Germacrone exerts anti-cancer effects on gastric cancer through induction of cell cycle arrest and promotion of apoptosis.

BACKGROUND: Germacrone is one of the natural bioactive compounds found in Rhizoma curcuma essential oils. In this study, the potential anti-cancer effect of germacrone in gastric cancer cell line BGC823 was investigated. METHODS: The cell viability and proliferative activity were assessed, and cell cycle analysis was also performed. Hoechst 33258 and Annexin V/PI double staining was used for detection of cell apoptosis. Protein profiles of cell cycle-related and apoptosis-related proteins were assessed. RESULTS: MTT assay revealed that germacrone had marked cytotoxicity on BGC823 cells. Germacrone induced cell cycle arrest in the G2/M phase via remarkably decreased expression levels of cyclin B1, cdc 2 and cdc 25c. In addition, the treatment with germacrone induced caspase-3 activity and PARP cleavage. These findings demonstrated the effects of germacrone on inhibiting cell proliferation through induction of G2/M phase cell cycle arrest and promotion of cell apoptosis. It also indicated that germacrone functioned through modulations of cell cycle-associated protein expression and mitochondria-mediated apoptosis. CONCLUSION: These findings will be valuable as the molecular basis for the germacrone-mediated anti-cancer effect against gastric cancer.

Prognostic value of multiple epithelial mesenchymal transition-associated proteins in intrahepatic cholangiocarcinoma.

The aim of the present study was to investigate the expression of epithelial mesenchymal transition (EMT)-associated proteins and their prognostic value in intrahepatic cholangiocarcinoma (ICC). The expression of six EMT-associated proteins, including E-cadherin, N-cadherin, Vimentin, Snail family transcriptional repressor 1 (Snail), Snail family transcriptional repressor 2 (Slug) and S100 calcium binding protein A4 (S100A4) was determined by immunohistochemistry in 109 patients with ICC who had received surgery. Survival analysis showed that patients with low E-cadherin expression (P<0.001) or high S100A4 (P<0.001) or Snail (P<0.001) expression had a reduced survival time. Based on the numbers of alterations in the expression of EMT-associated proteins as determined by immunohistochemical analysis, the patients were categorized as low (score, 0-3; n=75) or high (score, ≥4; n=34) EMT expression groups. The high EMT expression group was significantly associated with positive lymph node metastasis (P=0.023) and late Tumor-Node-Metastasis (TNM) stage (P<0.001). Furthermore, patients in the high EMT expression group had a significantly poorer overall survival time than those in the low EMT expression group (P<0.001). Multivariate analysis indicated that EMT status was a significant independent predictor for overall survival time (P=0.004), and was linked to surgical margin (P=0.013) and TNM stage (P<0.001). In conclusion, the reduced expression of E-cadherin and high expression of Snail and S100A4 were significantly associated with the poor survival of patients with ICC after surgery. The EMT protein expression status was associated with ICC progression, and may be considered as an independent prognostic indicator for patients with ICC.

PIVKA-II level is correlated to development of portal vein tumor thrombus in patients with HBV-related hepatocellular carcinoma.

AIM: To evaluate the correlation of serum PIVKA-II levels and development of portal vein tumor thrombus (PVTT) in hepatocellular carcinoma (HCC) patients. METHODS: One hundred and twenty-three patients with newly diagnosed HCC were included in this study between March 2016 and October 2018. Thirty-five of these patients were detected with PVTT and all subjects were randomly divided to analysis group (N = 73) and validation (N = 50) group. Serum levels of PIVKA-II, laboratory tests including serum aspartate aminotransferase, total bilirubin, platelet count, albumin levels were demonstrated in all the patients. T-test, chi-squared test and logistic regression was used for analyzing data. Diagnostic efficiency and cut-off value of PIVKA-II in PVTT development of HCC patients were calculated using receiver operator curve (ROC) analysis. RESULTS: Serum level of PIVKA-II in HCC patients with PVTT was significantly higher than that in HCC patients without PVTT (995.8 mAU/ml vs 94.87 mAU/ml; P = 0.003), as well as D-dimer levels (2.12 mg/L vs 0.56 mg/L P = 0.001). Univariate analysis showed that high serum D-dimer level was an independent risk factor for development of PVTT (OR = 1.22, 95%CI 1.02-1.45). ROC curve showed that among analysis group, the area under ROC curve (AUROC) of PIVKA-II was 0.73 (95%CI 0.59-0.86). For the detection of PVTT in HCC, PIVKA-II had a sensitivity of 83.7% and a specificity of 69.2% at a cutoff of 221.26 mAU/ml, which had a sensitivity of 85.71% and a specificity of 55.56% in validation group, respectively. CONCLUSION: Serum PIVKA-II level is a potential marker for diagnosis of PVTT in HCC patients, which may guide therapeutic strategy and assessment of tumor prognosis of HCC.

