Sodium selenite increases the activity of the tumor suppressor protein, PTEN, in DU-145 prostate cancer cells.

Epidemiological and clinical data suggest that selenium may prevent prostate cancer; however, the cellular effects of selenium in malignant prostate cells are not well understood. We previously reported that the activity of the tumor suppressor PTEN is modulated by thioredoxin (Trx) in a RedOx-dependent manner. In this study, we demonstrated that the activity of Trx reductase (TR) is increased by sevenfold in the human prostate cancer cell line, DU-145, after 5 days of sodium selenite (Se) treatment. The treatment of DU-145 cells with increasing concentrations of Se induced an increase in PTEN lipid phosphatase activity by twofold, which correlated with a decrease in phospho-ser(473)-Akt, and an increase in phospho-Ser(370)-PTEN levels. Se also increased casein kinase-2 (CK2) activity; and the use of apigenin, an inhibitor of CK2, revealed that the regulation of the tumor suppressor PTEN by Se may be achieved via both the Trx-TR system and the RedOx control of the kinase involved in the regulation of PTEN activity.

Discovery of a novel class of AKT pleckstrin homology domain inhibitors.

AKT, a phospholipid-binding serine/threonine kinase, is a key component of the phosphoinositide 3-kinase cell survival signaling pathway that is aberrantly activated in many human cancers. Many attempts have been made to inhibit AKT; however, selectivity remains to be achieved. We have developed a novel strategy to inhibit AKT by targeting the pleckstrin homology (PH) domain. Using in silico library screening and interactive molecular docking, we have identified a novel class of non-lipid-based compounds that bind selectively to the PH domain of AKT, with "in silico" calculated K(D) values ranging from 0.8 to 3.0 micromol/L. In order to determine the selectivity of these compounds for AKT, we used surface plasmon resonance to measure the binding characteristics of the compounds to the PH domains of AKT1, insulin receptor substrate-1, and 3-phosphoinositide-dependent protein kinase 1. There was excellent correlation between predicted in silico and measured in vitro K(D)s for binding to the PH domain of AKT, which were in the range 0.4 to 3.6 micromol/L. Some of the compounds exhibited PH domain-binding selectivity for AKT compared with insulin receptor substrate-1 and 3-phosphoinositide-dependent protein kinase 1. The compounds also inhibited AKT in cells, induced apoptosis, and inhibited cancer cell proliferation. In vivo, the lead compound failed to achieve the blood concentrations required to inhibit AKT in cells, most likely due to rapid metabolism and elimination, and did not show antitumor activity. These results show that these compounds are the first small molecules selectively targeting the PH domain of AKT.

The phosphatidylinositol-3-kinase inhibitor PX-866 overcomes resistance to the epidermal growth factor receptor inhibitor gefitinib in A-549 human non-small cell lung cancer xenografts.

Epidermal growth factor receptor (EGFR) inhibitors such as gefitinib show antitumor activity in a subset of non-small cell lung cancer (NSCLC) patients having mutated EGFR. Recent work shows that phosphatidylinositol-3-kinase (PI3-K) is coupled to the EGFR only in NSCLC cell lines expressing ErbB-3 and that EGFR inhibitors do not inhibit PI3-K signaling in these cells. The central role PI3-K plays in cell survival suggests that a PI3-K inhibitor offers a strategy to increase the antitumor activity of EGFR inhibitors in resistant NSCL tumors that do not express ErbB-3. We show that PX-866, a PI3-K inhibitor with selectivity for p110alpha, potentiates the antitumor activity of gefitinib against even large A-549 NSCL xenografts giving complete tumor growth control in the early stages of treatment. A-549 xenograft phospho-Akt was inhibited by PX-866 but not by gefitinib. A major toxicity of PX-866 administration was hyperglycemia with decreased glucose tolerance, which was reversed upon cessation of treatment. The decreased glucose tolerance caused by PX-866 was insensitive to the AMP-activated protein kinase inhibitor metformin but reversed by insulin and by the peroxisome proliferator-activated receptor-gamma activator pioglitazone. Prolonged PX-866 administration also caused increased neutrophil counts. Thus, PX-866, by inhibiting PI3-K signaling, may have clinical use in increasing the response to EGFR inhibitors such as gefitinib in patients with NSCLC and possibly in other cancers who do not respond to EGFR inhibition.

Thioredoxin-1 binds to the C2 domain of PTEN inhibiting PTEN's lipid phosphatase activity and membrane binding: a mechanism for the functional loss of PTEN's tumor suppressor activity.

Thioredoxin-1 (Trx-1) is a 12 kDa redox protein that is overexpressed in a large number of human tumors. Elevated Trx-1 is associated with increased tumor cell proliferation, inhibited apoptosis, aggressive tumor growth, and decreased patient survival. The molecular mechanisms for the promotion of tumorigenesis by Trx-1 are not known. PTEN is a major tumor suppressor of human cancer that acts by hydrolyzing membrane phosphatidylinositol (PtdIns)-3-phosphates, thus, preventing the activation of the survival signaling kinase Akt by PtdIns-3-kinase. We show that Trx-1 binds in a redox dependent manner to PTEN to inhibit its PtdIns-3-phosphatase activity which results in increased Akt activation in cells. Molecular docking and site-specific mutation studies show that the binding of Trx-1 to PTEN occurs through a disulfide bond between the active site Cys(32) of Trx-1 and Cys(212) of the C2 domain of PTEN leading to steric interference by bound Trx-1 of the catalytic site of PTEN and of the C2 lipid membrane-binding domain. The results of the study suggest that the increased levels of Trx-1 in human tumors could lead to functional inhibition of PTEN tumor suppressor activity providing an additional mechanism for tumorigenesis with loss of PTEN activity.

