Bianca N. Islam

Sildenafil suppresses inflammation-driven colorectal cancer in mice

Intestinal cyclic guanosine monophosphate (cGMP) signaling regulates epithelial homeostasis and has been implicated in the suppression of colitis and colon cancer. In this study, we investigated the cGMP-elevating ability of the phosphodiesterase-5 (PDE5) inhibitor sildenafil to prevent disease in the azoxymethane/dextran sulfate sodium (AOM/DSS) inflammation-driven colorectal cancer model. Treatment of mice with sildenafil activated cGMP signaling in the colon mucosa and protected against dextran-sulfate sodium (DSS)-induced barrier dysfunction. In mice treated with AOM/DSS, oral administration of sildenafil throughout the disease course reduced polyp multiplicity by 50% compared with untreated controls. Polyps that did form in sildenafil treated mice were less proliferative and more differentiated compared with polyps from untreated mice, but apoptosis was unaffected. Polyps in sildenafil treated mice were also less inflamed; they exhibited reduced myeloid-cell infiltration and reduced expression of iNOS, IFNγ, and IL6 compared with untreated controls. Most of the protection conferred by sildenafil was during the initiation stage of carcinogenesis (38% reduction in multiplicity). Administration of sildenafil during the later promotion stages did not affect multiplicity but had a similar effect on the polyp phenotype, including increased mucus production, and reduced proliferation and inflammation. In summary, the results demonstrate that oral administration of sildenafil suppresses polyp formation and inflammation in mice treated with AOM/DSS. This validation of PDE5 as a target highlights the potential therapeutic value of PDE5 inhibitors for the prevention of colitis-driven colon cancer in humans. Cancer Prev Res; 10(7); 377–88. ©2017 AACR. See related editorial by Piazza, p. 373.

Carbidopa, a drug in use for management of Parkinson disease inhibits T cell activation and autoimmunity

Carbidopa is a drug that blocks conversion of levodopa to dopamine outside of central nervous system (CNS) and thus inhibits unwanted side effects of levodopa on organs located outside of CNS during management of Parkinson’s Disease (PD). PD is associated with increased expression of inflammatory genes in peripheral and central nervous system (CNS), infiltration of immune cells into brain, and increased numbers of activated/memory T cells. Animal models of PD have shown a critical role of T cells in inducing pathology in CNS. However, the effect of carbidopa on T cell responses in vivo is unknown. In this report, we show that carbidopa strongly inhibited T cell activation in vitro and in vivo. Accordingly, carbidopa mitigated myelin oligodendrocyte glycoprotein peptide fragment 35–55 (MOG-35-55) induced experimental autoimmune encephalitis (EAE) and collagen induced arthritis in animal models. The data presented here suggest that in addition to blocking peripheral conversion of levodopa, carbidopa may inhibit T cell responses in PD individuals and implicate a potential therapeutic use of carbidopa in suppression of T cell mediated pathologies.

Cyclic-GMP elevating agents suppress polyposis in Apc min mice by targeting the preneoplastic epithelium.

The cGMP signaling axis has been implicated in the suppression of intestinal cancers, but the inhibitory mechanism and the extent to which this pathway can be targeted remains poorly understood. This study has tested the effect of cGMP-elevating agents on tumorigenesis in the ApcMin/+ mouse model of intestinal cancer. Treatment of ApcMin/+ mice with the receptor guanylyl-cyclase C (GCC) agonist linaclotide, or the phosphodiesterase-5 (PDE5) inhibitor sildenafil, significantly reduced the number of polyps per mouse (67% and 50%, respectively). Neither of the drugs affected mean polyp size, or the rates of apoptosis and proliferation. This was possibly due to increased PDE10 expression, as endogenous GCC ligands were not deficient in established polyps. These results indicated that the ability of these drugs to reduce polyp multiplicity was primarily due to an effect on nonneoplastic tissues. In support of this idea, ApcMin/+ mice exhibited reduced levels of endogenous GCC agonists in the nonneoplastic intestinal mucosa compared with wild-type animals, and this was associated with crypt hyperplasia and a loss of goblet cells. Administration of either sildenafil or linaclotide suppressed proliferation, and increased both goblet cell numbers and luminal apoptosis in the intestinal mucosa. Taken together, the results demonstrate that targeting cGMP with either PDE5 inhibitors or GCC agonists alters epithelial homeostasis in a manner that reduces neoplasia, and suggests that this could be a viable chemoprevention strategy for patients at high risk of developing colorectal cancer. Cancer Prev Res; 11(2); 81–92. ©2018 AACR.

