Selective cyclooxygenase-2 silencing mediated by engineered E. coli and RNA interference induces anti-tumour effects in human colon cancer cells.

BACKGROUND: Cyclooxygenase-2 (COX-2) overexpression is strongly associated with colorectal tumourigenesis. It has been demonstrated that the chronic use of non-steroidal anti-inflammatory drugs (COX inhibitors) partially protects patients from colorectal cancer (CRC) development and progression but induces severe cardiovascular side effects. New strategies for selective COX-2 blockade are required. METHODS: We developed an improved technique, based on RNA interference (RNAi), to gain a selective COX-2 silencing in CRC cells by a tumour-dependent expression of anti-COX-2 short-hairpin RNA (shCOX-2). Anti-COX-2 shRNA-expressing vectors were delivered in CRC cells (in vitro) and in colon tissues (ex vivo) using engineered Escherichia coli strains, capable of invading tumour cells (InvColi). RESULTS: A highly tumour-dependent shCOX-2 expression and a significant COX-2 silencing were observed in CRC cells following InvColi strain infection. Cyclooxygenase-2 silencing was associated with a strong reduction in both proliferative and invasive behaviour of tumour cells. We also demonstrated a pivotal role of COX-2 overexpression for the survival of CRC cells after bacterial infection. Moreover, COX-2 silencing was achieved ex vivo by infecting colon tissue samples with InvColi strains, leading to anti-inflammatory and anti-tumour effects. CONCLUSION: Our RNAi/InvColi-mediated approach offers a promising tool for a highly selective COX-2 blockade in vitro and in vivo.

Distinct proteomic profiles characterise non-erosive from erosive reflux disease.

BACKGROUND: Erosive reflux disease (ERD) and non-erosive reflux disease (NERD) are often regarded as part of the spectrum of the same disease. AIM: To elucidate molecular features that characterise NERD and ERD at the protein level. METHODS: A total of 56 consecutive subjects were enrolled: 10 healthy subjects, 24 with NERD and 22 with ERD. Eight specimens were taken from macroscopically normal mucosa at 5 cm of gastro-oesophageal junction. Four were processed for the proteins extraction and four for evaluation using haematoxylin-eosin and immunohistochemistry. We used shotgun proteomics to identify tentative disease molecular features for ERD or NERD. Candidate distinctive proteins were verified using immunohistochemistry. RESULTS: Shotgun proteomics analysis revealed 33 differentially expressed proteins in NERD vs. ERD samples, involved in cellular proliferation, keratinisation, stress responses and sugar metabolism. Based on a gene ontology meta-analysis, seven of them were further analysed using Western blotting (WB) and four also using immunohistochemistry. We identified novel candidate disease molecular features for GERD and few distinctive proteins to discriminate NERD and ERD. In particular, Transitional Endoplasmic Reticulum ATPase (TER ATPase), GAPDH, Alpha 1 Acid Glycoprotein 1, Annexin A1, Calmodulin and 14-3-3 proteins were confirmed at WB analysis. CONCLUSIONS: Non-erosive reflux disease and ERD are distinct disease entities at the protein level. This study proposes an array of candidate biomarkers possibly useful to discriminate between NERD and ERD.

Potential role of the cannabinoid receptor CB in the pathogenesis of erosive and non-erosive gastro-oesophageal reflux disease.

BACKGROUND: Cannabinoid (CB) receptors have been located in brain areas involved in the triggering of TLESRs as well as in the nodose ganglion from which vagal afferents emanate. The distribution of CB(1) receptors has been investigated in the human gastrointestinal mucosa, as expression of inflammatory process. AIM: To evaluate the CB(1) expression in oesophageal mucosa. METHODS: A total of 87 consecutive subjects were enrolled: 10 controls, 39 NERD and 38 erosive oesophagitis. Eight specimens were taken from macroscopically normal mucosa. Five were processed by haematoxylin-eosin, MIB1/CB(1) evaluation and three for the RNA and proteins extraction. RESULTS: The mean MIB1-LI value was 31% and 22% in NERD and ERD patients, respectively, compared to 68% in the healthy subjects. Mean CB(1)mRNA/GUSB mRNA value of the controls was 0.66, while in GERD patients, it was 0.28. In NERD and ERD, the mean values of CB(1)/GUSB were 0.38 and 0.17, respectively, with highly significant differences between the NERD vs. ERD groups. Semi-quantitative analysis of CB(1) expression, performed with WB, shows in NERD patients a higher CB(1) receptor expression than ERD patients. CONCLUSIONS: With this study, we showed for the first time the presence of CB(1) receptors in the human oesophageal epithelium.