Hepatoma-derived growth factor promotes growth and metastasis of hepatocellular carcinoma cells.

We aimed to elucidate the effects of hepatoma-derived growth factor (HDGF) on growth and metastasis of hepatocellular carcinoma (HCC) cells. Tissue microarrays with 236 HCC specimens and 18 extrahepatic metastases were utilized to detect the HDGF expression by immunohistochemistry. Meanwhile, HDGF expressions in HCC cell lines with different metastatic potentials were examined using immunofluorescence staining, real-time PCR and western blotting. After HDGF silencing, the growth and metastatic potentials of HCC cells were evaluated by soft agar assay, invasion assay, together with tumorigenicity assay in nude mice. The gelatin zymography was performed by detecting MMP-2 and MMP-9 levels. Additionally, western blotting was conducted to determine the levels of total and phosphorylated ERK1/2, JNK, p38 and Akt. The results showed that HDGF was overexpressed in HCC metastasis tumour, and the expression increased with the differentiation degree of tumours (Grade I 44.0%, Grade II 48.4% and Grade III 65.6%). Consistently, HDGF levels were positively associated with the metastatic capability of HCC cells (MHCC97L < MHCC97H < HCCLM3). The growth and metastasis were suppressed by HDGF-siRNA. Gelatinolytic activities were enhanced in the three metastatic HCC cell lines, but had no significant difference among them. The tumourigenicity and metastatic capability of HCCLM3 cells in nude mice were inhibited after silencing HDGF. Meanwhile, HDGF-siRNA specifically suppressed the total and phosphorylated protein levels of ERK1/2, while not JNK, p38 and Akt. In conclusion, HDGF was overexpressed in HCC patients and cells, and HDGF might be closely correlated with HCC metastasis via regulating ERK signalling pathway. Copyright © 2016 John Wiley & Sons, Ltd.

Surgical treatment of incidental gallbladder cancer discovered during or following laparoscopic cholecystectomy.

BACKGROUND: The optimal surgical management of patients with incidental gallbladder cancer (IGBC) and their long-term survival remains unclear. OBJECTIVE: The purpose of this study was to examine the long-term prognosis of patients with IGBC diagnosed during or after LC. METHODS: Between January 2002 and January 2012, a total of 7,582 consecutive patients underwent LC for presumed gallbladder benign disease in the Chinese PLA General Hospital, China. Among them, 69 patients (0.91%) were diagnosed to have IGBC. Their medical records, imaging data, surgery records, pathological findings, and survival data were retrospectively reviewed. RESULTS: Median age was 61 years (range: 34-83). After a median follow-up period of 61 months, the 1-, 3-, and 5-year survival rates of patients were 89.9, 78.3, and 76.8%, respectively. The 5-year survival rates of patients with T1a, T1b, T2, and T3 stages were 95.5, 93.8, 69.2, and 44.4%, respectively. The 5-year survival rates in simple LC (n = 45), converted to open extended cholecystectomy (n = 16), and radical second resection (n = 8) groups were 91.1, 37.5, and 75.0%, respectively. Local port-site tumor recurrence was identified in one patient. Prognostic factors including depth of invasion, lymph node status, vascular or neural invasion, tumor differentiation, extent of resection, bile spillage, and type of surgery were statistically significant (p < 0.05). CONCLUSIONS: Simple LC is appropriate for T1a patients with clear margin and unbroken gallbladder, whereas extended radical resection is recommended for patients with T1b or more advanced IGBC. An intact surgical specimen and the use of plastic retrieval bags are important to reduce the risk of port-site recurrences and disease relapse. Early diagnosis, meticulous perioperative assessment, and precise surgery are essential factors to obtain good results in IGBC treatment.

[Surgical management of incidental gallbladder cancer discovered during or after laparoscopic cholecystectomy].

