This study suggests that preconditioning with Nodosin perfusion provides a potential protective effect through inducing HO-1 expression to attenuate ischemia/reperfusion injury in liver transplantation.

Tissue factor pathway inhibitor-2 (TFPI-2) has been identified as a tumor suppressor gene in several types of cancers, but its role in gallbladder carcinoma (GBC) is yet to be determined. In the present study, TFPI-2 expression in GBC tissues was examined, and its inhibitory activities against GBC growth were evaluated in vitro and in vivo after adenovirus-mediated gene transfer of TFPI-2 (Ad5-TFPI-2) was constructed to restore the expression of TFPI-2 in GBC cell lines (GBC-SD, SGC-996, NOZ) and xenograft tumors. Immunohistochemical staining showed that TFPI-2 was significantly downregulated in GBC tissue specimens. Ad5-TFPI-2 could significantly inhibit GBC growth both in vitro and in vivo. Apoptosis analysis and western blotting assay demonstrated that Ad5-TFPI-2 could induce the apoptosis of both GBC cell lines and tissues by promoting the activities of cytochrome c, Bax, caspase-3 and -9 and suppressing Bcl-2 activity. These data indicated that TFPI-2 acts as a tumor suppressor in GBC, and may have a potential role in gene therapy for GBC. (Cancer Sci 2012; 103: 723–730)

To study the pharmacokinetics, distribution and excretion of m-THPC in rat models of liver cancer via orthotropic implantation using Walker-256.After an intravenous injection of m-THPC with 0.3 mg/kg, the concentrations of m-THPC in biological specimens were determined by a fluorescence method. The data obtained were processed with PK-GRAPH pharmacokinetic procedure.The disposition of m-THPC in rat models of liver cancer Walker-256 was conformed to a two compartment model with T(1/2)α = 1.18 h, T(1/2)β = 22.57 h at the dose of 0.3 mg/kg.m-THPC was shown to be widely distributed to the various tissues. There was a highest drug accumulation in liver and liver cancer, and lowest in skin and muscle. Ratio of m-THPC concentration in the Walker-256 tumor compared to normal tissue reach the peak 24 h after m-THPC administration.m-THPC is distributed widely and eliminated at a rapid rate in Walker-256 rats. Twenty four hours after m-THPC administration may be the best time for photodynamic therapy of liver cancer.

To investigate the possible mechanisms by which Somatostatin (SST) enhances the anti-tumor effect of doxorubicin (DOX) on gallbladder cancer cells.GBC-SD cells were grouped into 4 groups: SST-treated group, DOX-treated group, SST+DOX co-treated group and control group. The concentrations of SST and DOX were 75 µg/ml and 5 µg/ml based on our previous studies. In control group, cells were cultivated with phosphate buffered saline (PBS). In experimental groups, cells were cultivated with medium and the corresponding drugs. After drug treatment, cell viability was examined by MTT assay at 6, 12, 24 and 36 h respectively. Meanwhile, intracellular concentrations of doxorubicin in each group was determined by microspectrofluorimetry; Real-time polymerase chain reaction (RT-PCR) was used to determine the expressions of MDR1 mRNA in the cells at different time points and the expressions of P-gp protein, a product of MDR1 mRNA, were determined by Western blot analysis.SST did not exhibit significant inhibitory effect on the proliferation of GBC-SD cells as compared to that of control group (P0.05). SST+DOX co-treatment group and DOX showed significantly inhibitory effect on the growth of GBC-SD cells at Hour 12 post-treatment. However no statistical difference was found between SST+DOX and DOX groups. Interestingly, at Hour 24 post-treatment, SST+DOX group showed more robust inhibitory effect on GBC-SD cells as compared to DOX alone group. Moreover, SST could significantly down-regulate the expressions of MDR1 mRNA and P-gp protein. SST could increase intracellular DOX concentration. And the difference of intracellular DOX concentration between SST+DOX group and DOX group at Hour 24 was statistically significant.In our experiment, SST decreases the expression of MDR1 mRNA and P-gp protein so as to reduce the efflux of DOX and elevate DOX concentrations in GBC-SD cells. This eventually leads to enhanced cytotoxic effects of DOX on GBC-SD cells.

