Maor Lahav

Mutant KRAS is a druggable target for pancreatic cancer

Significance Pancreatic cancer is still one of the major challenges in clinical oncology. Mutant KRAS is a driving oncogene in the majority of human pancreatic cancer cases. We have made an effort to meet this challenge by developing a therapeutic platform for local and prolonged delivery of siRNA. Our results show that the siRNA targeted against KRAS mutations with a local prolonged release system knocks down KRAS expression in vitro and in vivo, leading to an antitumor effect. Our report describes an applicable and efficient delivery method of siRNA that overcomes the major obstacles of toxicity and organ accessibility. Notably our approach enabled the conversion of KRAS from a nondruggable to a potentially druggable cancer target. Pancreatic ductal adenocarcinoma (PDA) represents an unmet therapeutic challenge. PDA is addicted to the activity of the mutated KRAS oncogene which is considered so far an undruggable therapeutic target. We propose an approach to target KRAS effectively in patients using RNA interference. To meet this challenge, we have developed a local prolonged siRNA delivery system (Local Drug EluteR, LODER) shedding siRNA against the mutated KRAS (siG12D LODER). The siG12D LODER was assessed for its structural, release, and delivery properties in vitro and in vivo. The effect of the siG12D LODER on tumor growth was assessed in s.c. and orthotopic mouse models. KRAS silencing effect was further assessed on the KRAS downstream signaling pathway. The LODER-encapsulated siRNA was stable and active in vivo for 155 d. Treatment of PDA cells with siG12D LODER resulted in a significant decrease in KRAS levels, leading to inhibition of proliferation and epithelial–mesenchymal transition. In vivo, siG12D LODER impeded the growth of human pancreatic tumor cells and prolonged mouse survival. We report a reproducible and safe delivery platform based on a miniature biodegradable polymeric matrix, for the controlled and prolonged delivery of siRNA. This technology provides the following advantages: (i) siRNA is protected from degradation; (ii) the siRNA is slowly released locally within the tumor for prolonged periods; and (iii) the siG12D LODER elicits a therapeutic effect, thereby demonstrating that mutated KRAS is indeed a druggable target.

Somatostatin Through Its Specific Receptor Inhibits Spontaneous and TNF-α- and Bacteria-Induced IL-8 and IL-1β Secretion from Intestinal Epithelial Cells

Intestinal epithelial cells secrete proinflammatory cytokines and chemokines that are crucial in mucosal defense. However, this secretion must be tightly regulated, because uncontrolled secretion of proinflammatory mediators may lead to chronic inflammation and mucosal damage. The aim of this study was to determine whether somatostatin, secreted within the intestinal mucosa, regulates secretion of cytokines from intestinal epithelial cells. The spontaneous as well as TNF-α- and Salmonella-induced secretion of IL-8 and IL-1β derived from intestinal cell lines Caco-2 and HT-29 was measured after treatment with somatostatin or its synthetic analogue, octreotide. Somatostatin, at physiological nanomolar concentrations, markedly inhibited the spontaneous and TNF-α-induced secretion of IL-8 and IL-1β. This inhibition was dose dependent, reaching >90% blockage at 3 nM. Furthermore, somatostatin completely abrogated the increased secretion of IL-8 and IL-1β after invasion by Salmonella. Octreotide, which mainly stimulates somatostatin receptor subtypes 2 and 5, affected the secretion of IL-8 and IL-1β similarly, and the somatostatin antagonist cyclo-somatostatin completely blocked the somatostatin- and octreotide-induced inhibitory effects. This inhibition was correlated to a reduction of the mRNA concentrations of IL-8 and IL-1β. No effect was noted regarding cell viability. These results indicate that somatostatin, by directly interacting with its specific receptors that are expressed on intestinal epithelial cells, down-regulates proinflammatory mediator secretion by a mechanism involving the regulation of transcription. These findings suggest that somatostatin plays an active role in regulating the mucosal inflammatory response of intestinal epithelial cells after physiological and pathophysiological stimulations such as bacterial invasion.

Lidocaine inhibits secretion of IL-8 and IL-1β and stimulates secretion of IL-1 receptor antagonist by epithelial cells

Lidocaine and related local anaesthetics have been shown to be effective in the treatment of ulcerative colitis (UC). However, the mechanisms underlying their therapeutic effect are poorly defined. Intestinal epithelial cells play an important role in the mucosal inflammatory response that leads to tissue damage in UC via the secretion of pro‐inflammatory cytokines and chemokines. The aim of this study was to evaluate the direct immunoregulatory effect of lidocaine on pro‐inflammatory cytokine and chemokine secretion from intestinal epithelial cells. HT‐29 and Caco‐2 cell lines were used as a model system and treated with lidocaine and related drugs. The expression of IL‐8, IL‐1β and the IL‐1 receptor antagonist (RA) were assessed by ELISA and quantification of mRNA. In further experiments, the effect of lidocaine on the secretion of IL‐8 from freshly isolated epithelial cells stimulated with TNFα was tested. Lidocaine, in therapeutic concentrations, inhibited the spontaneous and TNFα‐stimulated secretion of IL‐8 and IL‐1β from HT‐29 and Caco‐2 cell lines in a dose‐dependent manner. Similarly, suppression of IL‐8 secretion was noted in the freshly isolated epithelial cells. Other local anaesthetics, bupivacaine and amethocaine, had comparable effects. Lidocaine stimulated the secretion of the anti‐inflammatory molecule IL‐1 RA. Both the inhibitory and the stimulatory effects of lidocaine involved regulation of transcription. The results imply that the therapeutic effect of lidocaine may be mediated, at least in part, by its direct effects on epithelial cells to inhibit the secretion of proinflammatory molecules on one hand while triggering the secretion of anti‐inflammatory mediators on the other.