OBJECTIVE: To analyze the surgical management of incidental gallbladder cancer (IGBC) discovered during or after laparoscopic cholecystectomy (LC) and to evaluate the associated factors of survival. METHODS: A retrospective analysis of patients with IGBC between January 2002 and December 2013 was performed. A total of 10 080 consecutive patients underwent LC operation for presumed gallbladder benign disease in Chinese People's Liberation Army General hospital. And among them, 83 patients were histologically diagnosed as IGBC. Data covering clinical characteristics, surgery records, local pathological stage, histological features and factors for long term survival were reviewed. The survival analysis was performed using Kaplan-Meier method, and the results were examined using the log-rank test.For multivariate statistical analyses of prognostic factors, a Cox proportional hazards model was carried out. RESULTS: A total of 83 patients with IGBC:68.7% females (57/83), median age of 61 years (range 34-83 years). There were 47 cases accepted the initial simple LC, 18 cases converted to open extended radical cholecystectomy, 16 cases with radical second resection, and 2 cases with re-laparotomy; the 5-year survival rates for each group were 89.4%, 38.9%, 87.5%, and 0, respectively. The 5-year survival rates in T1a, T1b, T2, and T3 stage patients were 95.7% (22/23), 90.0% (18/20), 75.0% (15/20), and 40.0% (8/20), respectively. Univariate analysis for prognostic factors associated with cancer-specific death showed that depth of invasion, lymph-node status, vascular or neural invasion, tumor differentiation, extent of resection, bile spillage during prior LC and type of surgery were statistically significant.In multivariate analysis, depth of invasion, extent of resection and bile spillage were the most important prognostic factors related to both cancer-specific mortality and disease relapse (P < 0.05). CONCLUSIONS: Simple LC is appropriate for T1a patients with clear margin and unbroken gallbladder. An extended radical resection in patients with T1b or more is highly recommended, and provided as a potentially curative R0 resection only if it is necessary.

STAT3-Activated Long Non-Coding RNA Lung Cancer Associated Transcript 1 Drives Cell Proliferation, Migration, and Invasion in Hepatoblastoma Through Regulation of the miR-301b/STAT3 Axis.

Hepatoblastoma (HB) usually occurs in infants and toddlers. Although long non-coding RNAs (lncRNAs) in various human cancers have been widely studied, the role of lncRNAs in HB remains unclear. This study aimed to investigate the biological role of the lncRNA lung cancer associated transcript 1 (LUCAT1) in HB. Analysis of data from The Cancer Genome Atlas indicated that upregulation of lncRNA LUCAT1 was closely associated with poor overall survival of HB patients. Quantitative reverse transcription polymerase chain reaction analysis showed that LUCAT1 was highly expressed in both HB tissues and cell lines. Loss-of function assays to identify the biological function of LUCAT1 in HB showed that LUCAT1 knockdown inhibited cell proliferation, migration, and invasion but reversed epithelial-mesenchymal transition. Luciferase assays indicated that STAT3 was a transcription activator of LUCAT1 and that LUCAT1 could increase STAT3 expression by competitively binding to miR-301b. In conclusion, it was found that LUCAT1 was activated by STAT3 and promoted cell proliferation, migration, and invasion in HB through modulation of the miR-301b/STAT3 axis.

Suppression of integrin alphaupsilonbeta6 by RNA interference in colon cancer cells inhibits extracellular matrix degradation through the MAPK pathway.

Integrin alphaupsilonbeta6 plays a very important role in the progression of colon cancer cells and is now defined as a novel, independent prognostic indicator for aggressive colon cancer in humans. Herein, we use the RNA interfering technology to downregulate the expression of alphaupsilonbeta6 in colon cancer cells. Our data demonstrate that plasmid vector based shRNA can effectively down-regulate alphaupsilonbeta6 expression in protein and mRNA levels. Supression of integrin alphaupsilonbeta6 inhibits the phosphorylation and nonphosphorylation level of ERK1/2, the secretion of uPA, pro-MMP-9 and pro-MMP-2 in tumor conditioned medium, and more important, inhibits MAPK-dependent [(3)H] labeled collagen IV degradation via the plasminogen activation cascade. Our study demonstrates in vitro that supression of integrin alphaupsilonbeta6 inhibits extracellular matrix degradation through the MAPK pathway.

