Heterodimerization of cholecystokinin 1 and cholecystokinin 2 receptors in gallbladder cancer: a new mechanism for carcinogenesis.

PURPOSE: Cholecystokinin is present in abundance in gallbladder tissue and mediates function through two structurally related receptors, CCK1R and CCK2R. Heterodimerization of these receptors is known to impact cell growth in vitro. However, the significance of these heterodimers in gallbladder carcinogenesis is relatively unknown. METHODS: Therefore, we evaluated the expression and the dimerization status of CCK1 and CCK2 receptors in human gallbladder carcinoma cell line (GBC-SD) and resected gallbladder tissue from normal (n = 10), cholelithiasis (n = 25) and gallbladder cancer (n = 25) by immunofluorescence/immunohistochemistry and western blot. The dimerization status of CCK1R and CCK2R was evaluated by co-immunoprecipitation. To understand the effect of heterodimerization of these receptors on growth-related signaling pathways, the expression of p-AKT, rictor, raptor and p-ERK was evaluated by western blot. RESULTS: We demonstrated the expression and heterodimerization of CCK1 and CCK2 receptor in GBC-SD gall bladder carcinoma cell line. Knockdown of CCK1R and CCK2R in the cell line led to significant reduction in p-AKT (P = 0.005; P = 0.0001) and rictor (P < 0.001; P < 0.001) levels. In tissue samples, significantly higher expression of CCK1R and CCK2R was observed in gallbladder cancer when compared to other groups both by immunohistochemistry (P = 0.008 and P = 0.013) and western blot (P = 0.009 and P = 0.003). An increase in heterodimer formation of CCK1R with CCK2R was observed in gallbladder cancer when compared to normal and cholelithiasis tissues. No significant difference in the expression of p-AKT and p-ERK was observed between the three groups. CONCLUSION: Our results provide the first evidence of heterodimerization of CCK1R and CCK2R in gallbladder tissue, and its association with development of gallbladder cancer. This finding has potential clinical and therapeutic significance.

Expression of Antimicrobial Peptides and Cytokines in Human Omentum Following Abdominal Surgery.

Introduction Omentum can secrete out biological agents like different growth factors, cytokines, and antimicrobial peptides. The aim of our study was to determine the expression of antimicrobial peptides and cytokines in human omentum tissue and its response to intra-abdominal infection. Methodology Omentum tissue was obtained from 60 patients: control (n=20) and cases (n=40). mRNA expression of antimicrobial peptides (LL-37, HBD-1, HBD-2, HNP1-3) and cytokines (TNF- α, IL-8, IL-10, IL1ß) was evaluated using Real-Time PCR. Protein quantification was done by Immunoblotting and ELISA. Results Significantly higher expression of antimicrobial peptides (LL-37, HBD-1, HBD-2, HNP1-3) and cytokines (TNF- α, IL-8, IL-10, IL1ß) was observed in cases as compared to control at both the transcriptional and translational level (p<0.0001). Conclusion Omentum governs a population of antimicrobial peptides with potent immunologic functions. The expression of antimicrobial peptides and cytokines is inducible and increases with the severity of infection. Omentum is thus an immunologically active and adaptable organ but its complete regulatory mechanism is still elusive.

Antibacterial spectrum of human omentum and differential expression of beta defensins.

BACKGROUND: Human ß defensins (hBD1 and hBD2) are cationic, cysteine-rich peptides and form an integral part of the mammalian innate immune system. hBD1 is constitutively expressed in epithelial cells, whereas hBD2 increases in response to bacterial infection. Human omentum is known for its anti-inflammatory properties and also possesses an antibacterial activity of its own. We hypothesized that antimicrobial peptides, ß defensins, may govern host defense mechanism in the microbe-rich environment of the peritoneal cavity. Therefore, we analyzed the expression of hBD1 and hBD2 in omentum tissue in vivo and also studied the antibacterial activity of omentum against common pathogens. METHODOLOGY: Omentum tissues were obtained from 30 patients (15 cases and 15 controls). Real-time polymerase chain reaction (PCR) was used to evaluate the mRNA expression of hBD1 and hBD2. Protein quantification was done using Western blotting technique. Antibacterial susceptibility was performed to check the antibacterial activity of omentum. RESULT: Significantly higher expression of hBD2 was observed in cases compared to controls at both the transcriptional and translational levels. In comparison with an array of antibiotics, activated omentum also showed antibacterial property even at lower concentration of its extract. CONCLUSION: Omentum directly responds to bacterial infection, which may be due to differential expression of hBD1 and hBD2 in human omental tissue. These peptides (hBD1 and hBD2) may be an ideal candidate for novel antibiotic class with a broad-spectrum activity.

