Correlation of S1P1 and ERp29 expression to progression, metastasis, and poor prognosis of gallbladder adenocarcinoma.

BACKGROUND: Gallbladder cancer (GBC) is one of the most aggressive malignant neoplasms with an extremely poor prognosis. Early diagnosis significantly increases the survival rate. The present study was undertaken to evaluate the diagnostic and prognostic value of sphingosine-1-phosphate receptor 1 (S1P1) and endoplasmic reticulum protein 29 (ERp29) in benign and malignant gallbladder lesions and to develop a possible alternative treatment for GBC. METHODS: A total of 100 gallbladder adenocarcinoma, 46 peritumoral, 30 gallbladder adenomatous, 15 gallbladder polyp, and 35 chronic cholecystitis tissues were included. S1P1 and ERp29 expressions were evaluated by immunohistochemistry. The correlation between S1P1 and ERp29 expression and tumor pathological features and prognosis was analyzed. RESULTS: S1P1 positive rate was significantly higher in gallbladder adenocarcinomas than that in peritumoral, adenomatous, polyp, and chronic cholecystitis tissues. On the contrary, ERp29 positive rate was significantly lower in adenocarcinomas than that in peritumoral, adenomatous, polyp, and chronic cholecystitis tissues. Benign lesions with positive S1P1 or negative ERp29 expression showed moderate or severe atypical hyperplasia in the gallbladder epithelium. The overexpression of S1P1 or non-expression of ERp29 was significantly associated with tumor differentiation, tumor mass, lymph node metastasis, and adenocarcinoma invasion. Univariate Kaplan-Meier analysis showed that the elevated S1P1 (P=0.008) or absence of ERp29 (P=0.043) was closely associated with decreased survival rate. Multivariate Cox regression analysis showed that S1P1 positive (P=0.004) or ERp29 negative (P=0.029) was an independent predictor of poor prognosis in gallbladder adenocarcinoma. CONCLUSION: S1P1 overexpression or ERp29 absence is related to the carcinogenesis and progression, and may be potential biomarkers for early detection of gallbladder adenocarcinoma.

Restoration of klotho gene expression induces apoptosis and autophagy in gastric cancer cells: tumor suppressive role of klotho in gastric cancer.

BACKGROUND: The loss of tumor suppressor gene expression is involved in the carcinogenesis of gastric cancer (GC). Klotho is a recently identified tumor suppressor gene that epigenetically inactivated in gastric cancer. However, the signaling pathways involved in the suppressive role of klotho have rarely been reported in gastric cancer. In this study, we investigated the involvement of klotho in gastric cancer cell proliferation, apoptosis, and autophagy as well as the associated signaling. METHODS: Methylation of klotho gene promoter in GC-7901, MNK-45 and AGS gastric cancer cells as well as GES-1 normal gastric epithelial cells was detected by bisulfate-based PCR. Restoration of klotho gene expression was established by applying a demethylating agent and delivering aklotho gene expression vector into GC-7901 cells. Cell viability was measured by CCK-8 assay. Cell apoptosis and cycling were analyzed by flow cytometry. Autophagy was measured by detecting LC3-I and LC3-II expression. Protein levels and phosphorylation were measured by Western blot assay. RESULTS: Methylation of klotho gene promoter and expression of the klotho gene were detected in GC cells. Restoration of klotho gene expression significantly inhibited cell proliferation, induced cell apoptosis, and increased LC3-I/LC3-II expression in GC cells. Restoration of klotho gene expression downregulated the phosphorylation levels of IGF-1 receptor, IRS-1, PI3K, Akt, and mTOR proteins. Both apoptosis and autophagy inhibitors blocked klotho-induced apoptosis and autophagy. CONCLUSION: Klotho is a tumor suppressor in gastric cancer, which regulates IGF-1R phosphorylation and the subsequent activation of IRS-1/PI3K/Akt/mTOR signaling, tumor cell proliferation, apoptosis, and autophagy.

