cGMP signaling as a target for the prevention and treatment of breast cancer.

One in eight women in the United States will be diagnosed with invasive breast cancer in her lifetime. Advances in therapeutic strategies, diagnosis, and improved awareness have resulted in a significant reduction in breast cancer related mortality. However, there is a continued need for more effective and less toxic drugs for both the prevention and the treatment of breast cancer in order to see a continued decline in the morbidity and mortality associated with this disease. Recent studies suggest that the cGMP signaling pathway may be aberrantly regulated in breast cancer. As such, this pathway may serve as a source of novel targets for future breast cancer drug discovery efforts. This review provides an overview of cGMP signaling in normal physiology and in breast cancer as well as current strategies being investigated for targeting this pathway in breast cancer.

Design and Synthesis of Substituted Pyridazinone-1-Acetylhydrazones as Novel Phosphodiesterase 4 Inhibitors.

A series of novel pyridazin-6-one-1-acetylhydrazone hybrids were rationally designed to inhibit phosphodiesterase 4 (PDE4B). The prepared compounds were evaluated for their in vitro ability to inhibit the PDE4B enzyme; several of these compounds showed moderate activity compared to the reference drug, rolipram. Compounds 6, 12, and 14 emerged as the most potent inhibitors in this series. The [3-(4-methoxyphenyl)-6-oxo-5,6-dihydro-4H-pyridazin-1-yl]acetic acid [1-(3,4,5-trimethoxyphenyl)ethylidene]hydrazide (12) showed an IC50 value of 13 µM against PDE4B. Docking of 6, 12, and 14 into the active site of PDE4B illustrates their possible binding mode and provides insights for further optimization of this drug scaffold.

New NSAID targets and derivatives for colorectal cancer chemoprevention.

Clinical and preclinical studies provide strong evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) can prevent numerous types of cancers, especially colorectal cancer. Unfortunately, the depletion of physiologically important prostaglandins due to cyclooxygenase (COX) inhibition results in potentially fatal toxicities that preclude the long-term use of NSAIDs for cancer chemoprevention. While studies have shown an involvement of COX-2 in colorectal tumorigenesis, other studies suggest that a COX-independent target may be at least partially responsible for the antineoplastic activity of NSAIDs. For example, certain NSAID derivatives have been identified that do not inhibit COX-2 but have demonstrated efficacy to suppress carcinogenesis with potential for reduced toxicity. A number of alternative targets have also been reported to account for the tumor cell growth inhibitory activity of NSAIDs, including the inhibition of cyclic guanosine monophosphate phosphodiesterases (cGMP PDEs), generation of reactive oxygen species (ROS), the suppression of the apoptosis inhibitor protein, survivin, and others. Here, we review several promising mechanisms that are being targeted to develop safer and more efficacious NSAID derivatives for colon cancer chemoprevention.

Novel Non-Cyclooxygenase Inhibitory Derivative of Sulindac Inhibits Breast Cancer Cell Growth In Vitro and Reduces Mammary Tumorigenesis in Rats.

The nonsteroidal anti-inflammatory drug (NSAID) sulindac demonstrates attractive anticancer activity, but the toxicity resulting from cyclooxygenase (COX) inhibition and the suppression of physiologically important prostaglandins precludes its long-term, high dose use in the clinic for cancer prevention or treatment. While inflammation is a known tumorigenic driver, evidence suggests that sulindac's antineoplastic activity is partially or fully independent of its COX inhibitory activity. One COX-independent target proposed for sulindac is cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) isozymes. Sulindac metabolites, i.e., sulfide and sulfone, inhibit cGMP PDE enzymatic activity at concentrations comparable with those associated with cancer cell growth inhibitory activity. Additionally, the cGMP PDE isozymes PDE5 and PDE10 are overexpressed during the early stages of carcinogenesis and appear essential for cancer cell proliferation and survival based on gene silencing experiments. Here, we describe a novel amide derivative of sulindac, sulindac sulfide amide (SSA), which was rationally designed to eliminate COX-inhibitory activity while enhancing cGMP PDE inhibitory activity. SSA was 68-fold and 10-fold less potent than sulindac sulfide (SS) in inhibiting COX-1 and COX-2, respectively, but 10-fold more potent in inhibiting growth and inducing apoptosis in breast cancer cells. The pro-apoptotic activity of SSA was associated with inhibition of cGMP PDE activity, elevation of intracellular cGMP levels, and activation of cGMP-dependent protein kinase (PKG) signaling, as well as the inhibition of ß-catenin/Tcf transcriptional activity. SSA displayed promising in vivo anticancer activity, resulting in a 57% reduction in the incidence and a 62% reduction in the multiplicity of tumors in the N-methyl-N-nitrosourea (MNU)-induced model of breast carcinogenesis. These findings provide strong evidence for cGMP/PKG signaling as a target for breast cancer prevention or treatment and the COX-independent anticancer properties of sulindac. Furthermore, this study validates the approach of optimizing off-target effects by reducing the COX-inhibitory activity of sulindac for future targeted drug discovery efforts to enhance both safety and efficacy.