Integrin alpha v beta 6 mediates the potential for colon cancer cells to colonize in and metastasize to the liver.

Integrin alpha v beta 6 (alpha v beta 6) is correlated with colon cancer progression. To detect the effects of alpha v beta 6 on liver metastasis, the specificity of alpha v beta 6 against the monoclonal antibody (mAb) 2G2 was examined by immunoprecipitation. Integrin alpha v beta 6-immunoreactivity (IR) in liver metastasis tissues (63 cases) and colon carcinoma (358 cases) were examined. These results showed that alpha v beta 6 was specifically recognized by the mAb 2G2, and that rates of alpha v beta 6 positivity in liver metastatic tissues (71.4%, 45/63) were higher than that for primary colon cancer (34.0%, 122/358) (P < 0.01). Patients who were alpha v beta 6-positive had higher liver metastasis rates (17%, 21/122) than those who were alpha v beta 6-negative (only 3%, 7/236) (P < 0.01). To examine the underlying mechanisms associated with alpha v beta 6 regulating colonic metastasis in the liver, experimental liver metastasis (intrasplenic injection of HT29 transfectants) and liver colonization assays (direct injection of WiDr transfectants into the liver) in nude mice were performed; these demonstrated that alpha v beta 6 contributed to the promotion of the metastatic potential and the survival of cancer cells in the liver. Matrix metalloproteinase-9 (MMP-9) levels in the cultures of both HT29 and WiDr cells were detected by the Biotrak MMP-9 activity assay system and gelatin zymography assay, and showed that suppression of alpha v beta 6-IR inhibited MMP-9 activity and secretion. Transwell migration assay in vitro also showed that alpha v beta 6 promoted migration on fibronectin for HT29/WiDr mock compared with HT29/WiDr antisense beta 6 transfects (P < 0.01). We concluded that alpha v beta 6 may mediate the potential for colon cancer cells to colonize in and metastasize to the liver. The mechanisms that alpha v beta 6 may be involved in include the promotion of MMP-9 secretion, the enhancement of migration on fibronectin, and the survival of cancer cells in the liver.

Modified method of hepatic portal choledochoplasty to treat benign strictures of hilar biliary ducts.

BACKGROUND AND AIM: Roux-en-Y hepaticojejunostomy (RYHJ) is usually used to treat benign strictures of hilar bile ducts. However, RYHJ might also induce ascending cholangitis and recurrent hepatolithiasis. The present study aims to introduce a modified hepatic portal choledochoplasty with a pedicled graft of gallbladder (HPC) to treat this disease. METHODS: One hundred and forty-nine patients, who had undergone HPC or RYHJ from January 1997 to January 2006 in our institutions, were included in this study, and the clinical data were retrospectively collected and analyzed. RESULTS: The incidences of perioperative bile leakage and inflammatory ileus in patients treated with HPC were slightly lower than RYHJ without significant difference (1.89% vs 2.08% and 3.77% vs 5.21%, both P > 0.05). However, in a long-term follow up, patients treated with HPC had significantly lower incidences of cholangitis and recurrent hepatolithiasis (5.66% and 3.77%, respectively) than those treated with RYHJ (cholangitis, 21.88%; hepatolithiasis, 16.67%; both P < 0.05). CONCLUSION: Compared to RYHJ, HPC is a safer and more efficient method to treat benign strictures of hilar biliary ducts. It preserves the sphincter of Oddi and normal biliary duct pressure, thus avoiding bile reflux into the bile duct.

[Effects of antisense 6 gene on colon cancer cells].