Clinicopathological correlation of cancer stem cell markers Oct-4 and CD133 expression as prognostic factor in malignant lesions of gallbladder: An immunohistochemical study.

BACKGROUND: Gallbladder cancer (GBC) is the most frequent biliary tract cancer, with high morbidity and poor prognosis, and shows early metastasis and invasiveness. No reliable biomarkers are available for detection of GBC progression. AIM: To investigate the immunohistochemical expression of Oct-4 and CD133 in malignant and nonneoplastic lesions of gallbladder and to analyze the clinical significance of the expressions related to clinicopathological parameters. SETTINGS AND DESIGN: This is a prospective case control study, conducted in medical college background. MATERIALS AND METHODS: A total of 103 cases of gallbladder were grouped into malignant lesions (n = 48) and nonneoplastic lesions (simple epithelial hyperplasia; n = 35 and chronic cholecystitis; n = 20). All tissue samples were evaluated for expression of Oct-4 and CD133 using immunohistochemistry in an effort to elucidate the correlation between their expressions with clinicopathological parameters. STATISTICAL ANALYSIS: The final score was calculated by multiplying the intensity to the percentage of positive cells. The scores ≥2 were considered as positive. RESULTS: Significant positive correlation of higher expression levels of Oct-4 and CD133 were observed in malignant as compared to nonneoplastic lesions of gallbladder (P < 0.0001). High expression of Oct-4 and CD133 were significantly associated with tumor grading (Oct-4, P = 0.04; CD133, P = 0.02), staging (Oct-4, P = 0.03; CD133, P = 0.02), and liver metastasis (Oct-4, P = 0.01; CD133, P = 0.007). Significantly reduced survival was observed with high expression of Oct-4 (P = 0.002). No significant correction was observed between CD 133 and survival. CONCLUSION: This study revealed that high expression level of Oct-4 may provide a new insight for the prognosis of the disease in terms of clinical staging and grade.

Low Expression of MicroRNA335-5p Is Associated with Malignant Behavior of Gallbladder Cancer: A Clinicopathological Study.

Background: MicroRNAs (miRNAs) are non-coding RNAs that regulate multiple cellular processes during cancer progression, identified to be involved in tumorgenesis of several cancers including cancers of digestive system. However its role in gallbladder inflammatory disease (GID) and gallbladder cancer (GBC) has not been well documented. The present study was aimed to investigate the clinical significance of hsa-miRNA-335-5p (miR-335) in GBC and GID. Subjects and Methods: This prospective case control study, conducted from July 1, 2014 to December 1, 2017 in Era's Lucknow Medical College & Hospital, India, evaluated miR-335 expression by real-time polymerase chain reaction. Hundred tissue samples GID (control; n=50) and GBC (case; n=50) were studied. Relative quantification of target miR-335 expression was examined using the comparative cycle threshold method. Their expression was correlated with different clinicopathological parameters. Fishers' exact test, Student's t-test, and Chi-square test were used as appropriate for data analysis. Kaplan-Meier methods were used to calculate overall and disease-free survival rate. Two sided P<0.05 was considered as significant. Results: miR-335 expression was found to be significantly low in GBC lesions when compared with GID lesions (P<0.001). The low expression level of miR-335 was correlated with histological grade (P=0.007), clinical stage (P<0.001), lymph node metastasis (P<0.001) and liver metastasis (P=0.016). Reduced expression of miRNA-335 was associated with a shorter median overall survival (7 months vs. 25 months) in GBC patients (P<0.001). Conclusions: Down regulation of miR-335 is associated with the severity of the disease and thus indicate that miR-335 expression may serve as prognostic marker for GBC.