Design, synthesis and biological evaluation of novel pyridine derivatives as anticancer agents and phosphodiesterase 3 inhibitors.

Two series of 4,6-diaryl-2-imino-1,2-dihydropyridine-3-carbonitriles and their isosteric 4,6-diaryl-2-oxo-1,2-dihydropyridine-3-carbonitriles were synthesized through a combinatorial approach. The prepared analogues were evaluated for their in vitro capacity to inhibit PDE3A and the growth of the human HT-29 colon adenocarcinoma tumor cell line. Compound 6-(4-bromophenyl)-4-(2-ethoxyphenyl)-2-imino-1,2-dihydropyridine-3-carbonitrile (Id) exhibited the strongest PDE3 inhibition when cGMP but not cAMP is the substrate with a IC(50)of 27microM, which indicates a highly selective mechanism of enzyme inhibition. On the other hand, compound 6-(1,3-benzodioxol-5-yl)-4-(2-ethoxyphenyl)-2-imino-1,2-dihydropyridine-3-carbonitrile (Ii) was the most active in inhibiting colon tumor cell growth with a IC(50) of 3microM. The electronic effects, steric effects and conformational aspects of Id seem to be the most crucial for the PDE3 inhibition. Meanwhile, steric factors and the H-bonding capability seem to be the most important factors for tumor cell growth inhibitory activity. Conversely, there is no direct correlation between PDE3 inhibition and anticancer activity for the prepared compounds. An in silico docking experiment indicates the potential involvement of other potential molecular targets such as PIM-1 kinase to explain its tumor cell growth inhibitory activity.

Ripped from the Headlines: Using Current Events and Deliberative Democracy to Improve Student Performance in and Perceptions of Nonmajors Biology Courses.

Despite the importance of scientific literacy, many foundational science courses are plagued by low student engagement and performance. In an attempt to improve student outcomes, an introductory biology course for nonscience majors was redesigned to present the course content within the framework of current events and deliberative democratic exercises. During each instructional unit of the redesigned course, students were presented with a highly publicized policy question rooted in biological principles and currently facing lawmakers. Working in diverse groups, students sought out the information that was needed to reach an educated, rationalized decision. This approach models civic engagement and demonstrates the real-life importance of science to nonscience majors. The outcomes from two semesters in which the redesign were taught were compared with sections of the course taught using traditional pedagogies. When compared with other versions of the same course, presenting the course content within a deliberative democratic framework proved to be superior for increasing students' knowledge gains and improving students' perceptions of biology and its relevance to their everyday lives. These findings establish deliberative democracy as an effective pedagogical strategy for nonmajors biology.

The role of cyclic nucleotide signaling pathways in cancer: targets for prevention and treatment.

For more than four decades, the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) have been recognized as important signaling molecules within cells. Under normal physiological conditions, cyclic nucleotides regulate a myriad of biological processes such as cell growth and adhesion, energy homeostasis, neuronal signaling, and muscle relaxation. In addition, altered cyclic nucleotide signaling has been observed in a number of pathophysiological conditions, including cancer. While the distinct molecular alterations responsible for these effects vary depending on the specific cancer type, several studies have demonstrated that activation of cyclic nucleotide signaling through one of three mechanisms-induction of cyclic nucleotide synthesis, inhibition of cyclic nucleotide degradation, or activation of cyclic nucleotide receptors-is sufficient to inhibit proliferation and activate apoptosis in many types of cancer cells. These findings suggest that targeting cyclic nucleotide signaling can provide a strategy for the discovery of novel agents for the prevention and/or treatment of selected cancers.