Anti-donor T cells mediate graft rejection and the frequency of anti-donor T cells directly correlates with transplant outcome. It has long been noted that long-term tolerance was occasionally observed in minor mismatched recipients, little is known about the mechanisms underlying this phenomenon. To quantitatively analyze the relationship between anti-donor T cells and target cells, long-term tolerant C57BL/6 mice were established by infusing 3 x 10(7) F1 splenocytes during the neonatal period. The removal of anti-donor T cells in these mice was demonstrated by unresponsiveness in mixed-lymphocyte reaction. A total of 2 x 10(7) or 5 x 10(7) syngenic naive cells were transferred into long-term tolerant mice; the dose of 5 x 10(7) syngenic cells destroyed chimerism and the skin grafts, while the dose of 2 x 10(7) syngenic cells led to loss of chimerism but the survival of the skin grafts. On Day 20 after the transfusion, a portion of 5 x 10(7) syngenic cells still remained in the mice, while no syngenic cells were detected in mice that received a total of 2 x 10(7) syngenic cells suggesting that these cells were completely exhausted. Syngenic CD4+ T cells proliferated and activated in both groups, while syngenic cells in the low-dose group were more susceptible to apoptosis than those in the high-dose group. Our results suggest that a given number of effector T cells could only kill a limited number of target cells. When that limit was reached, the T cells died. This novel concept not only provides a reasonable explanation for long-term tolerance in minor mismatched transplantation but also provides new insight into tolerance induction that depleting alloreactive T cells in recipients by donor cells or agents is a prerequisite for reconstitution of thymus by donor cells, the establishment of central tolerance is the key for successful tolerance induction.

Lymph node spread is the most common pattern of progression in gallbladder carcinoma (GBC) and is a prognostic factor. The purpose of this study was to evaluate the curative effects of radical surgery including different extent of regional lymphadenectomy for patients with different stage of GBC.A retrospective study was made of 91 patients who had undergone radical resection and regional lymphadenectomy from January 2001 to December 2006. The curative effects of radical surgery and survival rate were elucidated by different extent of regional lymphadenectomy according to the classification of lymph nodes according to the American Joint Committee on Cancer (AJCC) which is classified into two grades, standard regional lymphadenectomy and extended regional lymphadenectomy. The extent of standard regional lymphadenectomy includes lymph nodes around the cystic duct, pericholedochal and hepatoduodenal ligament. The extent of extended regional lymphadenectomy includes retroportal, posterosuperior pancreaticoduodenal, posteroinferior pancreaticoduodenal, along the common hepatic artery, celiac, superior mesenteric and interaorticocaval lymph nodes.There was no significant difference of survival rates in patients with stage II between the standard regional lymphadenectomy and extended regional lymphadenectomy groups (P=0.109). However, to the patients with stage III and stage IV without distant metastases, when extended regional lymphadenectomy was performed, survival rates were significantly higher than those who received standard regional lymphadenectomy (P=0.009 and P=0.029, respectively).The standard regional lymphadenectomy is enough for the patients with stage II. The extended regional lymphadenectomy should be performed to the patients with stage III and stage IV without distant metastases if the primary lesions can be dissected radically.

To investigate the mechanism of increasing chemosensitivity of gallbladder carcinoma stimulated by somatostatin.GBC-SD cells were divided into four groups: SST-alone-treated group, Doxorubicin (DOX)-alone-treated group and co-treated group (co-treatment of SST and DOX). In the control group, the cells were cultivated by medium only. In SST-alone-treated group, the cells were cultivated by medium with SST in the concentration of 75 microg/ml. In DOX-alone-treated group, the cells were cultivated by medium with DOX in the gradient concentrations of 5, 10, 20 microg/ml. In the co-treated group, cells were first cultivated by medium with 75 microg/ml SST for 24 h, followed by the addition of DOX in the gradient concentrations mentioned above. Cell viability curve was measured by MTT assay at 24, 48, 72 and 96 h, respectively. Meanwhile, the alterations of protein expressions of ICBP90 and Topo IIalpha after treatment of SST were examined by Western blot.The treatment of SST alone on GBC-SD cells did not exert significantly inhibitory effect compared to the control group (P0.05). However, 24 h after the treatment of SST, the protein expressions of ICBP90 and Topo IIalpha were both up-regulated (P0.05).Up-regulated the expression of ICBP90 by somatostatin maybe the cause of overexpression of Topo IIalpha, which leads to the enhanced lethal effect of DOX.