Propranolol prevents liver cirrhosis by inhibiting hepatic stellate cell activation mediated by the PDGFR/Akt pathway.

Propranolol is known to reduce portal pressure by decreasing blood flow to the splanchnic circulation and the liver. However, it is unknown if propranolol improves fibrogenesis and sinusoidal remodeling in the cirrhotic liver. The aim of this study was to investigate the therapeutic effects of propranolol on carbon tetrachloride (CCl4)-induced liver fibrosis in a mouse model and the intrinsic mechanisms underlying those effects. In this study, a hepatic cirrhosis mouse model was induced by CCl4 administration for 6 weeks. Propranolol was simultaneously administered orally in the experimental group. Liver tissue and blood samples were collected for histological and molecular analyses. LX-2 cells induced by platelet-derived growth factor-BB (PDGF-BB) were used to evaluate the anti-fibrogenic effect of propranolol in vitro. The results showed that treatment of mice with CCl4 induced hepatic fibrosis, as evidenced by inflammatory cell infiltration, collagen deposition and abnormal vascular formation in the liver tissue. All these changes were significantly attenuated by propranolol treatment. Furthermore, we also found that propranolol inhibited PDGF-BB-induced hepatic stellate cell migration, fibrogenesis, and PDGFR/Akt phosphorylation. Taken together, propranolol might prevent CCl4-induced liver injury and fibrosis at least partially through inhibiting the PDGF-BB-induced PDGFR/Akt pathway. The anti-fibrogenic effect of propranolol may support its status as a first-line treatment in patients with chronic liver disease.

Downregulation of esophageal cancer-related gene 4 promotes proliferation and migration of hepatocellular carcinoma.

Esophageal cancer-related gene 4 (ECRG4) is a candidate tumor suppressor gene, which is involved in cell apoptosis, migration, infection and inflammation responsiveness; however, its expression level and clinical significance in hepatocellular carcinoma (HCC) remains unclear. In the present study, the authors aim to evaluate the clinical significance and potential role of ECRG4 in HCC. Level of ECRG4 protein expression in HCC and peripheral tissues was investigated in tissue specimens obtained from 56 consecutive HCC patients by immunohistochemistry. Cell proliferation, cell migration and invasion regulations were examined by MTT curves, flow cytometry, Transwell assays and western blotting. ECRG4 expression was weak positive in normal liver cells but was downregulated in HCC cells in vivo or in vitro. A decreased expression of ECRG4 was associated with the age of the patients, metastasis and Ki-67 proliferation index. However, decreased ECRG4 expression was not associated with differentiation, tumor size, the presence of portal vein tumor thrombosis, satellite lesions, tumor relapse or mortality. Further investigations revealed that ectopic expression of ECRG4 inhibited cell proliferation, migration and invasion and promoted cell apoptosis in SMMC-7721 cells, which was mediated by the regulation of BAX and B cell lymphoma-2, in addition to the upregulation of epithelial-mesenchymal transition markers. In conclusion, the results of the present study indicated that ECRG4 was downregulated in HCC and served important roles in promoting cell proliferation and migration.

Valproic acid (VPA) promotes the epithelial mesenchymal transition of hepatocarcinoma cells via transcriptional and post-transcriptional up regulation of Snail.

