Adam B. Keeton

Suppression of Wnt/β-catenin signaling inhibits prostate cancer cell proliferation

Although mounting evidence has demonstrated an important role of Wnt/beta-catenin signaling in the development and progression of cancer, the therapeutic potential of small molecules that target this pathway for prostate cancer remains largely unknown. We reported herein that the highly invasive androgen-independent PC-3 and DU145 human prostate cancer cells exhibited higher levels of Wnt/beta-catenin signaling than the androgen-dependent LNCaP prostate cancer cells and non-cancerous PZ-HPV-7 and PWR-1E prostate cells, and that exogenous Wnt3A treatment exaggerated the difference of the Wnt/beta-catenin signaling levels among these prostate cells. Furthermore, we demonstrated that the non-steroidal anti-inflammatory drug, sulindac sulfide, the cyclooxygenase-2 (COX-2) selective inhibitor, celecoxib, and the nitric oxide-donating aspirin derivative, NO-ASA, blocked Wnt/beta-catenin signaling in PC-3 and DU145 cells. These effects occurred at concentrations comparable to those required to inhibit cell proliferation, indicating that the inhibitory effect of these drugs on prostate cancer cell proliferation may involve the suppression of Wnt/beta-catenin signaling. Finally, we showed that a novel small molecule inhibitor of Wnt/beta-catenin signaling, PKF118- 310, inhibited Wnt/beta-catenin signaling and proliferation in prostate cancer cells within the same concentration range. Together, these results suggest that small molecules that inhibit Wnt/beta-catenin signaling have therapeutic potential for the prevention or treatment of prostate cancer.

A Novel Sulindac Derivative That Does Not Inhibit Cyclooxygenases but Potently Inhibits Colon Tumor Cell Growth and Induces Apoptosis with Antitumor Activity

Nonsteroidal anti-inflammatory drugs such as sulindac have shown promising antineoplastic activity, although toxicity from cyclooxygenase (COX) inhibition and the suppression of prostaglandin synthesis limits their use for chemoprevention. Previous studies have concluded that the mechanism responsible for their antineoplastic activity may be COX independent. To selectively design out the COX inhibitory activity of sulindac sulfide (SS), in silico modeling studies were done that revealed the crucial role of the carboxylate moiety for COX-1 and COX-2 binding. These studies prompted the synthesis of a series of SS derivatives with carboxylate modifications that were screened for tumor cell growth and COX inhibitory activity. A SS amide (SSA) with a N,N-dimethylethyl amine substitution was found to lack COX-1 and COX-2 inhibitory activity, yet potently inhibit the growth of human colon tumor cell lines, HT-29, SW480, and HCT116 with IC50 values of 2 to 5 μmol/L compared with 73 to 85 μmol/L for SS. The mechanism of growth inhibition involved the suppression of DNA synthesis and apoptosis induction. Oral administration of SSA was well-tolerated in mice and generated plasma levels that exceeded its in vitro IC50 for tumor growth inhibition. In the human HT-29 colon tumor xenograft mouse model, SSA significantly inhibited tumor growth at a dosage of 250 mg/kg. Combined treatment of SSA with the chemotherapeutic drug, Camptosar, caused a more sustained suppression of tumor growth compared with Camptosar treatment alone. These results indicate that SSA has potential safety and efficacy advantages for colon cancer chemoprevention as well as utility for treating malignant disease if combined with chemotherapy.

Sulindac sulfide selectively inhibits growth and induces apoptosis of human breast tumor cells by phosphodiesterase 5 inhibition, elevation of cyclic GMP, and activation of protein kinase G

Sulindac displays promising antineoplastic activity, but toxicities from cyclooxygenase inhibition limit its use for chemoprevention. Previous reports suggest that its anticancer properties may be attributed to a cyclooxygenase-independent mechanism, although alternative targets have not been well defined. Here, we show that sulindac sulfide (SS) induces apoptosis and inhibits the growth of human breast tumor cells with IC50 values of 60 to 85 μmol/L. Within the same concentration range, SS inhibited cyclic GMP (cGMP) hydrolysis in tumor cell lysates but did not affect cyclic AMP hydrolysis. SS did not induce apoptosis of normal human mammary epithelial cells (HMEC) nor did it inhibit phosphodiesterase (PDE) activity in HMEC lysates. SS increased intracellular cGMP levels and activated protein kinase G in breast tumor cells but not HMEC. The guanylyl cyclase (GC) activator, NOR-3, and cGMP PDE inhibitors, trequinsin and MY5445, displayed similar growth-inhibitory activity as SS, but the adenylyl cyclase activator, forskolin, and other PDE inhibitors had no effect. Moreover, GC activation increased the sensitivity of tumor cells to SS, whereas GC inhibition reduced sensitivity. By comparing PDE isozyme profiles in breast tumor cells with HMEC and determining the sensitivity of recombinant PDE isozymes to SS, PDE5 was found to be overexpressed in breast tumor cells and selectively inhibited by SS. The mechanism of SS binding to the catalytic domain of PDE5 was revealed by molecular modeling. These data suggest that PDE5 inhibition is responsible for the breast tumor cell growth–inhibitory and apoptosis-inducing activity of SS and may contribute to the chemopreventive properties of sulindac. [Mol Cancer Ther 2009;8(12):3331–40]