Sulindac selectively inhibits colon tumor cell growth by activating the cGMP/PKG pathway to suppress Wnt/ß-catenin signaling.

Nonsteroidal anti-inflammatory drugs (NSAID) display promising antineoplastic activity for colorectal and other cancers, but toxicity from COX inhibition limits their long-term use for chemoprevention. Previous studies have concluded that the basis for their tumor cell growth inhibitory activity does not require COX inhibition, although the underlying mechanism is poorly understood. Here, we report that the NSAID sulindac sulfide inhibits cyclic guanosine 3',5'-monophosphate phosphodiesterase (cGMP PDE) activity to increase intracellular cGMP levels and activate cGMP-dependent protein kinase (PKG) at concentrations that inhibit proliferation and induce apoptosis of colon tumor cells. Sulindac sulfide did not activate the cGMP/PKG pathway, nor affect proliferation or apoptosis in normal colonocytes. Knockdown of the cGMP-specific PDE5 isozyme by siRNA and PDE5-specific inhibitors tadalafil and sildenafil also selectively inhibited the growth of colon tumor cells that expressed high levels of PDE5 compared with colonocytes. The mechanism by which sulindac sulfide and the cGMP/PKG pathway inhibits colon tumor cell growth involves the transcriptional suppression of ß-catenin to inhibit Wnt/ß-catenin T-cell factor transcriptional activity, leading to downregulation of cyclin D1 and survivin. These observations suggest that safer and more efficacious sulindac derivatives can be developed for colorectal cancer chemoprevention by targeting PDE5 and possibly other cGMP-degrading isozymes.

Novel Therapeutics: NSAIDs, Derivatives, and Phosphodiesterases.

The chemopreventive efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) for colorectal cancer has been well documented. However, long-term use of NSAIDs is precluded owing to potentially fatal toxicities associated with their mechanism of action involving cyclooxygenase (COX) inhibition. But studies have shown that their anticancer activity may be due, in part, to an off-target effect. Cyclic guanosine monophosphate (cGMP) phosphodiesterases (PDEs), which are responsible for negative regulation of cGMP signaling, are an attractive COX-independent target. cGMP signaling is aberrantly suppressed in cancer cells and its activation appears to be sufficient to inhibit tumor cell growth. Chemically modifying sulindac has produced a series of new derivatives that lack COX-inhibitory activity but have improved cGMP PDE inhibitory activity. This approach is proving to be a promising strategy for the discovery of improved agents for the prevention and/or treatment of colorectal cancer.

Exploring the PDE5 H-pocket by ensemble docking and structure-based design and synthesis of novel ß-carboline derivatives.

By studying the co-crystal information of interactions between PDE5 and its inhibitors, forty new tetrahydro-ß-carbolines based-analogues were synthesized, and tested for their PDE5 inhibition. Some compounds were as active as tadalafil in inhibiting PDE5 and of better selectivity profile particularly versus PDE11A, the nature of the terminal ring and its nitrogen substituent are the main determinants of selectivity. Ensemble docking confirmed the role of H-loop closed conformer in activity versus its occluded and open forms. Conformational studies showed the effect of bulkiness of the terminal ring N-alkyl substituent on the formation of stable enzyme ligands conformers. The difference in potencies of hydantoin and piperazinedione analogues, together with the necessity of C-5/C-6 R-absolute configuration has been revealed through molecular docking.