MTDH and EphA7 are markers for metastasis and poor prognosis of gallbladder adenocarcinoma.

Gallbladder cancer is an aggressive cancer with extremely poor prognosis. Over 90% of patients are diagnosed at an advanced, inoperable stage with metastasis and invasion to other organs. In this study, the expression of metadherin (MTDH) and erythropoietin-producing hepatoma-amplified sequence (Eph) receptor A7 (EphA7) in 96 benign and 108 malignant lesions of gallbladder was determined by immunohistochemistry, and their correlations with pathological features and prognosis were analyzed. Positive expression of EphA7 and MTDH was significantly higher in gallbladder adenocarcinoma than in benign lesions. In adenocarcinoma, the positive expression of EphA7 and MTDH was significantly associated with differentiation, tumor mass, lymphnode metastasis, invasion, and overall survival. Multivariate Cox regression analysis suggested that positive expression of EphA7 and MTDH was an independent poor-prognostic predictor in gallbladder adenocarcinoma. The elevated expression of EphA7 and/or MTDH is closely related to carcinogenesis, progression, clinical biological behaviors, and prognosis of gallbladder adenocarcinoma.

Epigenetic silencing of Klotho expression correlates with poor prognosis of human hepatocellular carcinoma.

Klotho is identified as a tumor suppressor in several tumors, but the expression of the Klotho gene (KL) and its regulative mechanism are not reported in hepatocellular carcinoma (HCC). The messenger RNA and protein levels of Klotho were measured in 64 HCC tumor tissues by real-time polymerase chain reaction and immunohistochemistry, respectively. The methylation of KL promoter DNA was examined by bisulfite-based polymerase chain reaction. The correlation of Klotho protein expression and methylation with survival of HCC was analyzed using Kaplan-Meier analysis. The interference of KL gene expression was conducted in HCC cells by DNA demethylating agent and/or histone deacetylase inhibitor. In HCC tissues, a significant loss of Klotho messenger RNA and protein expression was observed, which parallels the increased methylation in KL promoter DNA. Both Klotho expression and methylation correlated with the poor prognosis of HCC. Experiments with HCC cell lines showed that a combination of DNA demethylating agent and histone deacetylase inhibitor fully recovered Klotho expression and subsequently induced cell apoptosis. In conclusion, Klotho is a tumor suppressor in HCC. Both hypermethylation and acetylation are involved in the loss of Klotho expression in HCC cells. Both KL gene expression and its promoter DNA methylation are predictive factors for the poor prognosis of HCC. Our study also suggests that the Klotho gene could be a target for therapy of HCC.

Restoration of klotho expression induces apoptosis and autophagy in hepatocellular carcinoma cells.

PURPOSE: Klotho has been identified as a tumor suppressor in several human malignancies including hepatocellular carcinoma (HCC). However, the signaling pathways involved in the tumor suppressive role of klotho in HCC have not been reported. Here, we investigated the role of klotho in HCC cell proliferation, apoptosis, autophagy, and invasion, as well as its associated signal transduction pathways. METHODS: Restoration of klotho gene expression was established by delivering a klotho gene expression vector into the human HCC cell lines HepG2 and MHCC-97-H. Cell viability was measured using a cell counting (CCK-8) assay and apoptosis was analyzed through flow cytometry. Autophagy was measured via LC3-I and LC3-II protein expression levels and tumor cell invasion was assessed using a Matrigel invasion chamber assay. Expression and phosphorylation of several apoptosis and survival related proteins were assessed using Western blot assays. RESULTS: Exogenous klotho gene expression significantly inhibited HCC cell proliferation, induced HCC cell apoptosis, increased LC3-I and LC3-II protein expression in HCC cells, and decreased migration of HCC cells in a Matrigel invasion chamber assay. Exogenous klotho gene expression also down-regulated the phosphorylation levels of the IGF-1 receptor, and the downstream Akt, ERK, and p70S6K proteins. Both apoptosis and autophagy inhibitors decreased klotho-induced apoptosis and autophagy. CONCLUSION: Klotho is a tumor suppressor that, through the regulation of IGF-1R phosphorylation and subsequent activation of downstream Akt-p70S6K and ERK signaling, regulates HCC tumor cell proliferation, apoptosis, autophagy and invasion.