Inhibition of PDE5 by sulindac sulfide selectively induces apoptosis and attenuates oncogenic Wnt/β-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 β-catenin phosphorylation, decreased β-catenin mRNA and protein levels, reduced β-catenin nuclear localization, decreased T-cell factor/lymphoid enhancer factor (Tcf/Lef) promoter activity, and decreased expression of Wnt/β-catenin–regulated proteins. Suppression of PDE5 with siRNA or known PDE5 inhibitors was sufficient to selectively induce apoptosis and attenuate β-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/β-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. Cancer Prev Res; 4(8); 1275–84. ©2011 AACR.

Sulindac selectively inhibits colon tumor cell growth by activating the cGMP/PKG pathway to suppress Wnt/β-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 β-catenin to inhibit Wnt/β-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. Mol Cancer Ther; 12(9); 1848–59. ©2013 AACR.

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 β-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 β-catenin transcriptional activity may contribute to the chemopreventive properties of certain NSAIDs. Cancer Prev Res; 3(10); 1303–13. ©2010 AACR.

A Novel Sulindac Derivative that Potently Suppresses Colon Tumor Cell Growth by Inhibiting cGMP Phosphodiesterase and β-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 β-catenin as evident by its ability to reduce β-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. Cancer Prev Res; 5(6); 822–33. ©2012 AACR.

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.

Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell growth and β-catenin-dependent TCF transcriptional activity

The cyclic nucleotide phosphodiesterase 10A (PDE10) has been mostly studied as a therapeutic target for certain psychiatric and neurological conditions, although a potential role in tumorigenesis has not been reported. Here we show that PDE10 is elevated in human colon tumor cell lines compared with normal colonocytes, as well as in colon tumors from human clinical specimens and intestinal tumors from ApcMin/+ mice compared with normal intestinal mucosa, respectively. An isozyme and tumor-selective role of PDE10 were evident by the ability of small-molecule inhibitors and small interfering RNA knockdown to suppress colon tumor cell growth with reduced sensitivity of normal colonocytes. Stable knockdown of PDE10 by short hairpin RNA also inhibits colony formation and increases doubling time of colon tumor cells. PDE10 inhibition selectively activates cGMP/cGMP-dependent protein kinase signaling to suppress β-catenin levels and T-cell factor (TCF) transcriptional activity in colon tumor cells. Conversely, ectopic expression of PDE10 in normal and precancerous colonocytes increases proliferation and activates TCF transcriptional activity. These observations suggest a novel role of PDE10 in colon tumorigenesis and that inhibitors may be useful for the treatment or prevention of colorectal cancer.

A Novel Sulindac Derivative Inhibits Lung Adenocarcinoma Cell Growth through Suppression of Akt/mTOR Signaling and Induction of Autophagy

Nonsteroidal anti-inflammatory drugs such as sulindac sulfide have shown promising antineoplastic activity in multiple tumor types, but toxicities resulting from COX inhibition limit their use in cancer therapy. We recently described a N,N-dimethylethyl amine derivative of sulindac sulfide, sulindac sulfide amide (SSA), that does not inhibit COX-1 or -2, yet displays potent tumor cell growth–inhibitory activity. Here, we studied the basis for the growth-inhibitory effects of SSA on human lung adenocarcinoma cell lines. SSA potently inhibited the growth of lung tumor cells with IC50 values of 2 to 5 μmol/L compared with 44 to 52 μmol/L for sulindac sulfide. SSA also suppressed DNA synthesis and caused a G0–G1 cell-cycle arrest. SSA-induced cell death was associated with characteristics of autophagy, but significant caspase activation or PARP cleavage was not observed after treatment at its IC50 value. siRNA knockdown of Atg7 attenuated SSA-induced autophagy and cell death, whereas pan-caspase inhibitor ZVAD was not able to rescue viability. SSA treatment also inhibited Akt/mTOR signaling and the expression of downstream proteins that are regulated by this pathway. Overexpression of a constitutively active form of Akt was able to reduce autophagy markers and confer resistance to SSA-induced cell death. Our findings provide evidence that SSA inhibits lung tumor cell growth by a mechanism involving autophagy induction through the suppression of Akt/mTOR signaling. This unique mechanism of action, along with its increased potency and lack of COX inhibition, supports the development of SSA or related analogs for the prevention and/or treatment of lung cancer. Mol Cancer Ther; 12(5); 663–74. ©2013 AACR.