A let-7 microRNA binding site polymorphism in the KRAS 3'UTR is associated with increased risk and reduced survival for gallbladder cancer in North Indian population.

PURPOSE: Gallbladder cancer is a lethal malignancy of hepato-biliary system with high incidence in North India, especially along gangetic plain. The let-7 microRNAs play a key role in regulating KRAS expression and a polymorphism in 3' untranslated region (rs61764370, T/G) of KRAS leads to its higher expression. This polymorphism is known to be associated with increased risk and prognosis of various cancers but its association with gallbladder cancer has not been evaluated. To address this research question, we evaluated whether rs61764370 variant is associated with gallbladder cancer susceptibility and clinical outcomes. METHODS: In present case-control study, we enrolled 541 patients with gallbladder malignancy and 307 controls. Genomic DNA was obtained from peripheral blood and genotyping was performed using Taqman allelic discrimination assay. RESULTS: Heterozygous (TG) individuals are at a significant higher risk for GBC as compared with wild genotype (TT) (p = 0.007, odds ratio = 2.56, 95 % CI 1.27-5.18). At allelic level, allele G has significant higher risk for GBC as compared with T allele (p = 0.008, odds ratio = 2.5, 95 % CI 1.25-5.01). Survival analysis reveals decrease in overall survival for heterozygous genotype (p < 0.0001, hazard ratio = 3.42, 95 % CI 1.21-4.20). Also, significant decrease in overall survival was observed for patient carrying allele G (p < 0.0001, HR = 2.89, 95 % CI 1.21-4.20) as compared with allele C. CONCLUSIONS: We conclude that KRAS rs61764370 polymorphism is significantly associated with risk and prognosis of gallbladder malignancy in this endemic belt.

Polymorphism and Expression Profile of Cholecystokinin Type A Receptor in Relation to Gallstone Disease Susceptibility.

In the present study, we investigated expression pattern of Cholecystokinin type A receptor (CCKAR) in relation to its commonly studied polymorphism (rs1800857, T/C) in gallstone disease (GSD) patients and controls. A total of 502 subjects (272 GSD and 230 controls) were enrolled, and genotyping was performed by evaluating restriction fragments of PstI digested DNA. For analyzing expression pattern of CCKAR in relation to polymorphism, gallbladder tissue samples from 80 subjects (GSD-55; control-25) were studied. Expression of CCKAR mRNA was evaluated by reverse transcriptase-PCR and confirmed using real-time PCR. Protein expression was evaluated by enzyme-linked immunosorbent assay. We observed significantly (p < 0.0001) lower expression of CCKAR mRNA and protein in GSD tissues as compared with control. Significantly higher frequency of A1/A1 genotype (C/T transition) (p = 0.0005) was observed for GSD as compared with control. Expression of CCKAR protein was found to be significantly lower (p < 0.0001) in A1/A1 genotype as compared with other genotypes for GSD patients. Perhaps, this is the first report providing evidence of alteration in CCKAR expression in relation to its polymorphism elucidating the molecular pathway of the disease. Additional investigations with lager sample size are needed to confirm these findings.

Expression and clinicopathological significance of antiapoptotis protein survivin in gallbladder cancer.