Immunohistochemical study of Dicer and Drosha expression in the benign and malignant lesions of gallbladder and their clinicopathological significances.

Dicer and Drosha are two key enzymes that are involved in the processing of miRNA production. Their expressions in gallbladder cancer have not been investigated. In this study, we studied Dicer and Drosha expression in 21 non-dysplastic gallbladder epithelia and 108 gallbladder adenocarcinomas by immunohistochemical staining. The clinicopathological significance of Dicer and Drosha expressions was analyzed. We demonstrated that the percent of positive Dicer or Drosha expression was significantly lower in gallbladder adenocarcinoma than that in non-dysplastic gallbladder epithelia (p<0.01). The percent of positive Dicer and Drosha expression was significantly lower in poorly differentiated adenocarcinoma with lymph node metastasis, invasiveness, and no resection than in well-differentiated adenocarcinoma without metastasis, invasiveness, and with radical resection (p<0.05 or p<0.01). Univariate Kaplan-Meier analysis showed that loss of Dicer and Drosha expression was associated with decreased overall survival (p<0.05 or p<0.01). Multivariate Cox regression analysis showed that loss of Dicer and Drosha expression was an independent poor-prognostic predictor in patients with gallbladder adenocarcinoma. In conclusion, loss of Dicer and Drosha expression is closely related to the metastasis, invasion, and poor-prognosis in gallbladder adenocarcinoma.

Overexpression of EZH2 and loss of expression of PTEN is associated with invasion, metastasis, and poor progression of gallbladder adenocarcinoma.

Overexpression of EZH2 and inactivation or loss of PTEN expression was observed in invasive and metastatic tumors. However, their expressions and clinical significances in gallbladder cancer (GBC) have rarely been reported. In this study, we investigated EZH2 and PTEN expression in an extensive collection of human gallbladder cancer samples and benign lesions of gallbladder using immunohistochemistry. Overexpression of EZH2 was detected in 53.7% of gallbladder adenocarcinomas associated with poor differentiation, lymph node metastasis, and invasion, while loss of PTEN expression was identified in 51.8% of adenocarcinomas with high grade, metastatic, and invasive tumors. Univariate Kaplan-Meier analysis showed that overexpression of EZH2 (p=0.013) and loss of PTEN expression (p=0.008) were significantly associated with decreased overall survival. Multivariate Cox regression analysis revealed that overexpression of EZH2 (p=0.011) or loss of PTEN expression (p=0.009) is a predictor of poor prognosis in gallbladder adenocarcinoma. Our study suggests that overexpression of EZH2 and loss of PTEN expression might be closely related to the carcinogenesis, progression, clinical biological behavior, and prognosis of gallbladder adenocarcinoma.

[Expression of ephrin-A7 and metadherin and its clinicopathological significances in the benign and malignant lesions of gallbladder].