OBJECTIVE: To investigate the effect of antisense integrin beta6 gene on the growth of colon cancer cells. METHODS: Expressing vector of antisense alphavbeta6 was constructed. Human colon cancer cells of the line HT29 were cultured and divided into 3 groups: Group A, remaining wild type; Group B, transfected with antisense integrin beta6 gene; and Group C, transfected with blank vector. RT-PCR was used to detect the integrin beta6 mRNA expression of in the HT29 cells. The integrinbeta6 protein expression on the surface of the cells was detected by immunohistochemistry and flow cytometry. The binding between the cells and fibronectin was examined. (3)H-labeled thymidine (T) was added into the culture fluid of the cells, and then the radiation amount was detected every 6 days so as to determine the capacity to proliferation of the cells in vitro. Thirty female nude mice were divided into 3 groups to be injected subcutaneously with suspension of HT29 cells of Groups A, B, and C as mentioned above. Six weeks later the size of tumors was measured and part of the tumor nodules were resected 5 weeks after the inoculation to undergo pathological examination. RESULTS: Compared with Groups A and C, no corresponding band at 141 bp was found in Group B by RT-PCR. Flow cytometry showed that the expression level of beta6 protein had was (0.30 +/- 0.051, 30%), significantly lower than those of Groups A and C [(0.80 +/- 0.038, 80%) and (0.85 +/- 0.045, 85%), both P < 0.01]. The binding between the HT29 cells and fibronectin of Group B was significantly degraded after the further addition of anti-beta1 and anti-alphav in comparison of Groups A and C (both P < 0.01). The accumulation values of (3)H-labeled T of Group B 2, 4, and 6 days after addition were all significantly lower than those of Groups A and C (all P < 0.01). The tumors in 9 of the 10 mice injected with the HT29 cells of Group B disappeared and the tumor in the only one mice in Group B was only less than 1 mm(3), significantly smaller then those in Groups A and C (15 mm(3) on average, all P < 0.01). CONCLUSION: Antisense beta6 gene significantly inhibits the mRNA and protein expression of the beta6 gene, and then inhibits the growth and proliferation of colon cancer cells, thus proving that integrin beta6 plays an important role in the regulation of colon cancer cells.

Integrin αvß6 and matrix metalloproteinase 9 correlate with survival in gastric cancer.

AIM: To investigate the expression of integrin αvß6 and matrix metalloproteinase 9 (MMP-9), their association with prognostic factors and to assess their predictive role in gastric cancer patients. METHODS: Immunohistochemistry was used to determine the expressions of integrin αvß6 and MMP-9 in 126 specimens from patients with primary gastric carcinoma. Associations between immunohistochemical staining and various clinic pathologic variables of tissue specimens were evaluated by the χ(2) test and Fisher's exact test. Expression correlation of αvß6 and MMP-9 was assessed using bivariate correlation analysis. The patients were followed-up every 3 mo in the first two years and at least every 6 mo afterwards, with a median follow-up of 56 mo (ranging from 2 mo to 94 mo). Four different combinations of αvß6 and MMP-9 levels (that is, both markers positive, both markers negative, αvß6 positive with MMP-9 negative, and αvß6 negative with MMP-9 positive) were evaluated for their relative effect on survival. The difference in survival curves was evaluated with a log-rank test. Survival analysis was conducted using the Kaplan-Meier survival and Cox proportional hazards model analysis. RESULTS: The expressions of integrin αvß6 and MMP-9 were investigated in 126 cases, among which 34.92% were positive for αvß6 expression, and 42.06% for MMP-9 expression. The expression of αvß6 was associated with Lauren type, differentiation, N stage, and TNM stage (the P values were 0.006, 0.038, 0.016, and 0.002, respectively). While MMP-9 expression was associated with differentiation, T stage, N stage, and TNM stage (the P values were 0.039, 0.014, 0.033, and 0.008, respectively). The positive correlation between αvß6 and MMP-9 in gastric cancer was confirmed by a correlation analysis. The Kaplan-Meier survival analysis showed that patients with expression of αvß6 or MMP-9 alone died earlier than those with negative expression and that patients who were both αvß6 and MMP-9 positive had a shorter overall survival than those with the opposite pattern (both αvß6 and MMP-9 negative) (P = 0.000). A Cox model indicated that positive expression of αvß6 and MMP-9, diffuse Lauren type, as well as a senior grade of N stage, M stage, and TNM stage were predictors of a poor prognosis in univariate analysis. Only αvß6 and MMP-9 retained their significance when adjustments were made for other known prognostic factors in multivariate analysis (RR = 2.632, P = 0.003 and RR = 1.813, P = 0.007). CONCLUSION: The expression of αvß6 and MMP-9 are closely correlated, and the combinational pattern of αvß6 and MMP-9 can serve as a more effective prognostic index for gastric cancer patients.