AIM: To evaluate the feasibility and safety of No. 13 lymphadenectomy in radical gastrectomy for gastric carcinoma. METHODS: Medical records of the patients undergone No. 13 lymph node dissection during D2 gastrectomy for gastric carcinoma, were reviewed from March 2003 to May 2007. RESULTS: One hundred and fifty-eight patients underwent No. 13 lymph node dissection for D2 gastric carcinoma, of them, 4 (2.53%) were found to have metastasis in No. 13 lymph node. Metastasis to No. 12 lymph node was detected in 6 patients and 4 of them had positive No. 13 lymph node. The operative morbidity except for wound infection was 15.19% (24/158), and hospital death rate was 1.27% (2/158). No obstructive jaundice caused by No. 13 lymph node metastasis after No. 13 lymph node dissection in radical gastrectomy for gastric carcinoma was detected during the follow-up study to end of January 2007. CONCLUSION: Dissection of No. 13 lymph node in D2 gastrectomy for gastric carcinoma is safe with a low morbidity and mortality rate. Further study is needed to explore its long-term effect.

Background Gallbladder cancer is the most common billiary tract malignancy and carries a very poor prognosis. Somatostatin was recently shown to play an important role in the development of various tumors. In the current study, we evaluated the effect of doxorubicin on the chemosensitivity of gallbladder cancer cells and xenograft growth after treatment with somatostatin. Methods Twenty-four hours after somatostatin treatment, doxorubicin was gradually added and the growth curve of gallbladder cancer cells was determined. Exponential-phase gallbladder cancer cells were treated with doxorubicine or co-treated with doxorubicine and somastatine and the respective IC50 values were determined. In addition, the inhibitory effect on the growth of gallbladder cancer xenograft on nude mice was evaluated using the same treatments as those described above. Results Treatment of gallbladder cancer cells with somatostatin led to a block in the cell cycle at the S phase. Growth inhibition of gallbladder cancer cells by doxorubicin was concentration-dependent (P < 0.05). However, upon co-treatment with doxorubicin and somatostatin, the IC50 value significantly decreased as compared to that of cells treated with doxorubicine alone (P < 0.05). Interestingly, treatment with either doxorubicin or somatostatin did not significantly inhibit xenograft growth on nude mice, in contrast to a co-treatment with both drugs (P < 0.05). Conclusion Somatostatin most likely sensitizes the chemotherapeutic effect and diminishes the cytotoxicity of doxorubicin in a gallbladder cancer cell line and in mouse gallbladder cancer xenografts.

Protein-coding genes account for only ~2% of the human genome, whereas the vast majority of transcripts are non-coding RNAs (ncRNAs) including long ncRNAs (lncRNAs). A growing volume of literature has proposed that lncRNAs are important factors in cancer. Colon cancer-associated transcript-1 (CCAT1), an lncRNA, which was first identified in colon cancer, was previously shown to promote tumor development and be a negative prognostic factor in gastric cancer. However, the mechanism through which CCAT1 exerts its oncogenic activity remains largely unknown. Recently, a novel regulatory mechanism has been proposed in which RNAs can cross-talk with each other via competing shared for microRNAs (miRNAs). The proposed competitive endogenous RNAs could mediate the bioavailability of miRNAs on their targets, thus imposing another level of posttranscriptional regulation. In this study, we demonstrated that CCAT1 was upregulated in gallbladder cancer (GBC) tissues. CCAT1 silencing downregulated, whereas CCAT1 overexpression enhanced the expression of miRNA-218-5p target gene Bmi1 through competitively ‘spongeing’ miRNA-218-5p. Our data revealed that CCAT1 knockdown impaired the proliferation and invasiveness of GBC cells, at least in part through affecting miRNA-218-5p-mediated regulation of Bmi1. Moreover, CCAT1 transcript level was correlated with Bmi1 mRNA level in GBC tissues. Together, these results suggest that CCAT1 is a driver of malignancy, which acts in part through ‘spongeing’ miRNA-218-5p.

This study was conducted to evaluate the effects of postoperative immune enhancing enteral nutrition on the immune system, inflammatory responses, and clinical outcome of patients undergoing major abdominal surgery.This study was designed as a multicenter, prospective, randomized and controlled clinical trial. One hundred twenty-four patients undergoing major abdominal surgery were randomly assigned to receive either an immune enhancing enteral diet or an isocaloric and isonitrogenous control enteral diet for seven days. Enteral feeding was initiated 24 hours after surgery. Host immunity was evaluated by measuring levels of IgG, IgM, IgA, CD4, CD8, and CD4/CD8, and the inflammatory response was determined by assessing IL-1alpha, IL-2, IL-6, IL-10, and TNF-alpha levels. Infectious complications were also recorded.One hundred twenty patients completed the study and four patients were excluded. On postoperative day 9, among patients receiving an immune enhancing diet, IgG, IgA, CD4 and CD4/CD8 levels were significantly higher and TNF-alpha and IL-6 concentrations were significantly lower compared to the control group. Moreover, among patients receiving an immune enhancing diet, when comparing preoperation to day 9 postoperation levels, increases in IgA, CD4, and CD4/CD8 levels were significantly higher than in control patients and increases in TNF-alpha concentrations were significantly lower. No statistically significant differences were found between the two groups with regard to infectious complications.Postoperative administration of immune enhancing enteral nutrition in patients undergoing major abdominal surgery can positively modulate postoperative immunosuppressive and inflammatory responses.

