HMGA2 promotes the migration and invasion of gallbladder cancer cells and HMGA2 knockdown inhibits angiogenesis via targeting VEGFA.

The high mobility group AT­hook 2 (HMGA2) protein has been found to be upregulated in the majority of tumor types and is associated with a poor prognosis. Previous studies have suggested the oncogenic role of HMGA2 in gallbladder cancer (GBC). The present study aimed to investigate the effects of HMGA2 on the invasion, migration and angiogenesis of GBC cells. To achieve this aim, HMGA2 was overexpressed or silenced in the GBC cell line, EH­GB1, and then the proliferation, migration, invasion and epithelial­mesenchymal transition (EMT) abilities of EH­GB1 cells were investigated using Cell Counting Kit­8, wound healing, Transwell and western blotting assays. In addition, the expression levels of VEGFA were determined in EH­GB1 cells using western blotting and reverse transcription­quantitative PCR following HMGA2 overexpression or silencing. Furthermore, HMGA2­silenced EH­GB1 cells were transfected with VEGFA overexpression plasmids to evaluate the tube formation ability of HUVECs using tube formation assay. The results demonstrated that HMGA2 silencing inhibited GBC cell proliferation, migration, invasion and EMT, as evidenced by the downregulated expression of Ki67, proliferating cell nuclear antigen, MMP2, MMP9, N­cadherin, snail family transcriptional repressor 2 and zinc finger E­box­binding homeobox 1, and attenuated cell migration and invasion. However, the opposite results were obtained following HMGA2 overexpression. Moreover, HMGA2 knockdown and overexpression downregulated and upregulated VEGFA expression, respectively. In addition, the tube formation ability of HUVECs and the expression levels of CD31, VEGFR1 and VEGFR2 were downregulated following HMGA2 silencing. However, these effects were partially rescued by simultaneous VEGFA overexpression. In conclusion, the findings of the present study revealed that HMGA2 may promote GBC cell migration, invasion, EMT and angiogenesis. Therefore, inhibiting HMGA2 expression could be considered as a possible therapeutic approach for GBC.

Vascular evaluation using transabdominal ultrasound for gallbladder polyps.

Ultrasound (US) is a cost-effective and noninvasive procedure without radiation exposure, with real-time evaluation and high spatial resolution. Although it is useful for the detection of gallbladder (GB) polyps, including gallbladder cancer, adenoma, and benign polyps, conventional US is insufficient for differential diagnosis because it is not capable of evaluating hemodynamic information, unlike computed tomography or magnetic resonance imaging. With recent technological advances in US equipment and contrast agents, Doppler imaging and contrast-enhanced ultrasonography (CEUS) are being used to characterize GB polyps, and several reports on evaluation of the vascularity of GB polyp have been published. In this review, we aimed to report the latest developments in the hemodynamic diagnosis of GB polyps based on previous reports, with an emphasis on CEUS, and to evaluate the efficacy for differential diagnosis. The information in this article is expected to enable early diagnosis and prompt surgical treatment for gallbladder cancer.

Gallbladder cancer-associated fibroblasts promote vasculogenic mimicry formation and tumor growth in gallbladder cancer via upregulating the expression of NOX4, a poor prognosis factor, through IL-6-JAK-STAT3 signal pathway.