Integrin αvß6 sustains and promotes tumor invasive growth in colon cancer progression.

AIM: To detect the mechanism by which colon tumor escapes the growth constraints imposed on normal cells by cell crowding and dense pericellular matrices. METHODS: An immunohistochemical study of integrin αvß6 and matrix metalloproteinase-9 (MMP-9) was performed on tissue microarrays of 200 spots, including 100 cases of colon tumors. RESULTS: High immunoreactivity for αvß6 (73.7%; 28/38) and MMP-9 (76.5%; 52/68) was observed in invasive tumor portions. Furthermore, the effects of integrin αvß6 on tumor invasive growth in nude mice were detected. Tumor invasive growth and high expression of both αvß6 and MMP-9 were only seen in tumors resulting from WiDr cells expressing αvß6 in the tumorigenicity assay. Flow cytometry was applied to analyze αvß6 expression in colon cancer WiDr and SW480 cells. The effects of cell density on αvß6 expression and MMP-9 secretion were also detected by Biotrak MMP-9 activity assay and gelatin zymography assay. High cell density evidently enhanced αvß6 expression and promoted MMP-9 secretion compared with low density. CONCLUSION: Integrin αvß6 sustains and promotes tumor invasive growth in tumor progression via a self-perpetuating mechanism. Integrin ανß6-mediated MMP-9 secretion facilitates pericellular matrix degradation at high cell density, which provides the basis of invasive growth.

Downregulation of the expression of HDGF attenuates malignant biological behaviors of hilar cholangiocarcinoma cells.

Hepatoma-derived growth factor (HDGF) has been reported to be a potential predictive and prognostic marker for several types of cancer and important in malignant biological behaviors. However, its role in human hilar cholangiocarcinoma remains to be elucidated. Our previous study demonstrated that high expression levels of HDGF in hilar cholangiocarcinoma tissues correlates with tumor progression and patient outcome. The present study aimed to elucidate the detailed functions of the HDGF protein. This was performed by downregulating the protein expression of HDGF in the FRH0201 hilar cholangiocarcinoma cell line by RNA interference (RNAi) in vitro, and revealed that downregulation of the HDGF protein significantly inhibited the malignant biological behavior of the FRH0201 cells. In addition, further investigation revealed that downregulation of the protein expression of HDGF significantly decreased the secretion of vascular endothelial growth factor, which may be the mechanism partially responsible for the inhibition of malignant biological behaviors. These findings demonstrated that HDGF is important in promoting malignant biological behaviors, including proliferation, migration and invasion of hilar cholangiocarcinoma FRH0201 cells. Inhibition of the expression of HDGF downregulated the malignant biological behaviors, suggesting that downregulation of the protein expression of HDGF by RNAi may be a novel therapeutic approach to inhibit the progression of hilar cholangiocarcinoma.

Safety validation of decision trees for hepatocellular carcinoma.