A novel sulindac derivative that potently suppresses colon tumor cell growth by inhibiting cGMP phosphodiesterase and ß-catenin transcriptional activity.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely reported to inhibit tumor growth by a COX-independent mechanism, although alternative targets have not been well defined or used to develop improved drugs for cancer chemoprevention. Here, we characterize a novel sulindac derivative referred to as sulindac benzylamine (SBA) that does not inhibit COX-1 or COX-2, yet potently inhibits the growth and induces the apoptosis of human colon tumor cells. The basis for this activity appears to involve cyclic guanosine 3',5',-monophosphate phosphodiesterase (cGMP PDE) inhibition as evident by its ability to inhibit cGMP hydrolysis in colon tumor cell lysates and purified cGMP-specific PDE5, increase intracellular cGMP levels, and activate cGMP-dependent protein kinase G at concentrations that suppress tumor cell growth. PDE5 was found to be essential for colon tumor cell growth as determined by siRNA knockdown studies, elevated in colon tumor cells as compared with normal colonocytes, and associated with the tumor selectivity of SBA. SBA activation of PKG may suppress the oncogenic activity of ß-catenin as evident by its ability to reduce ß-catenin nuclear levels, Tcf (T-cell factor) transcriptional activity, and survivin levels. These events preceded apoptosis induction and appear to result from a rapid elevation of intracellular cGMP levels following cGMP PDE inhibition. We conclude that PDE5 and possibly other cGMP degrading isozymes can be targeted to develop safer and more efficacious NSAID derivatives for colorectal cancer chemoprevention.

Inhibition of PDE5 by sulindac sulfide selectively induces apoptosis and attenuates oncogenic Wnt/ß-catenin-mediated transcription in human breast tumor cells.

Nonsteroidal anti-inflammatory drugs (NSAID) such as sulindac sulfide (SS) display promising antineoplastic properties, but toxicities resulting from COX inhibition limit their clinical use. Although COX inhibition is responsible for the anti-inflammatory activity of SS, recent studies suggest that phosphodiesterase (PDE) 5 inhibition and activation of cyclic guanosine monophosphate (cGMP) signaling are closely associated with its ability to induce apoptosis of tumor cells. However, the underlying mechanisms responsible for apoptosis induction, factors that influence sensitivity of tumor cells to SS, and the importance of PDE5 for breast tumor cell growth have not been established. Here we show that SS can induce apoptosis of breast tumor cells, which predominantly rely on PDE5 for cGMP hydrolysis but not normal mammary epithelial cells, which rely on PDE isozymes other than PDE5 for cGMP hydrolysis. Inhibition of PDE5 and activation of protein kinase G (PKG) by SS was associated with increased ß-catenin phosphorylation, decreased ß-catenin mRNA and protein levels, reduced ß-catenin nuclear localization, decreased T-cell factor/lymphoid enhancer factor (Tcf/Lef) promoter activity, and decreased expression of Wnt/ß-catenin-regulated proteins. Suppression of PDE5 with siRNA or known PDE5 inhibitors was sufficient to selectively induce apoptosis and attenuate ß-catenin-mediated transcription in breast tumor cells with minimal effects on normal mammary epithelial cells. These findings provide evidence that SS induces apoptosis of breast tumor cells through a mechanism involving inhibition of PDE5 and attenuation of oncogenic Wnt/ß-catenin-mediated transcription. We conclude that PDE5 represents a novel molecular target for the discovery of safer and more efficacious drugs for breast cancer chemoprevention.

Synthesis and molecular modeling of novel tetrahydro-ß-carboline derivatives with phosphodiesterase 5 inhibitory and anticancer properties.

New derivatives based upon the tetrahydro-ß-carboline-hydantoin and tetrahydro-ß-carboline-piperazinedione scaffolds were synthesized. All compounds were evaluated for their ability to inhibit PDE5 in vitro, and numerous compounds with IC(50) values in the low nanomolar range were identified including compounds derived from l-tryptophan. Compounds with high potency versus PDE5 were then evaluated for inhibitory activity against other PDEs to assess isozyme selectivity. Compound 5R,11aS-5-(3,4-dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)dione 14 showed a selectivity index of >200 for cGMP hydrolysis by PDE5 versus PDE11. Meanwhile, 6R,12aR-6-(2,4-dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4dione 45 demonstrated strong potency for inhibition of PDE11 with an IC(50) value of 11 nM, representing the most potent PDE11 inhibitor thus far reported. Docking experiments differentiated between active and inactive analogues and revealing the conformational, steric, and lipophilic necessities for potent PDE5 inhibition. Many derivatives, including potent PDE5 inhibitors, were able to inhibit the growth of the MDA-MB-231 breast tumor cell line with low micromolar potency.