BACKGROUND: Cancer-associated fibroblasts (CAFs) and vasculogenic mimicry (VM) play important roles in the occurrence and development of tumors. However, the relationship between CAFs and VM formation, especially in gallbladder cancer (GBC) has not been clarified. In this study, we investigated whether gallbladder CAFs (GCAFs) can promote VM formation and tumor growth and explored the underlying molecular mechanism. METHODS: A co-culture system of human GBC cells and fibroblasts or HUVECs was established. VM formation, proliferation, invasion, migration, tube formation assays, CD31-PAS double staining, optic/electron microscopy and tumor xenograft assay were used to detect VM formation and malignant phenotypes of 3-D co-culture matrices in vitro, as well as the VM formation and tumor growth of xenografts in vivo, respectively. Microarray analysis was used to analyze gene expression profile in GCAFs/NFs and VM (+)/VM (-) in vitro. QRT-PCR, western blotting, IHC and CIF were used to detected NOX4 expression in GCAFs/NFs, 3-D culture/co-culture matrices in vitro, the xenografts in vivo and human gallbladder tissue/stroma samples. The correlation between NOX4 expression and clinicopathological and prognostic factors of GBC patients was analyzed. And, the underlying molecular mechanism of GCAFs promoting VM formation and tumor growth in GBC was explored. RESULTS: GCAFs promote VM formation and tumor growth in GBC; and the finding was confirmed by facts that GCAFs induced proliferation, invasion, migration and tube formation of GBC cells in vitro, and promoted VM formation and tumor growth of xenografts in vivo. NOX4 is highly expressed in GBC and its stroma, which is the key gene for VM formation, and is correlated with tumor aggression and survival of GBC patients. The GBC patients with high NOX4 expression in tumor cells and stroma have a poor prognosis. The underlying molecular mechanism may be related to the upregulation of NOX4 expression through paracrine IL-6 mediated IL-6/JAK/STAT3 signaling pathway. CONCLUSIONS: GCAFs promote VM formation and tumor growth in GBC via upregulating NOX4 expression through the activation of IL-6-JAK-STAT3 signal pathway. NOX4, as a VM-related gene in GBC, is overexpressed in GBC cells and GCAFs, which is related to aggression and unfavorable prognosis of GBC patients.

Vascular Resection During Hepatectomy for Liver Malignancies. Results from a Tertiary Center using Autologous Peritoneal Patch for Venous Reconstruction.

BACKGROUND: To evaluate early outcomes of venous reconstruction with peritoneal patch (PP) during resection for hepatic malignancies. METHODS: Since May 2015, PP was considered as the first option for venous reconstruction in the case of lateral resection. Between May 2015 and June 2019, 579 consecutive hepatectomies for malignancies were performed at our institution. Among 27 patients requiring venous resection, PP was used in 22, who were included in the present study. Data from a prospectively collected database were analysed. RESULTS: Tumour types were ten colorectal metastases (CRLM), six intrahepatic cholangiocarcinomas, four hilar cholangiocarcinomas, one hepatocellular carcinoma and one gallbladder carcinoma. Hepatectomies were major in 50% of cases. Eleven patients had hepatic vein resections, eight portal vein and three inferior vena cava. Venous reconstruction enabled resection in 12 (54.5%) patients, otherwise non-resectable. Among CRLM, the venous reconstruction allowed avoidance of major resection in eight (80%) cases. Median operative time was 456 min (range 270-960). Blood loss was a median 300 cc (range 40-1500), and blood transfusions were required in three patients (13.6%). At pathological examination, venous infiltration was confirmed in 14 (63.6%) patients. No vascular complications related to the patch were recorded. Post-operative major (Dindo III/IV) complications were observed in two (9%) patients. One patient died because of liver failure without vascular thrombosis and one due to biliary fistula complicated by arterial bleeding. Overall, post-operative mortality was 9% (2/22). CONCLUSIONS: Venous reconstruction with peritoneal patch during hepatectomy for malignancies can feasibly allow resection in otherwise unresectable patients and decrease the rate of major resection in colorectal liver metastases.

miR-143-3p targeting of ITGA6 suppresses tumour growth and angiogenesis by downregulating PLGF expression via the PI3K/AKT pathway in gallbladder carcinoma.

Gallbladder cancer (GBC) is the most common malignant tumour of the biliary track system. Angiogenesis plays a pivotal role in the development and progression of malignant tumours. miR-143-3p acts as a tumour suppressor in various cancers. Their role in GBC is however less well defined. Here we show that the expression levels of miR-143-3p were decreased in human GBC tissues compared with the non-tumour adjacent tissue (NAT) counterparts and were closely associated with overall survival. We discovered that miR-143-3p was a novel inhibitor of tumour growth and angiogenesis in vivo and in vitro. Our antibody array, ELISA and PLGF rescue analyses indicated that PLGF played an essential role in the antiangiogenic effect of miR-143-3p. Furthermore, we used miRNA target-prediction software and dual-luciferase assays to confirm that integrin α6 (ITGA6) acted as a direct target of miR-143-3p. Our ELISA and western blot analyses confirmed that the expression of PLGF was decreased via the ITGA6/PI3K/AKT pathway. In conclusion, miR-143-3p suppresses tumour angiogenesis and growth of GBC through the ITGA6/PI3K/AKT/PLGF pathways and may be a novel molecular therapeutic target for GBC.

