Activation of Guanylate Cyclase-C With Heat-Stable Enterotoxin Fails to Potentiate Celecoxib-Induced Reduction in Colorectal Cancer Cell Growth in Vitro

This study evaluated the use of heat-stable enterotoxin (ST), a guanylate cyclase receptor type C (GC-C) agonist, in combination with celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, for the prevention of colorectal cancer (CRC) growth in vitro. GC-C is a membrane-bound enzyme found in the lumen of the intestines and is responsible for the conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). Activation of GC-C elevates intracellular cGMP which controls fluid-ion homeostasis and enterocyte differentiation along the crypt-villus axis. GC-C function can regulate colonic cell proliferation and induce cell cycle arrest. COX-2 is a cytosolic enzyme that catalyzes the formation of prostaglandins from arachidonic acid and inhibition of this enzyme is known to inhibit proliferation of human cancer cells. Previous research demonstrated that inhibition of the primary enzyme responsible for degradation of cGMP potentiated the cytostatic effects of celecoxib. This thesis tested if GC-C activation can enhance the antiproliferative effects of celecoxib. Cell proliferation was assessed using a thymidine incorporation protocol, in which cells are cultured with tritiated thymidine after treatment and the amount of measured incorporated radioactivity correlates with cell division. Celecoxib was found to inhibit proliferation of T84 cells, which are derived from a human colorectal adenocarcinoma. This was consistent with previous studies that demonstrated an anti-proliferative effect of celecoxib in other human cancer cell lines. However, the addition of ST to celecoxib was unable to potentiate the anti-proliferative effects of celecoxib. ST also had no effect on proliferation when used as a monotherapy. Changes in COX-2 activity, as well as the presence of COX-2 enzyme, were measured to assess whether the effect of celecoxib in T84 cells was dependent on its inhibitory activity on COX-2. No changes in cyclooxygenase activity were observed and COX-2 was not detected in T84 cells. In addition, celecoxib and the combination of celecoxib and ST were unable to produce a detectable amount of Caspase-3, a marker of cell death, suggesting that these drugs do not have a cytotoxic effect in T84 cells. This research showed that ST does not have an effect on proliferation of T84 cells, which conflicts with previous research conducted by Pitari et al. (2001). It is possible that an effect of ST on adenocarcinomas in the gastrointestinal tract may vary based on the location of the targeted cells. Therefore, while ST did not reduce proliferation on cells derived from the colon, it may have an effect on proliferation of epithelial cells that are located in the small intestine upstream from the colon. Celecoxib was confirmed to have an anti-proliferative effect, and this was shown to be independent of the drugs enzymatic target, implying that there are additional mechanisms through which celecoxib exerts its activity. Additional research is needed to clarify these mechanisms

Valganciclovir inhibits human adenovirus replication and pathology in permissive immunosuppressed female and male Syrian hamsters

Adenovirus infections of immunocompromised pediatric hematopoietic stem cell transplant patients can develop into serious and often deadly multi-organ disease. There are no drugs approved for adenovirus infections. Cidofovir (an analog of 2-deoxycytidine monophosphate) is used at times but it can be nephrotoxic and its efficacy has not been proven in clinical trials. Brincidofovir, a promising lipid-linked derivative of cidofovir, is in clinical trials. Ganciclovir, an analog of 2-deoxyguanosine, has been employed occasionally but with unknown efficacy in the clinic. In this study, we evaluated valganciclovir against disseminated adenovirus type 5 (Ad5) infection in our permissive immunosuppressed Syrian hamster model. We administered valganciclovir prophylactically, beginning 12 h pre-infection or therapeutically starting at Day 1, 2, 3, or 4 post-infection. Valganciclovir significantly increased survival, reduced viral replication in the liver, and mitigated the pathology associated with Ad5 infection. In cultured cells, valganciclovir inhibited Ad5 DNA replication and blocked the transition from early to late stage of infection. Valganciclovir directly inhibited Ad5 DNA polymerase in vitro, which may explain, at least in part, its mechanism of action. Ganciclovir and valganciclovir are approved to treat infections by certain herpesviruses. Our results support the use of valganciclovir to treat disseminated adenovirus infections in immunosuppressed patients

Overexpression of BH3-Only Protein BNIP3 Leads to Enhanced Tumor Growth

BCL-2/E1B-19 kDa–interacting protein 3 (BNIP3) is a BH3-only mitochondrial protein. Expression of BNIP3 is strongly stimulated by hypoxia. Up-regulation of BNIP3 has been detected in several human carcinomas including carcinomas of the lung and breast. The significance of BNIP3 overexpression in these cancers is not known. To determine whether BNIP3 plays a role in tumor growth, we generated A549 lung carcinoma cells that overexpressed BNIP3 and examined their ability to form tumors in the mouse xenograft model. All cell lines that overexpressed BNIP3 formed larger tumors compared to the parental or vector-transformed A549 cells. Breast carcinoma cell lines that overexpressed BNIP3 also induced tumors in athymic mice in the absence of hormone administration, while the parental cell line did not. Stable shRNA-mediated knockdown of endogenous BNIP3 severely impaired the tumorigenic activity of A549 cells. The tumor growth-enhancing activity was reduced by deletion of the BH3 domain of BNIP3. Expression of a dominant-negative mutant of BNIP3 lacking the C-terminal transmembrane domain also inhibited the tumorigenic potential of A549 cells. These results suggest that BNIP3 plays a fundamental role in the development of certain solid tumors such as the lung and breast carcinomas

Correction: STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control.

[This corrects the article DOI: 10.1371/journal.ppat.1005084.]

STAT2 Knockout Syrian Hamsters Support Enhanced Replication and Pathogenicity of Human Adenovirus, Revealing an Important Role of Type I Interferon Response in Viral Control.

Human adenoviruses have been studied extensively in cell culture and have been a model for studies in molecular, cellular, and medical biology. However, much less is known about adenovirus replication and pathogenesis in vivo in a permissive host because of the lack of an adequate animal model. Presently, the most frequently used permissive immunocompetent animal model for human adenovirus infection is the Syrian hamster. Species C human adenoviruses replicate in these animals and cause pathology that is similar to that seen with humans. Here, we report findings with a new Syrian hamster strain in which the STAT2 gene was functionally knocked out by site-specific gene targeting. Adenovirus-infected STAT2 knockout hamsters demonstrated an accentuated pathology compared to the wild-type control animals, and the virus load in the organs of STAT2 knockout animals was 100- to 1000-fold higher than that in wild-type hamsters. Notably, the adaptive immune response to adenovirus is not adversely affected in STAT2 knockout hamsters, and surviving hamsters cleared the infection by 7 to 10 days post challenge. We show that the Type I interferon pathway is disrupted in these hamsters, revealing the critical role of interferon-stimulated genes in controlling adenovirus infection. This is the first study to report findings with a genetically modified Syrian hamster infected with a virus. Further, this is the first study to show that the Type I interferon pathway plays a role in inhibiting human adenovirus replication in a permissive animal model. Besides providing an insight into adenovirus infection in humans, our results are also interesting from the perspective of the animal model: STAT2 knockout Syrian hamster may also be an important animal model for studying other viral infections, including Ebola-, hanta-, and dengue viruses, where Type I interferon-mediated innate immunity prevents wild type hamsters from being effectively infected to be used as animal models