OBJECTIVE: To study the expression of ephrin-A7 (EphA7) and metadherin (MTDH) and their clinicopathological significances in the benign and malignant lesions of gallbladder. METHODS: EnVisiom immunohistochemical methods was used for determining the expressions of EphA7 and MTDH in routinely paraffin-embedded sections of surgically-resected specimens from 108 cases with gallbladder adenocarcinoma, 15 cases with adenomatous polyp and 35 cases with chronic cholecystitis treated from June 1996 to June 2006. And 46 cases of peritumoral tissues were also harvested as controls (n = 35). RESULTS: The positive expression rates of EphA7 and MTDH were significantly higher in gallbladder adenocarcinoma than those in peritumoral tissues (χ(2)(EphA7) = 12.65, χ(2)(MTDH) = 13.00; P < 0.01), adenomatous polyp (χ(2)(EphA7) = 8.21, χ(2)(MTDH) = 9.39; P < 0.01) and chronic cholecystitis (χ(2)(EphA7) = 21.21, χ(2)(MTDH) = 23.68; P < 0.01); Moderately-or severely-atypical hyperplasia of gallbladder epithelium was found in the benign lesions with positive expression of EphA7 and/or MTDH. The positive rates of EphA7 and MTDH were significantly lower in the cases of well-differentiated adenocarcinoma, maximal diameter of tumor < 2 cm, no-metastasis of lymph node, and tumor with no-invasiveness of regional tissues than those in the poorly-differentiated adenocarcinoma (χ(2)(EphA7) = 12.34, χ(2)(MTDH) = 12.80; P < 0.01), maximal diameter of tumor ≥ 2 cm (χ(2)(EphA7) = 5.22, χ(2)(MTDH) = 5.00; P < 0.05), cases with metastasis of lymph node (χ(2)(EphA7) = 5.15, χ(2)(MTDH) = 5.86; P < 0.05) and cases with invasiveness of regional tissues (χ(2)(EphA7) = 7.06, P < 0.01; χ(2)(MTDH) = 4.13; P < 0.05) in gallbladder adenocarcinoma (P < 0.05). The high consistency was found between the expressive levels of EphA7 and MTDH in gallbladder adenocarcinoma (χ(2) = 13.11, P < 0.01). The univariate Kaplan-Meier analysis showed that the increased expression of EphA7 (P = 0.023) and MTDH (P = 0.034) was negatively associated with the overall survival. The multivariate Cox regression analysis showed that increased expression of EphA7 and/or MTDH (P(EphA2) = 0.023, P(MTDH) = 0.034) was an independent poor-prognostic predictor for gallbladder adenocarcinoma. CONCLUSIONS: The expression of EphA7 and/or MTDH might be closely related to the carcinogenesis, progression, clinical biological behaviors and prognosis of gallbladder adenocarcinoma. The positive expression of EphA7 and/or MTDH may predict bad-prognosis in gallbladder adenocarcinoma.

Clinicopathologic significance of minichromosome maintenance protein 2 and Tat-interacting protein 30 expression in benign and malignant lesions of the gallbladder.

Gallbladder cancers are aggressive tumors with a poor prognosis and high mortality rate. To find specific biological markers for early diagnosis and prognosis and to develop possible alternative treatment strategies, we examined minichromosome maintenance protein 2 (MCM2) and Tat-interacting protein 30 (TIP30) expression in 108 gallbladder adenocarcinomas, 15 gallbladder polyps, 35 chronic cholecystitis tissues, and 46 peritumoral tissues using immunohistochemistry. Expression of MCM2 was significantly higher in adenocarcinomas than in peritumoral tissues (χ² = 8.41; P < .01), adenomatous polyps (χ² = 6.81; P < .01), and chronic cholecystitis (χ² = 21.00; P < .01). In contrast, Tat-interacting protein 30 expression was significantly less in adenocarcinomas than in peritumoral tissues (χ² = 13.26; P < .01), adenomatous polyps (χ² = 4.76; P < .05), and chronic cholecystitis (χ² = 18.93; P < .01). The benign lesions in gallbladder epithelium with positive MCM2 or negative Tat-interacting protein 30 expression showed moderate to severe atypical hyperplasia. Expression of MCM2 and absence of Tat-interacting protein 30 were significantly associated with poor differentiation, large tumor mass, lymph node metastasis, and invasion of adenocarcinoma. Univariate Kaplan-Meier analysis showed that either elevated MCM2 (P = .006) or lowered Tat-interacting protein 30 (P = .006) expression was closely associated with shorter overall survival. Multivariate Cox regression analysis revealed that expression of MCM2 (P = .007) or nonexpression of Tat-interacting protein 30 (P = .009) was an independent predictor of a poor prognosis in adenocarcinoma. Our results suggest that overexpression of MCM2 or loss of expression of Tat-interacting protein 30 is closely related to carcinogenesis, progression, biological behavior, and prognosis of gallbladder adenocarcinoma.