DUSP1 inhibits cell proliferation, metastasis and invasion and angiogenesis in gallbladder cancer.

DUSP1/MKP1 is a dual-specific phosphatase that regulates MAPK activity and is known to play a key role in tumor biology. Its function in gallbladder cancer (GBC) remains largely unknown, however. By exploring its activities in two GBC cell lines (SGC996 and GBC-SD), DUSP1 was found to inhibit GBC cell proliferation, migration and invasion. Moreover, DUSP1 inhibited GBC growth and metastasis in nude mice subcutaneously xenografted with SGC996 cells. The tumor suppression appeared to be mediated via the DUSP1-pERK/MAPK-MMP2 signal pathway. Angiogenesis was associated with the tumor metastasis in the mouse model and was impaired by DUSP1, which suppressed VEGF expression. These results suggest that DUSP1 suppresses GBC growth and metastasis by targeting the DUSP1-pERK-MMP2/VEGF axis. Identification of the DUSP1-pERK-MMP2/VEGF signals may provide new biomarkers and/or therapeutic targets to better suppress GBC metastasis in the future.

Intraarterial 5-fluorouracil and interferon therapy is safe and effective for nonresectable biliary tract adenocarcinoma.

PURPOSE: The prognosis in advanced biliary carcinoma has remained poor. The purpose of this study was to investigate the efficacy of intraarterial 5-fluorouracil and interferon therapy against unresectable biliary carcinoma. METHODS: Patients with unresectable biliary carcinoma with performance status 0 or 1 were enrolled between January 2002 and September 2012. They received pegylated interferon-α 2a and intraarterial 5-FU every 4 weeks. The therapy was either terminated at the end of the first cycle for the patients with progressive disease or continued for at least three cycles. Patients' characteristics (physical, laboratory and radiographic) at the time of starting intraarterial 5-FU therapy were investigated. The relationship between the patients' characteristics and outcome, i.e., survival time and radiographic therapeutic evaluation of patients, was statistically analyzed. RESULTS: Tumor sites were the intrahepatic bile ducts in 23 patients and gallbladder in 2 patients. Previous treatment had been administered in ten patients. The overall response rate was 24% (6 partial responses in 25 patients). Stable disease was observed in 13 patients. The median overall survival was 358 days. Among the six partial responses, three patients received surgery, and one patient received radiofrequency ablation because clinical downstaging was obtained. The treatment was well tolerated. The survival analyses revealed that two factors (serum albumin ≥ 3.5 and hypovascular tumor) were significantly associated with overall survival. CONCLUSIONS: Combination therapy with 5-FU and interferon-α was safe and may improve the prognosis of advanced biliary carcinomas.

Inhibition of tumor angiogenesis by interferon-γ by suppression of tumor-associated macrophage differentiation.

Tumor-associated macrophages (TAMs) differentiate from monocytes and are the M2-polarized macrophages in most human tumors, secreting generous vascular endothelial growth factor (VEGF) to promote angiogenesis. Although it has been shown in vitro that interferon-γ (IFN-γ) can inhibit monocytes differentiating to M2 macrophages in the tumor microenvironment and switch TAMs from M2 into M1, suppressing the ability of secreting VEGF, its effects on TAMs in vivo remains unknown. Here we tried to examine the effects of IFN-γ on the recruitment of monocyte/macrophage differentiation of TAMs and tumor angiogenesis in vivo. We built a gallbladder cancer model by inoculating subcutaneously the human gallbladder cancer cell line (GBC-SD) into BALB/C nude mice and injected the recombinant mouse IFN-γ intratumorally. We found that in the IFN-γ group, the number of monocytes/macrophages was significantly higher than that in the control group (p < 0.01), and TAM differentiation rate, which we defined as the number of TAMs / the number of monocytes/macrophages × 100%, mice-VEGF concentration, and microvessels density (MVD) were significantly lower than those in the control group (p < 0.01, p < 0.05, and p < 0.01). Our results suggest that IFN-γ can induce monocytes/macrophages recruiting into the tumor microenvironment, but inhibit them, differentiating to TAMs in vivo, which may reduce the concentration of VEGF and angiogenesis in tumor.