Chronic exposure to a high-fat diet induces hepatic steatosis, impairs nitric oxide bioavailability, and modifies the mitochondrial proteome in mice.

Obesity-related pathologies, such as nonalcoholic fatty liver disease, are linked to mitochondrial dysfunction and nitric oxide (NO) deficiency. Herein, we tested the hypothesis that a high-fat diet (HFD) modifies the liver mitochondrial proteome and alters proteins involved in NO metabolism, namely arginase 1 and endothelial NO synthase. Male C57BL/6 mice were fed a control or HFD and liver mitochondria were isolated for proteomics and reactive oxygen species measurements. Steatosis and hepatocyte ballooning were present in livers of HFD mice, with no pathology observed in the controls. HFD mice had increased serum glucose and decreased adiponectin. Mitochondrial reactive oxygen species was increased after 8 weeks in the HFD mice, but decreased at 16 weeks compared with the control, which was accompanied by increased uncoupling protein 2. Using proteomics, 22 proteins were altered as a consequence of the HFD. This cohort consists of oxidative phosphorylation, lipid metabolism, sulfur amino acid metabolism, and chaperone proteins. We observed a HFD-dependent increase in arginase 1 and decrease in activated endothelial NO synthase. Serum and liver nitrate + nitrite were decreased by HFD. In summary, these data demonstrate that a HFD causes steatosis, alters NO metabolism, and modifies the liver mitochondrial proteome; thus, NO may play an important role in the processes responsible for nonalcoholic fatty liver disease.

A novel access to arylated and heteroarylated beta-carboline based PDE5 inhibitors.

Starting from a previously reported lead compound GR30040X (a hydantoin tetrahydro-ß-carboline derivative with a 4- pyridinyl ring at C- 5), a series of structurally related tetrahydro-ß-carboline derivatives were prepared. The tetrahydro-ß-carboline skeleton was fused either to a hydantoin or to a piperazindione ring, the pendant aryl group attached to C-5 or C-6 was changed to a 3, 4-dimethoxyphenyl or a 3-pyridinyl ring; different N-substituents on the terminal ring were introduced, a straight chain ethyl group, a branched tert. butyl and P-chlorophenyl group rather than n-butyl group of the lead compound. All four possible diastereomers of target tetrahydro-ß-carboline derivatives were prepared, separated by column chromatography and the significance of these stereochemical manipulations were studied. Synthesized compounds were evaluated for their inhibitory effect versus PDE5. Seven hits were obtained with appreciable inhibitory activity versus PDE5 with IC50s 0.14 - 4.99 µM.

Synthesis, molecular modeling and biological evaluation of novel tadalafil analogues as phosphodiesterase 5 and colon tumor cell growth inhibitors, new stereochemical perspective.

The synthesis of novel tadalafil analogues in which the benzodioxole moiety is replaced by 2-bromophenyl; the chiral carbons swing from R,R to R,S, S,R and S,S; the piperazinedione ring is maintained or reduced to the 5-membered imidazolidinedione or thioxoimidazolinone is described. The prepared analogues were evaluated for their capacity to inhibit the cyclic guanosine monophosphate (cGMP) selective phosphodiesterase 5 (PDE5) isozyme and the growth of human HT-29 colon adenocarcinoma cells. The R absolute configuration of C-5 in the beta-carboline-hydantoin and C-6 in the beta-carboline-piperazinedione derivatives was found to be essential for the PDE5 inhibition. In addition, tadalafil analogues that were synthesized from l-tryptophan were more active than those derived from d-tryptophan, which is of economic value and expands the horizon for the discovery of new carbolines as PDE5 inhibitors. While some analogues displayed potent tumor cell growth inhibitory activity, there was no apparent correlation with their PDE5 inhibitory activity, which leads us to conclude that other PDE isozymes or PDE5 splice variants may be involved.