Due to the low cost and favorable safety profile, valproic acid (VPA) has been considered as a potential candidate drug for therapy of various cancers. Our present study revealed that VPA, at the concentration (1mM) which has no effect on cell proliferation, can significantly increase the in vitro migration and invasion of hepatocarcinoma (HCC) HepG2 and Huh7 cells via induction of epithelial mesenchymal transition (EMT). VPA treatment can significantly increase the mRNA and protein expression of Snail, the key transcription factor of EMT. While knockdown of Snail can abolish VPA induced EMT of HCC cells. It suggested that Snail is essential for VPA induced EMT of HCC cells. VPA treatment also increased the phosphorylation of NF-κB p65. BAY 11-7082, the inhibitor of NF-κB, can significantly abolish VPA induced up regulation of Snail mRNA. Furthermore, VPA can increase the protein expression of Snail since 1h treatment via up regulation of half-lives of Snail protein. The increased protein stabilization of Snail can be attributed to VPA induced phosphorylation of Akt and GSK-3ß. Collectively, our present study revealed that VPA can promote the EMT of HCC cells via up regulation of Snail through activation of NF-κB and Akt/GSK-3ß signals.

Carvedilol may attenuate liver cirrhosis by inhibiting angiogenesis through the VEGF-Src-ERK signaling pathway.

AIM: To investigate the effect of carvedilol on angiogenesis and the underlying signaling pathways. METHODS: The effect of carvedilol on angiogenesis was examined using a human umbilical vascular endothelial cell (HUVEC) model. The effect of carvedilol on cell viability was measured by CCK8 assay. Flow cytometry was used to assess the effect of carvedilol on cell cycle progression. Cell migration, transwell migration and tube formation assays were performed to analyze the effect of carvedilol on HUVEC function. Vascular endothelial growth factor (VEGF) induced activation of HUVECs, which were pretreated with different carvedilol concentrations or none. Western blot analysis detected the phosphorylation levels of three cell signaling pathway proteins, VEGFR-2, Src, and extracellular signal-regulated kinase (ERK). The specific Src inhibitor PP2 was used to assess the role of Src in the VEGF-induced angiogenic pathway. RESULTS: Carvedilol inhibited HUVEC proliferation in a dose-dependent manner (IC50 = 38.5 mmol/L). The distribution of cells in the S phase decreased from 43.6% to 37.2%, 35.6% and 17.8% by 1, 5 and 10 µmol/L carvedilol for 24 h, respectively. Carvedilol (10 µmol/L) reduced VEGF-induced HUVEC migration from 67.54 ± 7.83 to 37.11 ± 3.533 (P < 0.001). Carvedilol concentrations of 5 µmol/L and 10 µmol/L reduced cell invasion from 196.3% ± 18.76% to 114.0% ± 12.20% and 51.68% ± 8.28%, respectively. VEGF-induced tube formation was also reduced significantly by 5 µmol/L and 10 µmol/L carvedilol from 286.0 ± 36.72 to 135.7 ± 18.13 (P < 0.05) and 80.27 ± 11.16 (P < 0.01) respectively. We investigated several intracellular protein levels to determine the reason for these reductions. Treatment with 10 µmol/L carvedilol reduced VEGF-induced tyrosine phosphorylation of VEGFR-2 from 175.5% ± 8.54% to 52.67% ± 5.33% (P < 0.01). Additionally, 10 µmol/L carvedilol reduced VEGF-induced ERK 1/2 phosphorylation from 181.9% ± 18.61% to 56.45% ± 7.64% (P < 0.01). The VEGF-induced increase in Src kinase activity was alleviated by carvedilol [decreased from 141.8% ± 15.37% to 53.57 ± 7.18% (P < 0.01) and 47.04% ± 9.74% (P < 0.01) at concentrations of 5 and 10 µmol/L, respectively]. Pretreatment of HUVECs with Src kinase inhibitor almost completely prevented the VEGF-induced ERK upregulation [decreased from 213.2% ± 27.68% to 90.96% ± 17.16% (P < 0.01)]. CONCLUSION: Carvedilol has an anti-angiogenic effect on HUVECs. This inhibitory effect is mediated by VEGF-induced Src-ERK signaling pathways.