Inhibition of tumor vasculogenic mimicry and prolongation of host survival in highly aggressive gallbladder cancers by norcantharidin via blocking the ephrin type a receptor 2/focal adhesion kinase/paxillin signaling pathway.

Vasculogenic mimicry (VM) is a newly-defined tumor microcirculation pattern in highly aggressive malignant tumors. We recently reported tumor growth and VM formation of gallbladder cancers through the contribution of the ephrin type a receptor 2 (EphA2)/focal adhesion kinase (FAK)/Paxillin signaling pathways. In this study, we further investigated the anti-VM activity of norcantharidin (NCTD) as a VM inhibitor for gallbladder cancers and the underlying mechanisms. In vivo and in vitro experiments to determine the effects of NCTD on tumor growth, host survival, VM formation of GBC-SD nude mouse xenografts, and vasculogenic-like networks, malignant phenotypes i.e., proliferation, apoptosis, invasion and migration of GBC-SD cells. Expression of VM signaling-related markers EphA2, FAK and Paxillin in vivo and in vitro were examined by immunofluorescence, western blotting and real-time polymerase chain reaction (RT-PCR), respectively. The results showed that after treatment with NCTD, GBC-SD cells were unable to form VM structures when injecting into nude mouse, growth of the xenograft was inhibited and these observations were confirmed by facts that VM formation by three-dimensional (3-D) matrix, proliferation, apoptosis, invasion, migration of GBC-SD cells were affected; and survival time of the xenograft mice was prolonged. Furthermore, expression of EphA2, FAK and Paxillin proteins/mRNAs of the xenografts was downregulated. Thus, we concluded that NCTD has potential anti-VM activity against human gallbladder cancers; one of the underlying mechanisms may be via blocking the EphA2/FAK/Paxillin signaling pathway.

Expression of N-acetylglucosaminyltransferase V in the subserosal layer correlates with postsurgical survival of pathological tumor stage 2 carcinoma of the gallbladder.

BACKGROUND: N-Acetylglucosaminyltransferase V (GnT-V), an enzyme that catalyzes the ß1-6 branching of N-acetylglucosamine on asparagine-linked oligosaccharides of cellular proteins, enhances the malignant behaviors of carcinoma cells in experimental models. The aim of this study was to determine clinical significance of GnT-V expression in human pT2 gallbladder carcinoma with simple in vitro experiments. METHODS: Ninety patients with pT2 gallbladder carcinoma were included for this study. The in vitro and in vivo biological effects of GnT-V were investigated using gallbladder carcinoma cells with variable GnT-V expression levels induced by a small interfering RNA. RESULTS: Of the 90 cases, 57 showed positive staining and the remaining 33 demonstrated negative staining, the subcellular localization in the 57 cases was classified into the granular-type in 31 cases and the diffuse-type in 26 cases. In 76 cases with curative resection, postsurgical survival was significantly poorer in those showing positive staining than in those showing negative staining (P = 0.028). In all of the 76 cases, postsurgical recurrence was significantly more frequent in those showing diffuse-type localization than in those showing negative staining. Experimental analyses demonstrated that the down-regulation of GnT-V expression in gallbladder carcinoma cells induced suppression of cell growth in vitro. The expression levels of GnT-V in the cells were highly correlated with the rapid in vivo growth coupled with the enhanced angiogenesis, and the tendency to form liver metastasis. CONCLUSIONS: GnT-V expression in the subserosal layer of pT2 gallbladder carcinoma is correlated with the aggressiveness of the disease.

Utility of contrast-enhanced harmonic EUS in the diagnosis of malignant gallbladder polyps (with videos).