Identification of PEG10 and TSG101 as carcinogenesis, progression, and poor-prognosis related biomarkers for gallbladder adenocarcinoma.

PEG10 is a transcriptional factor while TSG101 is involved in numerous cellular processes, including apoptotic resistance. Overexpression of PEG10 and TSG101 were observed in a variety of human cancers. However, their expression and clinical significance in gallbladder cancer (GBC) have not yet been identified. To understand the tumor biology of GBC at the molecular level, we examined PEG10 and TSG101 expression in 108 adenocarcinomas, 15 gallbladder polyps, 35 chronic cholecystitis tissues, and 46 peritumoral tissues by using immunohistochemistry. Overexpression of PEG10 and TSG101 was detected in gallbladder adenocarcinoma (48.1% and 47.2%, respectively). Conversely, there was less expression detected in the peritumoral tissues (19.6%), adenomatous polyps (13.3%), and gallbladder epithelium with chronic cholecystitis (5.1%) (p < 0.01, p < 0.05, and p < 0.01, respectively). Notably, the benign lesions with positive PEG10 and/or TSG101 expression showed moderately or severely atypical hyperplasia in gallbladder epithelium. The overexpression of PEG10 and TSG101 was significantly associated with differentiation, tumor mass, lymph node metastasis and invasion of adenocarcinoma. Univariate Kaplan-Meier analysis showed that overexpression of PEG10 (p = 0.041) and TSG101 (p = 0.025) was closely associated with decreased overall survival. Multivariate Cox regression analysis revealed that positive expression of PEG10 (p = 0.036) or TSG101 (p = 0.022) is a predictor of poor prognosis in gallbladder adenocarcinoma. Our study suggested that overexpression of PEG10 and TSG101 might be closely related to the carcinogenesis, progression, clinical biological behaviors, and prognosis of gallbladder adenocarcinoma.

Identification of musashi-1 and ALDH1 as carcinogenesis, progression, and poor-prognosis related biomarkers for gallbladder adenocarcinoma.

In this study, we investigated the expressions of Msi-1 and ALDH1 in gallbladder adenocarcinoma (n=100), peritumoral tissues (n=46), adenomatous polyp (n=15), and chronic cholecystitis (n=35) using immunohistochemical method. The percentage of cases with positive Msi-1 and ALDH1 expression were significantly higher in gallbladder adenocarcinoma than that in peritumoral tissues, adenomatous polyp and chronic cholecystitis (ps < 0.01). The expression of Msi-1 and ALDH1 was significantly associated with differentiation, tumor mass, lymph node metastasis and invasion of adenocarcinoma. The expression of Msi-1 and ALDH1 was found to be highly consistent in gallbladder adenocarcinoma (p < 0.01). Univariate Kaplan-Meier analysis showed a negative correlation between Msi-1 (p=0.042) or ALDH1 (p< 0.001) expression with overall survival. The average survival time of patients with both low or no Msi-1 expression and ALDH1 expression was significantly longer than patients with the other three subtypes (p< 0.001). Multivariate Cox regression analysis showed that positive expression of Msi-1 or ALDH1 (p=0.016, p=0.006, respectively) was an independent bad-prognostic predictor in gallbladder adenocarcinoma. Our study suggested that Msi-1 and/or ALDH1 expression might be closely related to the carcinogenesis, progression, clinical biological behaviors, and prognosis of gallbladder adenocarcinoma.

[Expression of galectin 3 and Sambucus nigra agglutinin and their clinicopathological significance in benign and malignant lesions of stomach].