AIM: To evaluate a different decision tree for safe liver resection and verify its efficiency. METHODS: A total of 2457 patients underwent hepatic resection between January 2004 and December 2010 at the Chinese PLA General Hospital, and 634 hepatocellular carcinoma (HCC) patients were eligible for the final analyses. Post-hepatectomy liver failure (PHLF) was identified by the association of prothrombin time < 50% and serum bilirubin > 50 µmol/L (the "50-50" criteria), which were assessed at day 5 postoperatively or later. The Swiss-Clavien decision tree, Tokyo University-Makuuchi decision tree, and Chinese consensus decision tree were adopted to divide patients into two groups based on those decision trees in sequence, and the PHLF rates were recorded. RESULTS: The overall mortality and PHLF rate were 0.16% and 3.0%. A total of 19 patients experienced PHLF. The numbers of patients to whom the Swiss-Clavien, Tokyo University-Makuuchi, and Chinese consensus decision trees were applied were 581, 573, and 622, and the PHLF rates were 2.75%, 2.62%, and 2.73%, respectively. Significantly more cases satisfied the Chinese consensus decision tree than the Swiss-Clavien decision tree and Tokyo University-Makuuchi decision tree (P < 0.01，P < 0.01); nevertheless, the latter two shared no difference (P = 0.147). The PHLF rate exhibited no significant difference with respect to the three decision trees. CONCLUSION: The Chinese consensus decision tree expands the indications for hepatic resection for HCC patients and does not increase the PHLF rate compared to the Swiss-Clavien and Tokyo University-Makuuchi decision trees. It would be a safe and effective algorithm for hepatectomy in patients with hepatocellular carcinoma.

Effects of substance P on growth of fibroblast-like cells derived from bile duct: an in vitro cell culture study.

BACKGROUND: The possible role of substance P (SP) during wound healing has been the primary research focus in recent years, but its effect on the healing process after bile duct injury is little understood. This study aimed to investigate the effects of SP on growth of fibroblast-like cells derived from rabbit bile duct. METHODS: Fibroblast-like cells derived from rabbit bile duct were identified and divided randomly into control and experimental groups. SP-treated cells at different concentrations of 10(-9)-10(-5) mol/L and control group were incubated, respectively, for 48 hours. After incubating, the effects of SP on cell proliferation were assessed by cell counts and MTT test. Apoptosis rate (AR) of cells was measured by flow cytometry. RESULTS: Cultured rabbit bile duct cells were fibroblast-like in morphology, and these cells were stained positively for vimentin and negatively for desmin. After SP was added to nonconfluent cells for 48 hours, cell numbers were significantly increased in experimental groups than in controls (P < 0.05). The maximum stimulation of cell proliferation was achieved at SP of 10(-5) mol/L. Bile duct fibroblast-like cells in the SP group showed a higher proliferating activity and lower AR than those in the control group or in the SP + Spantide group (P < 0.05). Spantide partly inhibited the effects of SP on fibroblast-like cells. Examination under transmission electron microscopy revealed rough endoplasmic reticulum and prominent Golgi complexes after SP treatment. CONCLUSIONS: SP has a growth regulatory property on cultivated bile duct fibroblast-like cells in vitro, suggesting that SP may involve in wound healing after bile duct injury by promoting wound fibroblast proliferation and inhibiting apoptosis and participate in pathological scar formation.

Vascular endothelial growth factor (VEGF) enhances gastric carcinoma invasiveness via integrin alpha(v)beta6.

Integrins play an important role in tumor metastasis induced by vascular endothelial growth factor (VEGF). However, in the case of gastric cancer, the precise role of VEGF in regulating integrin alphavbeta6 is unclear. In this study, we found that most of the alphavbeta6 integrin-positive gastric cancer tissues were also VEGF-positive. Furthermore, when gastric carcinoma cells were exposed to VEGF, expression of alphavbeta6 integrin was up-regulated and the extracellular signal-related kinase (ERK) pathway was activated. When integrin alphavbeta6 was blocked either with beta6 siRNA or anti-alphavbeta6 antibody, the migration of tumor cells normally induced by VEGF, as well as the activation of ERK, were markedly inhibited. Blocking the ERK signaling pathway significantly inhibited cell mobility. Taken together, the data suggest that VEGF is critical to the invasive process in human gastric cancer and that this occurs via up-regulation of integrin alphavbeta6 expression and activation of ERK.