BACKGROUND: The differential diagnosis between benign and malignant polyps of the gallbladder (GB) is often challenging. OBJECTIVES: To evaluate whether contrast-enhanced harmonic EUS (CEH-EUS) might be an accurate method for discriminating malignant GB polyps from benign polyps. DESIGN: Observational study. SETTING: Tertiary care medical center. PATIENTS: Ninety-three patients with GB polyps larger than 10 mm in diameter that were detected by conventional EUS underwent CEH-EUS for evaluation of microvasculature. INTERVENTION: CEH-EUS was performed using a radial echoendoscope and the extended pure harmonic detection mode. MAIN OUTCOME MEASUREMENTS: The abilities of conventional EUS and CEH-EUS to diagnose malignant polyp were compared. Two blinded reviewers classified the perfusion images into 3 categories: diffuse enhancement, perfusion defect, or nonenhancement. The vessel images were categorized as having a regular spotty vessel, an irregular vessel, or no vessels. RESULTS: An irregular vessel pattern determined by CEH-EUS aided in the diagnosis of malignant polyps with a sensitivity and specificity of 90.3% and 96.6%, respectively. The presence of perfusion defects, determined by CEH-EUS, was calculated to diagnose malignant polyps with a sensitivity and specificity of 90.3% and 94.9%, respectively. Based on the definitely determined diagnosis, sensitivity and specificity for CEH-EUS were 93.5% and 93.2% versus 90.0% and 91.1% for conventional EUS. In 8 cases, management changed after CEH-EUS. LIMITATIONS: A tertiary medical center with a limited number of patients. CONCLUSIONS: The presence of irregular intratumoral vessels or perfusion defects seen on CEH-EUS may be sensitive and accurate predictors of malignant GB polyps. CEH-EUS offers slightly improved diagnostic accuracy compared with EUS.

Contribution of the PI3K/MMPs/Ln-5γ2 and EphA2/FAK/Paxillin signaling pathways to tumor growth and vasculogenic mimicry of gallbladder carcinomas.

Vasculogenic mimicry (VM) is a new tumor blood supply in some highly aggressive malignant tumors. We previously reported VM in human gallbladder carcinomas, 3-D matrices in vitro and nude mouse xenografts in vivo of highly aggressive GBC-SD cells and its clinical significance. In this study, we further studied the underlying mechanisms of VM in gallbladder carcinomas via the 3-D matrix in vitro, the nude mouse xenografts in vivo of GBC-SD or SGC-996 cells, immunohistochemistry (H&E staining and CD31-PAS double staining), electron microscopy, expression of MMP-2, MT1-MMP, PI3K, Ln-5γ2, EphA2, FAK and Paxillin-P proteins/mRNAs determined by SABC, ELISA, immunofluorescence, western blotting and qRT-PCR, respectively. It was shown that all of untreated highly aggressive GBC-SD cells and xenografts formed vasculogenic-like structures within 2 weeks of seeding and injecting, and facilitated the growth of tumor cells or xenografts; whereas poorly aggressive SGC-996 cells or GBC-SD cells treated by TIMP-2 were unable to form the vasculogenic-like structures with the same conditions; and tumor xenograft growth was inhibited. Expression of MMP-2, MT1-MMP proteins/mRNAs from sections and supernates of 3-D matrix in vitro, expression of PI3K, MMP-2, MT1-MMP, Ln-5γ2, EphA2, FAK and Paxillin-P proteins/mRNAs from sections of xenografts in vivo in untreated GBC-SD group was upregulated significantly (all P<0.001); however, expression of these VM signal-related proteins/mRNAs in the SGC-996 group and GBC-SD treated by the TIMP-2 group was significantly downregulated (all P<0.001). Thus, we identified for the first time that highly aggressive GBC-SD cells formed VM in vitro and in vivo through the upregulation of PI3K/MMPs/Ln-5γ2 and/or EphA2/FAK/Paxillin signaling. PI3K/MMPs/Ln-5γ2 and EphA2/FAK/Paxillin as key signaling pathways in a coordinated manner contributed to tumor growth and VM of gallbladder carcinomas and provided novel targets that could be potentially exploited for therapeutic intervention of human gallbladder carcinomas.

Overexpression of HIF-1α in primary gallbladder carcinoma and its relation to vasculogenic mimicry and unfavourable prognosis.