Background Gallbladder carcinoma (GBC) is highly lethal, and effective treatment will require synergistic anti-tumor management. The study is aimed at investigating the oncolytic value of myxoma virus (MYXV) infection against GBC and optimizing MYXV oncolytic efficiency. Methods We examined the permissiveness of GBC cell lines to MYXV infection and compared the effects of MYXV on cell viability among GBC and control permissive glioma cells in vitro and in vivo after MYXV + rapamycin (Rap) treatment, which is known to enhance cell permissiveness to MYXV by upregulating p-Akt levels. We also assessed MYXV + hyaluronan (HA) therapy efficiency by examinating Akt activation status, MMP-9 expression, cell viability, and collagen distribution. We further compared hydraulic conductivity, tumor area, and survival of tumor-bearing mice between the MYXV + Rap and MYXV + HA therapeutic regimens. Results MYXV + Rap treatment could considerably increase the oncolytic ability of MYXV against GBC cell lines in vitro but not against GBC xenografts in vivo. We found higher levels of collagen IV in GBC tumors than in glioma tumors. Diffusion analysis demonstrated that collagen IV could physically hinder MYXV intratumoral distribution. HA–CD44 interplay was found to activate the Akt signaling pathway, which increases oncolytic rates. HA was also found to enhance the MMP-9 secretion, which contributes to collagen IV degradation. Conclusions Unlike MYXV + Rap, MYXV + HA therapy significantly enhanced the anti-tumor effects of MYXV in vivo and prolonged survival of GBC tumor-bearing mice. HA may optimize the oncolytic effects of MYXV on GBC via the HA–CD44 interaction which can promote viral infection and diffusion.

AIM: To describe the distribution of micrometastases in the surrounding liver of patients with primary liver cancer (PLC), and to describe the minimal length of resection margin (RM) for hepatectomy. METHODS: From November 2001 to March 2003, 120 histologically verfied PLC patients without macroscopic tumor thrombi or macrosatellites or extrahepatic metastases underwent curative hepatectomy. Six hundreds and twenty-nine routine pathological sections from these patients were re-examined retrospectively by light microscopy. In the prospective study, curative hepatectomy was performed from November 2001 to March 2003 for 76 histologically verfied PLC patients without definite macroscopic tumor thrombi or macrosatellites or extrahepatic metastases in preoperative imaging. Six hundreds and forty-five pathological sections from these patients were examined by light microscopy. The resected liver specimens were minutely examined to measure the resection margin and to detect the number of daughter tumor nodules, dominant lesions, and macroscopic tumor thrombi inside the lumens of the major venous system. The paraffin sections were microscopically examined to detect the microsatellites, microscopic tumor thrombi, fibrosis tumor capsules, as well as capsule invasion and the distance of histological spread of the micrometastases. RESULTS: In the retrospective study, 70 micrometastases were found in surrounding liver in 26 of the 120 cases (21.7%). The farthest distance of histological micrometastasis was 3.5 mm, 5.3 mm and 6.0 mm in 95%, 99% and 100% cases, respectively. Macroscopic tumor thrombi or macrosatellites were observed in 18 of 76 cases, and 149 micrometastases were found in the surrounding live in 25 (43.1%) of 58 cases with no macroscopic tumor thrombi. The farthest distance of histological micrometastasis was 4.5 mm, 5.5 mm and 6.0 mm in 95%, 99% and 100% cases, respectively. Two hundred and sixty-seven micrometastases were found in surrounding liver in 14 (77.8%) out of 18 cases with macroscopic tumor thrombi or macrosatellites. The farthest distance of histological micrometastasis was 18.5 mm, 18.5 mm and 19.0 mm in 95%, 99% and 100% cases, respectively. CONCLUSION: The required minimal length of RM is 5.5 mm and 6 mm respectively to achieve 99% and 100% micrometastasis clearance in surrounding liver of PLC patients without macroscopic tumor thrombi or macrosatellites, and should be greater than 18.5 mm to obtain 99% micrometastasis clearance in surrounding liver of patients with macroscopic tumor thrombi or macrosatellites.