The expression of S100A4 protein in human intrahepatic cholangiocarcinoma: clinicopathologic significance and prognostic value.

Intrahepatic cholangiocarcinoma(ICC) is a highly malignant adenocarcinoma arising from bile duct epithelial cells of the intrahepatic biliary system with early hematogenous and lymphatic extrahepatic spread. The current treatment methods for ICC are far from ideal. Identifying novel effective prognostic biomarkers which might be related to the development and progression of ICC may help provide new therapeutic strategies. Both calcium-binding protein S100A4 and Matrix metalloproteinase-9(MMP-9) are correlated with development and progression of many carcinomas. In the present study, we investigated expression of S100A4 as well as MMP-9 in ICC tissues from 65 patients using immunohistochemistry. The correlation of S100A4 and MMP-9 expression with clinicopathological features and prognosis of patients were analyzed. S100A4 and MMP-9 were positively expressed in 32(49.2 %) and 35(53.8%) patients, respectively. The positive correlation between S100A4 and MMP-9 expression was statistically significant (P = 0.018). S100A4 positive expression was significantly correlated with vascular invasion (P = 0.008), lymph node metastasis (P = 0.029) and the TNM stage (P = 0.008). MMP-9 expression was not found to be correlated with any clinicopathological parameter. Patients with S100A4 positive expression had a significantly poorer overall survival rate than those with S100A4 negative expression (P = 0.000). MMP-9 positive expression was also correlated with poor survival (P = 0.044). However, only S100A4 expression (P = 0.004) and the surgical margin (P = 0.024) were significantly independent prognostic predictors by multivariate analysis. In conclusion, expression of S100A4 is correlated with MMP-9 expression and it could be a useful marker for predicting the progression, metastasis and prognosis of ICC.

MDCT angiography to evaluate the therapeutic effect of PTVE for esophageal varices.

AIM: To evaluate the role of multi-detector row computed tomography (MDCT) angiography for assessing the therapeutic effects of percutaneous transhepatic variceal embolization (PTVE) for esophageal varices (EVs). METHODS: The subjects of this prospective study were 156 patients who underwent PTVE with cyanoacrylate for EVs. Patients were divided into three groups according to the filling range of cyanoacrylate in EVs and their feeding vessels: (1) group A, complete obliteration, with at least 3 cm of the lower EVs and peri-/EVs, as well as the adventitial plexus of the gastric cardia and fundus filled with cyanoacrylate; (2) group B, partial obliteration of varices surrounding the gastric cardia and fundus, with their feeding vessels being obliterated with cyanoacrylate, but without reaching lower EVs; and (3) group C, trunk obliteration, with the main branch of the left gastric vein being filled with cyanoacrylate, but without reaching varices surrounding the gastric cardia or fundus. We performed chart reviews and a prospective follow-up using MDCT images, angiography, and gastrointestinal endoscopy. RESULTS: The median follow-up period was 34 mo. The rate of eradication of varices for all patients was 56.4% (88/156) and the rate of relapse was 31.3% (41/131). The rates of variceal eradication at 1, 3, and 5 years after PTVE were 90.2%, 84.1% and 81.7%, respectively, for the complete group; 61.2%, 49% and 42.9%, respectively, for the partial group; with no varices disappearing in the trunk group. The relapse-free rates at 1, 3 and 5 years after PTVE were 91.5%, 86.6% and 81.7%, respectively, for the complete group; 71.1%, 55.6% and 51.1%, respectively, for the partial group; and all EVs recurred in the trunk group. Kaplan-Meier analysis showed P values of 0.000 and 0.000, and odds ratios of 3.824 and 3.603 for the rates of variceal eradication and relapse free rates, respectively. Cyanoacrylate in EVs disappeared with time, but those in the EVs and other feeding vessels remained permanently in the vessels without a decrease with time, which is important for the continued obliteration of the feeding vessels and prevention of EV relapse. CONCLUSION: MDCT provides excellent visualization of cyanoacrylate obliteration in EV and their feeding veins after PTVE. It confirms that PTVE is effective for treating EVs.