BACKGROUND: Clinical significance of survivin (antiapoptosis protein) in gallbladder cancer is not yet established. AIMS: This study was performed to assess the expression pattern of survivin in benign and malignant gallbladder lesions using immunohistochemistry (IHC), and to assess its clinicopathological significance. SETTINGS AND DESIGN: Prospective study from July 2012 to July 2014 was performed as a part of intramural research project. MATERIALS AND METHODS: Tissue samples from resected gallbladder for cholelithiasis (n = 27) and carcinoma gallbladder (n= 24) were evaluated for survivin expression by IHC using a scoring system. Their expression was correlated with different clinicopathological parameters. STATISTICAL ANALYSIS: Fisher's exact test, Student's t-test, and Chi-square test were used as appropriate for data analysis. Kaplan-Meier methods were used to calculate overall and disease-free survival rates among different groups. Two-sided P< 0.05 was considered as significant. RESULTS: Benign group (19 females, age [mean ± standard deviation [SD] 45 ± 14 years) and malignant group (20 females, age [mean ± SD] 48.9 ± 13.4 years) were comparable with respect to menopausal status, presence, size and types of stones. However, survivin expression was significantly higher (66.7%, 95% confidence interval [CI] 24-75) in gallbladder cancer than in cholelithiasis group (33%, CI 46-83), P= 0.025). Its expression did not correlate with gender, age, menopausal status, presence of gallstones or their size, number and type, tumor differentiation, and tumor stage. CONCLUSIONS: Significantly higher expression of survivin protein in gallbladder cancer as compared to cholelithiasis group suggests its role in gallbladder carcinogenesis though it may not have prognostic value.

Prognostic significance of survivin in resected gallbladder cancer.

BACKGROUND: Survivin, a novel inhibitor of apoptosis, plays a role in oncogenesis and has been correlated with poor prognosis. We investigated its expression in gallbladder tissues of control, cholelithiasis, and gallbladder cancer (GBC). Survivin expression was correlated with different clinicopathologic parameters including prognosis in patients with GBC. MATERIALS AND METHODS: Gallbladder tissue samples were collected from GBC (n = 39), cholelithiasis (n = 30), and control (n = 25). Expression of survivin messenger RNA (mRNA) was evaluated by real time polymerase chain reaction. Protein quantification was done by enzyme-linked immunosorbent assay. RESULTS: Significantly higher expression of survivin mRNA was observed in GBC (2.9-fold) and cholelithiasis (1.85-fold) as compared with control (P < 0.0001). In GBC, increased survivin expression (mRNA and protein) was significantly associated with higher tumor stage (stage III versus stage II) (P < 0.0001) and poor tumor differentiation (poor and moderate versus well) (P < 0.0001). No significant correlation was observed with any of the other clinicopathologic factors studied. Increased expression of survivin was associated with shorter survival (median survival 11.5 mo versus 18 mo). CONCLUSIONS: Differential expression of survivin in GBC suggests its possible role in gallbladder carcinogenesis. Its overexpression is associated with poor prognosis. Assessment of survivin might be used to stratify GBC patients for optimal treatment modalities, including targeted therapy.

Expression of serum survivin protein in diagnosis and prognosis of gallbladder cancer: a comparative study.

The role of survivin in gallbladder cancer (GBC) has not been evaluated. We investigated survivin protein expression in serum of patients with gallbladder diseases (cholelithiasis, n = 30; GBC, n = 39) and compared with healthy controls (n = 25). Clinicopathological parameters, diagnosis and prognosis of patients with GBC were correlated with the expression of serum survivin by enzyme-linked immunosorbent assay. Significantly higher (P < 0.0001) expression of survivin protein was observed in GBC as compared to cholelithiasis and control. Increased survivin expression was significantly associated with higher tumor stage (stage III vs. stage II; P < 0.0001) and cellular differentiation (poor and moderate vs. well differentiated; P < 0.0001) in GBC. No significant correlation was observed with any of the other clinico-pathological parameters studied. The cutoff value of survivin protein of 79 pg/ml with sensitivity of 81.16 % and specificity of 80 % differentiated the diseased group (cholelithiasis or GBC) from control group were as the cutoff value of 109 pg/ml differentiated GBC from cholelithiasis with a sensitivity of 82.05 % and specificity of 93.33 %. Though not significant, increased expression of survivin was associated with median overall survival (12 vs. 18 months; P = 0.05) in GBC patients. Our study suggests that survivin protein in serum could be both a useful diagnostic marker and an important prognostic factor for GBC.

Differential expression of cholecystokinin A receptor in gallbladder cancer in the young and elderly suggests two subsets of the same disease?