As a novel mode of tumor neovascularization, vasculogenic mimicry (VM) has been reported to increase tumor-related mortality in many different solid tumors. In the present study, two established human gallbladder carcinoma (GBC) cell lines (highly aggressive GBC-SD and poorly aggressive SGC-996) cultured on a three-dimensional matrix were assessed for the ability of VM channel formation under normoxic or hypoxic conditions. In addition, the relationship between HIF-1α gene expression and VM channel formation of GBC cells in vitro was measured using the small interfering RNA (siRNA) technique, western blotting and real-time reverse transcription (RT)-PCR analysis. Furthermore, H&E and CD31/periodic acid-Schiff (PAS) staining were used to observe VM in GBC tissue samples. Additionally, all seventy-one specimens with VM and non-VM were stained for hypoxia inducible factor-1 α (HIF-1α) and its correlation with clinicopathological features and prognosis was analyzed simultaneously. We found that hypoxia could induce more VM channel formation and elevated HIF-1α expression in highly aggressive GBC-SD cells. HIF-1α siRNA efficiently knocked down HIF-1α expression and GBC VM networks under either normoxic or hypoxic conditions. VM was present in human primary GBC and overexpression of HIF-1α was significantly correlated with depth of invasion and perineural involvement in the non-VM group. Moreover, VM and HIF-1α were independent factors for the overall survival of GBC patients and correlated with decreased survival. In conclusion, VM was present in human GBC. As a critical mediator in VM formation, high expression of HIF-1α was associated with VM and tumor progression in GBC patients.

Portal vein embolization before extended hepatectomy for biliary cancer: current technique and review of 494 consecutive embolizations.

BACKGROUNDS: Portal vein embolization (PVE) has been widely applied before extended hepatectomy; however, its clinical utility for patients with biliary cancer has not been fully addressed. METHODS: Between 1991 and 2010, 494 patients with cholangiocarcinoma (n = 353) or gallbladder cancer (n = 141) underwent PVE before extended hepatectomy. PVE was performed by a transhepatic ipsilateral approach using fibrin glue or absolute ethanol with steel coils. Surgical outcomes of this cohort were retrospectively reviewed. RESULTS: PVE-related complications requiring interventions were found in 3 (0.6%) of the 494 patients; no patient died of these complications. Among the 494 patients, 122 (24.7%) did not undergo subsequent hepatectomy. The unresectability rate was significantly higher in patients with gallbladder cancer than in those with cholangiocarcinoma [43.2% (61/141) and 17.3% (61/353), respectively, p < 0.001]. The remaining 372 patients underwent hepatectomy, and 24 (6.5%) died of postoperative complications [13 of 80 (16.3%) with gallbladder cancer vs. 11 of 292 (3.8%) with cholangiocarcinoma, p < 0.05]. The overall survival for patients with cholangiocarcinoma was significantly better than that for patients with gallbladder cancer, where the 5-year survival rate was 39 and 23%, respectively (p < 0.001). Thirty-six patients with cholangiocarcinoma and 10 patients with gallbladder cancer survived more than 5 years after extended surgery. CONCLUSION: PVE can be performed safely in patients with cholestatic liver, and it has a potential benefit for patients with advanced biliary cancer who are to undergo extended, difficult hepatectomy.

Identification of CD146 expression, angiogenesis, and lymphangiogenesis as progression, metastasis, and poor-prognosis related markers for gallbladder adenocarcinoma.