Sildenafil normalizes bowel transit in preclinical models of constipation

Guanylyl cyclase-C (GC-C) agonists increase cGMP levels in the intestinal epithelium to promote secretion. This process underlies the utility of exogenous GC-C agonists such as linaclotide for the treatment of chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C). Because GC-C agonists have limited use in pediatric patients, there is a need for alternative cGMP-elevating agents that are effective in the intestine. The present study aimed to determine whether the PDE-5 inhibitor sildenafil has similar effects as linaclotide on preclinical models of constipation. Oral administration of sildenafil caused increased cGMP levels in mouse intestinal epithelium demonstrating that blocking cGMP-breakdown is an alternative approach to increase cGMP in the gut. Both linaclotide and sildenafil reduced proliferation and increased differentiation in colon mucosa, indicating common target pathways. The homeostatic effects of cGMP required gut turnover since maximal effects were observed after 3 days of treatment. Neither linaclotide nor sildenafil treatment affected intestinal transit or water content of fecal pellets in healthy mice. To test the effectiveness of cGMP elevation in a functional motility disorder model, mice were treated with dextran sulfate sodium (DSS) to induce colitis and were allowed to recover for several weeks. The recovered animals exhibited slower transit, but increased fecal water content. An acute dose of sildenafil was able to normalize transit and fecal water content in the DSS-recovery animal model, and also in loperamide-induced constipation. The higher fecal water content in the recovered animals was due to a compromised epithelial barrier, which was normalized by sildenafil treatment. Taken together our results show that sildenafil can have similar effects as linaclotide on the intestine, and may have therapeutic benefit to patients with CIC, IBS-C, and post-infectious IBS.

Abstract 1918: Type 2 cGMP-dependent protein kinase activates antineoplastic signaling in the colon

Signaling through cGMP has emerged as a potentially important suppressor of tumorigenesis in the colon but the mechanisms are poorly defined. Studies of guanylyl-cyclase (GCC) knockout mice indicate that cGMP signaling inhibits proliferation in the colon epithelium and that AKT may have an important role. Type-2 cGMP-dependent protein kinase (PKG2) is a central effector of cGMP in the colon epithelium and likely mediates the suppressive signaling. An important growth-inhibitory effect of AKT is the inactivation of forkhead transcription factors (FoxO). FoxO proteins are tumor suppressors that regulate growth-inhibitory and pro-apoptotic target gene expression but these proteins have not previously been examined in the intestinal tract. The present study sought to determine whether PKG2 can activate FoxO in colon cancer cells and in the colon epithelium. Activation of PKG2 in LS174T colon cancer cells inhibited cell proliferation but did not significantly affect apoptosis. Suggestive of a mechanism, it was found that PKG2 inhibited the activity of AKT but not ERK in these cells. Activation of PKG2 also significantly increased luciferase reporter activity driven by FoxO-responsive elements in LS174T cells. In support of FoxO activation, PKG2 also increased the expression of several FoxO target genes, including catalase, GADD45, and p27kip. Treatment of colon explants with 8Br-cGMP also activated FoxO target gene expression at both RNA and protein levels, and this was associated with reduced redox stress. The regulation of FoxO by cGMP in vivo most likely requires PKG2, because target gene expression was reduced in the colon mucosa of Prkg2-/- mice compared to wild type siblings. Since FoxO has not previously been studied in the colon, we first examined expression using IHC. These data showed that FoxO3a is the most prominent isoform, and was concentrated almost exclusively at the lumenal border. While FoxO1 showed negligible staining, FoxO4 was observed deeper in the crypt. In order to determine whether cGMP could activate FoxO in vivo, the PDE-5 inhibitor vardenafil (Levitra™) was used. Mice treated with vardenafil showed a dramatic mobilization of FoxO3a to the nucleus of lumenal epithelial cells. Analysis of the colon mucosa from treated animals showed increased levels of FoxO target genes and reduced redox stress. This was likely to be mediated by FoxO3a since vardenafil did not significantly affect the localization of FoxO1 or FoxO4. Taken together these data define a novel signaling pathway in the colon epithelium, where PKG2 leads to activation of FoxO. The established tumor suppressor role of FoxO proteins highlights the potential therapeutic role for cGMP signaling in colon cancer prevention. Citation Format: Allison Bridges, Bianca Islam, Sarah Sharman, Rui Wang, Subbaramiah Sridhar, Darren Douglas Browning. Type 2 cGMP-dependent protein kinase activates antineoplastic signaling in the colon. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1918. doi:10.1158/1538-7445.AM2015-1918

Clinical utility of plecanatide in the treatment of chronic idiopathic constipation

Constipation is an important health burden that reduces the quality of life for countless millions of people. Symptom-centric therapeutics are often used to treat constipation due to unknown etiology, but in many cases, these drugs are either inadequate or have significant side effects. More recently, synthetic peptide agonists for epithelial guanylyl cyclase C (GC-C) have been developed which are effective at treating constipation in a sub-population of adult constipation patients. The first to market was linaclotide that is structurally related to the diarrheagenic enterotoxin, but this was followed by plecanatide, which more closely resembles endogenous uroguanylin. Both the drugs exhibit almost identical clinical efficacy in about 20% of patients, with diarrhea being a common side effect. Despite the potential for reduced side effects with plecanatide, detailed analysis suggests that clinically, they are very similar. Ongoing clinical and preclinical studies with these drugs suggest that treating constipation might be the tip of the iceberg in terms of clinical utility. The expression of cGMP signaling components could be diagnostic for functional bowel disorders, and increasing cGMP using GC-C agonists or phosphodiesterase inhibitors has huge potential for treating enteric pain, ulcerative colitis, and for the chemoprevention of colorectal cancer.