OBJECTIVE: To study the expressive levels of galectin-3(gal-3) and Sambucus nigra agglutinin(SNA) and their clinicopathological significance in the benign and malignant lesions of stomach. METHODS: EnVision immunohistochemistry for assaying gal-3 expressive level and ABC cytochemistry for determining SNA expressive level were used in conventional paraffin-embedded sections from specimens of gastric cancer(n=49), peritumoral tissues(n=20), metastatic foci of lymph nodes(n=36), and different types of benign lesions(n=80). RESULTS: The positive rates of gal-3 and SNA were significantly higher in gastric cancer tissues than those in peritumoral tissues and different types of benign lesions(P<0.05, P<0.01). The positive cases of gal-3 and/or SNA in peritumoral tissues and benign lesions showed mild- to severe-atypical hyperplasia of mucous epithelial cells. No difference was found between the primary foci and metastatic foci in gal-3 and SNA expressions(P>0.05). The positive rates of gal-3 and SNA were significantly lower in histologic grade II(, infiltrating depth T1,T2 and no-metastasis of regional lymph node than those in histologic grade III(,IIII(, infiltrating depth T3,T4 and metastasis of lymph node in gastric cancer(P<0.05). The positive rates of gal-3 and SNA were higher in lymphnode metastatic site N1 and no-metastasis of distant organs than those in lymphnode metastatic site N2,N3 and metastasis of distant organs, but no significant difference was found(P>0.05). The consistency was found between the expression of gal-3 and SNA in gastric cancer tissues(chi(2)=6.59,P<0.05). CONCLUSIONS: The expressive levels of gal-3 and SNA may be important molecular markers of lectins for reflecting the carcinogenesis, progression and biological behaviors in gastric cancer.

[Expression of enhancer of zesle homolog 2 and phosphatase and tension homolog and its clinicopathological significance in benign and malignant lesion of gallbladder].

OBJECTIVE: To examine the expressive level of enhancer of zesle homolog 2 (EZH2) and phosphatase and tension homolog (PTEN), and to explore its clinicopathological significance in benign and malignant lesion of gallbladder. METHODS: EnVision immunohistochemical method was used to detect the expressive levels of EZH2 and PTEN in routinely paraffin-embedded sections in the resected specimens of gallbladder adenocarcinoma (n = 108), peritumoral tissues (n = 46), adenomatous polyp(n = 15), and chronic cholecystitis (n = 35). RESULTS: The positive rate of EZH2 was significantly higher in gallbladder adenocarcinoma than that in peritumoral tissues (chi(2) = 24.49, P < 0.01), adenomatous polyp(chi(2) = 11.68, P < 0.01), and chronic cholecystitis (chi(2) = 31.62, P < 0.01). The benign lesions in the positive cases of EZH2 and (or) the negative ones of PTEN showed the moderately- or severely- atypical hyperplasia of gallbladder epithelium. The positive rate of PTEN was significantly lower in gallbladder adenocarcinoma than that in peritumoral tissues(n = 20.20, P < 0.01), adenomatous polyp(chi(2)=10.81, P<0.01), and chronic cholecystitis (n = 29.83, P < 0.01).The positive rates of EZH2 were significantly lower in the highly-differentiated adenocarcinoma, the maximal diameter of mass < 2 cm, non-metastasis of lymphnodes, and non-infiltration of regional tissues than those in the moderately or low-differentiated adenocarcinoma, the maximal diameter > or = 2 cm, metastasis of lymphnode, and infiltration of regional tissues (P < 0.05 or P < 0.01). High inconsistency was found between the expression of EZH2 and PTEN in gallbladder adenocarcinoma (P < 0.05). CONCLUSION: Expression of EZH2 and/or PTEN might be important biological markers in the carcinogenesis, progression, biological behaviors and prognosis of gallbladder adenocarcinoma.