The utility of capsule endoscopy in the diagnosis of Crohn's disease based on patient's symptoms.

Background and Goals Video capsule endoscopy (VCE) enables visualization of the entire small bowel and can identify lesions that may go undetected by conventional endoscopy and radiography. In this study, we assessed whether patients selection based on symptoms may increase the yield of VCE in the diagnosis of Crohns disease (CD). Study Findings of 125 consecutive patients referred for VCE in whom CD may be suspected, were analyzed. Indications for VCE included iron-deficiency anemia, abdominal pain, diarrhea, or a combination of symptoms. Capsule endoscopy (CE) results were defined positive if 4 or more obvious clear ulcers, erosions, or a region with clear exudate and mucosal hyperemia and edema were identified. Results One hundred twelve patients were included in the final analysis. Mean age of patients was 44±22 years and median follow-up 36±15 months. Findings on CE were considered compatible with a diagnosis of CD in 7 patients (6%). In general, CE yielded a diagnosis of CD in a very small portion of the patients (0% to 4%), except in patients undergoing the test for a combination of abdominal pain and diarrhea. In this group, findings suggestive of inflammatory bowel disease were encountered in one-third of the patients (P=0.002). Conclusions The greatest yield of CE in diagnosing CD is achieved in young patients who present with symptoms of abdominal pain plus diarrhea.

Clinical and demographic characterization of Jewish Crohn's disease patients in Israel.

Background Crohns disease (CD) is a chronic transmural inflammatory disease of unknown etiology. Genetic background may influence the nature of disease. The Jewish population in Israel is composed of Ashkenazi and Sephardic Jews, who differ in their genetic background. Previous studies have reported that the prevalence of CD in Sephardic Jews is lower than in Ashkenazi Jews; however, no information is available regarding disease characteristics in these patient populations. Goals To assess the demographic and clinical characteristics of CD in Israeli Jewish patients. Study We studied 189 CD patients who were observed in our department. Demographic and clinical data were collected and analyzed. Results Forty-eight percent of the patients had ileum-only disease. Fistulizing disease was more frequently seen in ileocolonic than in ileal (p = 0.04) or colonic (p = 0.01) disease, whereas strictures occurred more often in ileal than in colonic disease (22% and 3%, respectively, p = 0.007). No association was found between current smoking and disease severity. Sephardic patients more frequently had extraintestinal disease than did Ashkenazi patients (35% vs. 17%, respectively, p = 0.019). Conclusion Jewish Israeli patients show a predilection for ileum-only disease. Disease severity appears not to be influenced by smoking. These differences in disease behavior may be related to genetic factors.

Lidocaine inhibits secretion of pro-inflammatory mediators from intestinal epithelial cells

Background: Lidocaine has been shown to b e effective in the treatment of ulcerative colitis (UC). It was suggested that lidocaine regulated the function of immune cells indirectly, by mediating the secretion of immunologically active neuropeptides. Secretion of pro-inflammatory mediators such a s IL-8 and IL-I J3 by intestinal epithelial cells was shown to be important in the inflammatory process of UC. However, no information is available regarding the possible direct effect of lidocaine on chemokine and cytokine secretion from intestinal epithelial cells. Study aims: To assess the direct effect of lidocaine on the secretion of pro-inflammatory mediators from intestinal epithelial cells. Methods: HT-29 epithelial cells were incubated with escalating lidocaine concentrations. Spontaneous and TNFa -stimulated secretion of IL-8 and IL-I J3 were determined using ELISA. Expression of mRNA levels was assessed by an RNA protection assay and quantitated using a phospho-imager. Results : Lidocaine inhibited the secretion of IL-8 and IL-I J3 from intestinal epithelial cells in a dosedependent manner. Secretion of spontaneous IL-8 was reduced from 22.4 ng/ml to 5.45 ng/ml using a concentration of 10 JL g/ml lidocaine. No further inhibition was shown using lidocaine concentrations up to 100 JL g/ml. Similarly, IL-If3 levels were reduced from 0.792 ng/ml to 0.179 ng/ml at a similar dose range. Inhibition of IL-8 and IL-If3 secretion occurred at therapeutic levels. TNFa stimulation resulted in a significant increase of IL-8 secretion from the cells. Using Lidocaine at a concentration of 10 JL g/ml, IL-8levels were reduced from 67.5 ng/ml to 13.7 ng/ml. IL-If3levels were reduced from 1.446 ng/ml to 0.285 ng/ml. Quantitation of IL-8 mRNA by an RNA protection assay, using TNFa -stimulated cells and cells in which lidocaine was added prior to the addition of TNFa, demonstrated a marked reduction in the levels of IL-8 mRNA in the lidocaine -treated cells . Conclusions : Lidocaine directly inhibits proinflammatory mediator secretion from intestinal epithelial cells. Suppression occurs at doses within the therapeutic range. Suppression of IL-8 mRNA levels suggest that the effect is mediated by intracellular signal transduction pathways which regulate transcription.