Gallbladder cancers (GBC) are associated with high disease-specific mortality rates because of no means of early detection and effective therapies. In this study, we investigated CD146 expression, microvessel densities, and lymph vessel densities in 108 adenocarcinomas, 15 gallbladder polyps, 35 chronic cholecystitis tissues, and 46 peritumoral tissues using immunohistochemistry. We demonstrated that positive CD146 expression, and average microvessel and lymph vessel counts in gallbladder adenocarcinomas were significantly higher than those in peritumoral tissues, polyps, and chronic cholecystitis (ps < 0.01). Positive CD146 expression, and average microvessel and lymph vessel counts were also significantly lower in cases with well-differentiated adenocarcinoma, maximal tumor diameter <2 cm, no metastasis of lymph node, and no invasion of regional tissues than in cases with poorly differentiated adenocarcinoma, maximal tumor diameter ≥ 2 cm, metastasis in lymph nodes, and invasion of regional tissues (p < 0.05 or p < 0.01). Univariate Kaplan-Meier analysis showed that increased expression of CD146 (p = 0.056), higher average microvessel counts (p < 0.05), and lymph vessel counts (p < 0.05) were associated with decreased overall survival. Multivariate Cox regression analysis showed that average microvessel and lymph vessel counts (ps < 0.05) were independent prognostic predictors in gallbladder adenocarcinoma. Our study suggested that the elevated expression of CD146, angiogenesis, and lymphangiogenesis might be closely related to progression, invasion, metastasis, and prognosis of gallbladder adenocarcinoma.

Norcantharidin: a potential antiangiogenic agent for gallbladder cancers in vitro and in vivo.

Our objective was to explore the antiangiogenic activity of norcantharidin (NCTD) as an angiogenic inhibitor for gallbladder cancers. In vitro and in vivo experiments to determine the effects of NCTD on HUVECs, chicken CAM capillaries and gallbladder cancer xenograft angiogenesis in nude mice were respectively done. The MTT method was used to assay the cytotoxicity of NCTD on HUVECs. Immunofluorescence was used to evaluate HUVEC apoptosis. The scraping line method, matrigel invasion assay and tube formation assay were used to detect the migration, invasion and tube formation of HUVECs. A digital camera was used to observe chicken CAM capillaries. Experiments with NCTD in a xenograft model were used to observe the effect of NCTD on xenograft growth and survival of mice with xenografts. CD34 immunohistochemistry, flow cytometry and micro-MRA were used, respectively, to determine MVD, cell apoptosis and hemodynamic analysis of the xenografts. Immunohistochemistry and RT-PCR were used, respectively, to detect the expression of VEGF, Ang-2, TSP, TIMP-2 proteins/mRNAs of the xenografts. The xenograft MVD associated with tumor volume, the PCNA/apoptosis ratio and related-protein expression was evaluated simultaneously. We found that NCTD effectively inhibited the proliferation, migration, invasion and capillary-like tube formation of HUVECs in vitro; it reduced angiogenesis and directly destroyed the formed CAM capillaries in vivo. In the experiments in mice, NCTD not only inhibited significantly xenograft proliferation and growth, prolonged survival time of mice with xenografts, decreased the xenograft MVD and vascular perfusion, but also, similarly to ES, decreased significantly the expression of VEGF or Ang-2 protein/mRNA, increased the expression of TSP or TIMP-2 protein/mRNA. Moreover, the xenograft MVD was positively related with tumor volume, PCNA/apoptosis ratio, and VEGF or Ang-2 expression, respectively (all P<0.05), but negatively correlated with TSP or TIMP-2 expression (both P<0.05). These data showed that NCTD could serve as a potential antiangiogenic agent for gallbladder cancers.

Expression pattern of tumor endothelial marker 8 protein in gallbladder carcinomas.

Tumor endothelial marker 8 protein (TEM8) is highly specific to tumor angiogenesis and is not required for normal adult angiogenesis and hence might prove to be a target for anti-angiogenic therapies in the future. We here evaluated protein and gene expression patterns in human endothelial cells of benign gallbladder - gallstone diseases (GSDs) and gallbladder carcinomas (GBCs) using immunostaining, immunofluorescence and western blotting techniques. Subjects comprised 175 GBC patients, 38 males and 137 females, aged 30-85 years (mean age 50.3 ± 13.4 years) and twenty with GSDs, aged 30-75 years, (51.4 ± 10.0 years) for comparison (male 4/20 and females 16/20). TEM8 protein expression increased significantly (p < 0.0001) with increasing stage of GBC and was mostly limited to endothelial cells, although there was no significant change with the grade. Interestingly, only 80-85 kDa and 60 kDa isoforms of TEM8 increased significantly whereas 45 kDa isoform was absent in GBCs. Conclusions- These results suggest that TEM8 plays an unknown important biological role to promote tumor angiogenesis in GBC.