Colon tumor cell growth-inhibitory activity of sulindac sulfide and other nonsteroidal anti-inflammatory drugs is associated with phosphodiesterase 5 inhibition.

Nonsteroidal anti-inflammatory drugs (NSAID) display promising antineoplastic activity, but toxicity resulting from cyclooxygenase (COX) inhibition limits their clinical use for chemoprevention. Studies suggest that the mechanism may be COX independent, although alternative targets have not been well defined. Here, we show that the NSAID sulindac sulfide (SS) inhibits cyclic guanosine 3',5'-monophosphate (cGMP) phosphodiesterase (PDE) activity in colon tumor cell lysates at concentrations that inhibit colon tumor cell growth in vitro and in vivo. A series of chemically diverse NSAIDs also inhibited cGMP hydrolysis at concentrations that correlate with their potency to inhibit colon tumor cell growth, whereas no correlation was observed with COX-2 inhibition. Consistent with its selectivity for inhibiting cGMP hydrolysis compared with cyclic AMP hydrolysis, SS inhibited the cGMP-specific PDE5 isozyme and increased cGMP levels in colon tumor cells. Of numerous PDE isozyme-specific inhibitors evaluated, only the PDE5-selective inhibitor MY5445 inhibited colon tumor cell growth. The effects of SS and MY5445 on cell growth were associated with inhibition of ß-catenin-mediated transcriptional activity to suppress the synthesis of cyclin D and survivin, which regulate tumor cell proliferation and apoptosis, respectively. SS had minimal effects on cGMP PDE activity in normal colonocytes, which displayed reduced sensitivity to SS and did not express PDE5. PDE5 was found to be overexpressed in colon tumor cell lines as well as in colon adenomas and adenocarcinomas compared with normal colonic mucosa. These results suggest that PDE5 inhibition, cGMP elevation, and inhibition of ß-catenin transcriptional activity may contribute to the chemopreventive properties of certain NSAIDs.

NSAIDs: Old Drugs Reveal New Anticancer Targets.

There is compelling evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 selective inhibitors have antineoplastic activity, but toxicity from cyclooxygenase (COX) inhibition and the suppression of physiologically important prostaglandins limits their use for cancer chemoprevention. Previous studies as reviewed here suggest that the mechanism for their anticancer properties does not require COX inhibition, but instead involves an off-target effect. In support of this possibility, recent molecular modeling studies have shown that the NSAID sulindac can be chemically modified to selectively design out its COX-1 and COX-2 inhibitory activity. Unexpectedly, certain derivatives that were synthesized based on in silico modeling displayed increased potency to inhibit tumor cell growth. Other experiments have shown that sulindac can inhibit phosphodiesterase to increase intracellular cyclic GMP levels and that this activity is closely associated with its ability to selectively induce apoptosis of tumor cells. Together, these studies suggest that COX-independent mechanisms can be targeted to develop safer and more efficacious drugs for cancer chemoprevention.

Discovery of colon tumor cell growth inhibitory agents through a combinatorial approach.

Two series with the general formula of 4,6-diaryl-2-oxo-1,2 dihydropyridine-3-carbonitriles and their isosteric 4,6-diaryl-2-imino-1,2-dihydropyridine-3-carbonitrile were synthesized through one pot reaction of the appropriate acetophenone, aldehyde, ammonium acetate with ethyl cyanoacetate or malononitrile, respectively. The synthesized compounds were evaluated for their tumor cell growth inhibitory activity against the human HT-29 colon tumor cell line, as well as their PDE3 inhibitory activity. Compound 4-(2-Ethoxyphenyl)-2-oxo-6-thiophen-3-yl-1,2-dihydropyridine-3 carbonitrile (21) showed tumor cell growth inhibitory activity with an IC50 value of 1.25 microM. Meanwhile, 4-(4-Ethoxyphenyl)-2-imino-6-(thiophen-3-yl)-1,2-dihydropyridine-3-carbonitrile (26) showed inhibitory effect upon PDE3 using cAMP or cGMP as substrate. No correlation exists between PDE3 inhibition and the tumor cell growth inhibitory activity. Docking compound 21 to other possible molecular targets showed the potential to bind PIM1 Kinase.