BACKGROUND: Cholecystokinin type A receptor (CCKAR) is known to be overexpressed in variety of human malignancies but information regarding its expression in gallbladder cancer (GBC) is limited. Attempts were now made to investigate expression pattern of CCKAR mRNA and protein in controls and GBC patients and correlate it with various clinicopathological parameters following surgical resection. MATERIALS AND METHODS: Gallbladder tissue samples from 64 subjects (GBC: 39; control: 25) were studied. Expression of CCKAR mRNA was evaluated by reverse transcriptase-polymerase chain reaction and confirmed using real-time polymerase chain reaction. Protein expression was studied by enzyme-linked immunosorbent assay. RESULTS: Significantly higher expression of CCKAR mRNA (P < 0.0001) and protein (P < 0.0001) was observed in GBC tissues. Overexpression was also observed for stage III and in moderately and poorly differentiated tumors. When the clinicopathological parameters were compared, we found age dependent decrease in CCKAR expression. Relatively higher expression of CCKAR was observed in younger patients (age < 45 years) having more aggressive disease when compared with elderly ones (age ≥ 45 years). CONCLUSIONS: Age related differential expression of CCKAR in GBC may suggest two possible variants of the disease in this endemic belt.

Expression of survivin mRNA in gallbladder cancer: a diagnostic and prognostic marker?

Survivin, an inhibitor of apoptosis, has been shown to be expressed in various malignancies. However, its role in gallbladder cancer (GBC) has not been evaluated yet. We investigated its expression in peripheral blood of patients with gallbladder diseases (gallstone disease (GSD), n = 30; GBC, n = 39) and compared with healthy controls (n = 25). Survivin expression was correlated with clinicopathological parameters, diagnosis, and prognosis of patients with GBC. Expression of survivin messenger RNA (mRNA) in blood was evaluated by real-time PCR. Significantly higher (P < 0.0001) expression of survivin mRNA was observed in GBC (2.2-fold) and GSD (1.52-fold) as compared to control. In GBC, increased survivin expression was significantly associated with higher tumor stage (stage III vs. stage II; P < 0.0001) and tumor differentiation (poor and moderate vs. well differentiated; P < 0.0001). No significant correlation was observed with any of the other clinicopathological parameters (age, gender, and presence or absence of gallstones) studied. Cutoff value of survivin mRNA relative quantification (RQ) was 1.08, with a sensitivity of 98.55 % and specificity of 100 % for the diseased group (GSD or GBC). RQ value of 1.71 differentiated GBC from GSD with a sensitivity of 89.74 % and specificity of 100 %. Increased expression of survivin was associated with a shorter median overall survival (12 vs. 18 months) in GBC patients. Differential expression of survivin in GBC suggests its possible role and association with poor prognosis. Expression of survivin in peripheral blood could be useful both in the diagnosis and prognosis of GBC.

Prognostic significance of K-ras codon 12 mutation in patients with resected gallbladder cancer.

BACKGROUND: Point mutation of K-ras is associated with carcinogenesis and overall survival in various cancers. We investigated the mutational spectrum of K-ras codon 12 in resected normal and gallbladder cancer tissue samples in a Northern Indian population and correlated it with different clinicopathological parameters. PATIENTS AND METHODS: Gallbladder tissues from normal (n = 24) and cancer patients (n = 39) were analyzed for K-ras codon 12 mutation by restriction fragment length polymorphism. Statistical analysis was carried out using the χ(2) test or Fisher's exact test. Survival was estimated using the Kaplan-Meier method, and the difference between survival curves was analyzed by the log-rank test. RESULTS: The frequency of K-ras mutation was significantly higher (p = 0.001) in gallbladder cancer tissue samples (16/39) compared to normal samples (1/24). Patients with K-ras mutation had a significantly decreased overall survival (p = 0.003), particularly for stage II (p = 0.021) and III (p = 0.009) cancers. No significant correlation was observed with any of the other clinicopathological factors studied. CONCLUSIONS: Gallbladder cancer has a high frequency of K-ras codon 12 mutation with poorer outcomes in resected stage II and III disease. K-ras mutational analysis has important prognostic implications that need to be investigated further.