Upregulation of vasohibin-1 expression with angiogenesis and poor prognosis of hepatocellular carcinoma after curative surgery.

Vasohibin-1 has recently been found and is known as an endogenous angiogenesis inhibitor, but the role of vasohibin-1 in hepatocellular carcinoma (HCC) is unknown. This study investigated the expression pattern of vasohibin-1, its correlation with clinicopathological features, and its potential role in tumor angiogenesis and prognosis of HCC. Expression of vasohibin-1, vascular endothelial growth factor-A (VEGF-A), and intratumoral microvessel density (MVD, labeled by CD34) were assessed by immunohistochemistry in 117 HCC specimens and adjacent nontumor liver tissues (ANLT). Correlation between vasohibin-1 and VEGF-A, MVD, and clinicopathological features was then investigated. Prognostic value of these factors was determined using Kaplan-Meier analysis and a Cox proportional hazards regression model. Cytoplasm high expression of vasohibin-1 was detected in 38.5% (45/117) of the HCC tissues, which was significantly higher than that in 16.2% (19/117) of ANLT (P<0.001). Vasohibin-1 was statistically correlated with VEGF-A, MVD, and microvascular invasion in HCC (P=0.014, 0.035, and 0.002, respectively). Patients with vasohibin-1 high expression had significantly poor disease-free survival (DFS) and overall survival (OS) at 5 years after curative hepatectomy (P<0.001 for each). Multivariate analysis confirmed that vasohibin-1 high expression was an independent prognosticator for unfavorable DFS (HR=2.554, P<0.001) and OS (HR=2.232, P=0.002), along with VEGF-A and TNM stage. Upregulation of vasohibin-1 expression is associated with angiogenesis and poor prognosis of HCC. Vasohibin-1 and VEGF-A are the most important factors influencing the dismal prognosis based on the modulation of angiogenesis in HCC, which provides a rational approach for treatment in the future.

Modified percutaneous transhepatic variceal embolization with 2-octylcyanoacrylate for bleeding gastric varices: long-term follow-up outcomes.

OBJECTIVE: The objective of our study was to evaluate the long-term efficacy and safety of a modified percutaneous transhepatic variceal embolization procedure with 2-octylcyanoacrylate (2-OCA) in the treatment of gastric variceal bleeding. MATERIALS AND METHODS: From January 2003 to December 2008, 71 patients with a history of gastric variceal bleeding underwent modified percutaneous transhepatic variceal embolization with 2-OCA in our hospital: 12 patients with acute gastric variceal bleeding underwent emergency obliteration and the remaining 59 patients with recent variceal bleeding underwent modified percutaneous transhepatic variceal embolization as a secondary prophylaxis. The initial hemostasis rate, rebleeding rate, survival rate, and complications were evaluated. RESULTS: Complete obliteration--that is, all the gastric varices and their feeding veins were obliterated--was achieved after the percutaneous transhepatic variceal embolization procedure in 67 patients (94.4%). Acute variceal bleeding was arrested after the procedure in all 12 patients (100%). The mean follow-up period was 24.2 ± 12.4 (SD) months (range, 6-62 months). During the follow-up period, the cumulative probability of remaining free of gastric variceal rebleeding in patients with complete obliteration was 100%, 88.2%, and 88.2% at 1, 3, and 5 years after the procedure, respectively. Follow-up CT revealed that the modified percutaneous transhepatic variceal embolization procedure with 2-OCA can achieve long-lasting obliteration in the entire varices and in all the feeding veins. The cumulative survival rates at 1, 3, and 5 years after the procedure were 96.9%, 68.9%, and 53.7%. No severe complications occurred after the procedure. CONCLUSION: The modified percutaneous transhepatic variceal embolization with 2-OCA is considered to be an effective and safe method for the extensive and permanent obliteration of both gastric varices and their feeding veins.