[Expressions of VEGF-C and VEGF-D and their correlation with lymphangiogenesis and angiogenesis in gallbladder carcinoma].

OBJECTIVE: To investigate the expression of VEGF-C and VEGF-D and their correlations with lymphangiogenesis and angiogenesis in gallbladder carcinoma. METHODS: Fifty cases of gallbladder carcinoma with complete clinical and pathological data were analyzed. The expression of VEGF-C and -D, D2-40, CD31 was assayed by immunohistochemical staining, with 10 samples of normal gallbladder tissues away from cancer and 19 samples of chronic cholecystitis as controls, and their correlation with clinicopathological findings were analyzed retrospectively. RESULTS: Thirty-two (64.0%) of the 50 gallbladder cancers were positive for VEGF-C protein expression by immunohistochemistry and the positive rate of VEGF-D protein expression was 62.0% (31/50). The protein expression of VEGF-C and VEGF-D in tumor tissues was significantly higher than that in normal gallbladder tissues away from the tumor (P < 0.05), but no correlation with that in chronic cholecystitis (P < 0.05). The VEGF-C expression correlated with the patient age and lymph node metastasis (both P < 0.05). The VEGF-D expression only correlated with lymph node metastasis (P < 0.05). In the 50 gallbladder cancers, the MLVD was 6.9 + or - 3.6 and the MVD was 36.1 + or - 12.8. The MLVD in both VEGF-C and -D positive groups was significantly higher than that in the negative groups (P = 0.000), and the lymph node metastasis also increased. MVD in both VEGF-C and -D positive groups was higher than that in the negative groups (P < 0.05), and it was also correlated with tumor differentiation (P < 0.05). A significant positive correlation was also found between VEGF-C and VEGF-D expression (r = 0.498, P < 0.01). CONCLUSION: VEGF-C and VEGF-D are involved in the lymphangiogenesis and angiogenesis in gallbladder carcinoma, promote lymph node metastasis of the tumor, and both are important in the regulation of lymphangiogenesis and angiogenesis in this cancer. VEGF-C and VEGF-D are of clinical significance in evaluating lymph node metastatic potency and estimation of prognosis in gallbladder carcinoma.

Evaluation of cholecystic venous flow using indocyanine green fluorescence angiography.

BACKGROUND/PURPOSE: The cholecystic veins are thought to be an important metastatic route of gallbladder carcinoma to the liver. In the present study we evaluated the cholecystic venous drainage area, utilizing a novel method, indocyanine green (ICG) fluorescence angiography after superselective catheterization of the cholecystic artery, to detect and elucidate cholecystic venous flow. METHODS: Cannulation of the cholecystic artery was performed under laparotomy in nine patients who required a cholecystectomy. After ICG injection into the cholecystic artery, the cholecystic venous flow images were visualized with a near-infrared camera system and were analyzed according to site, shape, and time of fluorescence. RESULTS: Fluorescence images of the cholecystic venous flow could be viewed as real-time images in all patients. We demonstrated that the route of the cholecystic venous flow could be classified into two patterns: type 1, in which the cholecystic veins flowed directly into the hepatic parenchyma adjacent to the gallbladder; and type 2, in which the veins flowed into sites separate from the gallbladder. In the type 1 pattern, fluorescence was observed in segment (S; defined according to Couinaud's nomenclature) 4a or S5 adjacent to the gallbladder in all cases. On the other hand, in the type-2 pattern, fluorescence was observed in S4a (6/9), S5 (8/9), S4b (2/9), S3 (2/9), S1 (1/9), S2 (1/9), and S8 (1/9) distant from the gallbladder. Overall, two-thirds of the cases showed fluorescence in segments other than S4a or S5. CONCLUSIONS: Indocyanine green (ICG) fluorescence angiography is considered to be a useful method to detect and elucidate cholecystic venous flow in real time. This study showed that the cholecystic venous flow spread to the liver through two different pathways, one that flowed directly into the hepatic parenchyma adjacent to the gallbladder, while the other flowed into sites separate from the gallbladder. Taking these findings into consideration, we may therefore need to reconsider the preventive effects of a hepatic resection.