Molecular pharmacology and antitumor activity of PX-866, a novel inhibitor of phosphoinositide-3-kinase signaling.

We have developed biologically stable semisynthetic viridins as inhibitors of phosphoinositide (PtdIns)-3-kinases. The most active compound was PX-866 (acetic acid (1S,4E,10R,11R,13S,14R)-[4-diallylaminomethylene-6-hydroxy-1-methoxymethyl-10,13-dimethyl-3,7,17-trioxo-1,3,4,7,10,11,12,13,14,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-11-yl ester), which inhibited purified PtdIns-3-kinase with an IC50 of 0.1 nmol/L and PtdIns-3-kinase signaling measured by phospho-Ser473-Akt levels in HT-29 colon cancer cells with an IC50 of 20 nmol/L. PX-866 administered to mice at 10 mg/kg inhibited phospho-Ser473-Akt in HT-29 colon tumor xenografts up to 80% with recovery taking >48 hours after p.o. administration but more rapidly after i.v. or i.p. administration. PX-866 was eliminated from mouse plasma with a half-life of 18 minutes and a clearance of 360 mL/min/kg following i.v. administration and, when administered i.p. or p.o., showed first-pass metabolism with sequential N-deallylation. Synthetic standards of the N-deallylated metabolites of PX-866 inhibited PtdIns-3-kinase at low nanomolar per liter concentrations. PX-866 exhibited in vivo antitumor activity against s.c. OvCar-3 human ovarian cancer and A-549 human lung cancer xenografts in immunodeficient mice with log cell kills up to 1.2. PX-866 also increased the antitumor activity of cisplatin against A-549 xenografts and radiation treatment against OvCar-3 xenografts. The results show that PX-866 is a biologically stable broad-spectrum PtdIns-3-kinase inhibitor with good pharmacokinetics that causes prolonged inhibition of PtdIns-3-kinase signaling in human tumor xenografts. PX-866 exhibits single agent in vivo antitumor activity and increases the antitumor effects of cisplatin and radiation treatment.

Synthesis and biological evaluation of synthetic viridins derived from C(20)-heteroalkylation of the steroidal PI-3-kinase inhibitor wortmannin.

A series of viridin analogs was prepared from wortmannin by nucleophilic ring opening at C(20) and evaluated against the signaling kinases PI-3-kinase and mTOR. Several subnanomolar enzyme inhibitors with orders of magnitude selectivity for PI-3-kinase and strong cytotoxic activity against four cancer cell lines were identified. Among the ten most promising derivatives, six demonstrated lower liver toxicity and greater promise for inhibition of tumor cell growth than the lead structure wortmannin.

Increased expression of mitochondrial peroxiredoxin-3 (thioredoxin peroxidase-2) protects cancer cells against hypoxia and drug-induced hydrogen peroxide-dependent apoptosis.

Peroxiredoxin-3 (Prdx3) is a mitochondrial member of the antioxidant family of thioredoxin peroxidases that uses mitochondrial thioredoxin-2 (Trx2) as a source of reducing equivalents to scavenge hydrogen peroxide (H(2)O(2)). Low levels of H(2)O(2) produced by the mitochondria regulate physiological processes, including cell proliferation, while high levels of H(2)O(2) are toxic to the cell and cause apoptosis. WEHI7.2 thymoma cells with stable overexpression of Prdx3 displayed decreased levels of cellular H(2)O(2) and decreased cell proliferation without a change in basal levels of apoptosis. Prdx3-transfected cells showed a marked resistance to hypoxia-induced H(2)O(2) formation and apoptosis. Prdx3 overexpression also protected the cells against apoptosis caused by H(2)O(2), t-butylhydroperoxide, and the anticancer drug imexon, but not by dexamethasone. Thus, mitochondrial Prdx3 is an important cellular antioxidant that regulates physiological levels of H(2)O(2), leading to decreased cell growth while protecting cells from the apoptosis-inducing effects of high levels of H(2)O(2).

Specific inhibition of the Akt1 pleckstrin homology domain by D-3-deoxy-phosphatidyl-myo-inositol analogues.

Activation of Akt (protein kinase B), a Ser/Thr protein kinase that promotes cell survival, has been linked to tumorigenesis. Akt is activated by phosphorylation after binding of its pleckstrin homology (PH) domain to plasma membrane phosphatidyl-myo-inositol-3-phosphates, formed by phosphoinositide-3-kinase. We report a novel strategy to inhibit Akt activation based on the use of D-3-deoxy-phosphatidyl-myo-inositols (DPIs) that cannot be phosphorylated on the 3-position of the myo-inositol ring. We have studied the DPIs, DPI 1-[(R)-2,3-bis(hexadecanoyloxy)propyl hydrogen phosphate], its ether lipid derivative DPI 1-[(R)-2-methoxy-3-octadecyloxypropyl hydrogen phosphate] (DPIEL), and its carbonate derivative DPI 1-[(R)-2-methoxy-3-octadecyloxypropyl carbonate]. We demonstrate in platelet-derived growth factor-stimulated mouse NIH3T3 cells that the DPIs bind to the PH domain of Akt, trapping it in the cytoplasm and thus preventing Akt activation. DPIEL did not inhibit myristylated-Akt, a constitutively active membrane-bound Akt expressed in NIH3T3 cells, and cell growth was not inhibited, unlike in wild-type NIH3T3 cells. Molecular modeling and docking studies show that DPIEL binds with much higher affinity to Akt's PH domain as compared with DPI and DPI 1-[(R)-2-methoxy-3-octadecyloxypropyl carbonate]. This study shows that the DPIs are a novel class of growth inhibitory agents with a novel mechanism of action through binding to the PH domain of Akt and inhibition of Akt activation.