Development of highly sensitive cell-based AKT kinase ELISA for monitoring PI3K beta activity and compound efficacy.

Phosphatidylinositol-3 kinase (PI3K) pathway regulates multiple cellular functions involving cell survival, growth, motility proliferation, apoptosis, and adhesion. These are deregulated in various diseases such as cancer, atherosclerosis, and inflammation. PI3Ks phosphorylate phosphatidylinositol 4,5-biphosphate (PIP2) yielding phosphatidylinositol 3, 4, 5 triphosphate (PIP3) which in turn activate AKT kinase (serine/threonine kinase), the central enzyme in regulation of metabolic functions. Due to their implications in disease pathophysiology, PI3K/AKT inhibitors became attractive targets for pharmaceutical industries. In order to assess the functional response generated by PI3K inhibitors, an appropriate cell-based screening system is essential in any screening cascade. Here we report the development of highly sensitive in-vitro cell-based kinase ELISA which quantifies the phosphorylated AKT kinase (serine 473) and total AKT kinase directly within the cells upon compound treatment. PI3Kß overexpressing NIH3T3 cells stimulated by lysophosphatidic acid was used for PI3K/Akt pathway activation. Assay performance reliability and robustness were determined by percentage coefficient of variation (%CV) and Z factor which demonstrated an excellent agreement with assay guidelines. This 96-well plate medium throughput assay methodology was used to screen novel molecules and proved a commendable tool to study the mechanism of action property and target engagement of novel PI3K inhibitors in drug discovery.

Pioglitazone, an anti-diabetic drug requires sustained MAPK activation for its anti-tumor activity in MCF7 breast cancer cells, independent of PPAR-γ pathway.

BACKGROUND: The thiazolidinedione (TZD) class of peroxisome proliferator-activated receptor gamma (PPAR-γ) ligands are known for their ability to induce adipocyte differentiation, to increase insulin sensitivity including anticancer properties. But, whether or not upstream events like MAPK activation or PPAR-γ signaling are involved or associated with this anticancer activity is not well understood in breast cancer cells. The role of MAPK and PPAR pathways during the pioglitazone (Pio) induced PPAR-γ independent anticancer activity in MCF7 cells has been focused here. METHODS: The anticancer activity of Pio has been investigated in breast cancer cells in vitro. Anti-tumor effects were assessed by alamar blue assay, Western blot analysis, cell cycle analysis, and annexin V-FITC/PI binding assay by flow cytometry, Hoechst staining and luciferase assay. RESULTS: The anticancer activity of Pio is found to be correlating with the up regulation of CDKIs (p21/p27) and down regulation of CDK-4. This study demonstrates that the induction of CDKIs by Pio is due to the sustained activation of MAPK. The Pio-mediated activation of MAPK is transmitted to activate ELK-1 and the related anti-proliferation is blocked by MEK inhibitor (PD-184352). CONCLUSIONS: Pio suppresses the proliferation of MCF7 cells, at least partly by a PPAR-γ-independent mechanism involving the induction of p21 which in turn requires sustained activation of MAPK. These findings implicate the utility of Pio in the treatment of PPAR positive or negative human cancers and the development of a new class of compounds to enhance the effectiveness of Pio.

Development of phosphocellulose paper-based screening of inhibitors of lipid kinases: case study with PI3Kß.

The phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate the cellular signal transduction pathways involved in cell growth, proliferation, survival, apoptosis, and adhesion. Deregulation of these pathways are common in oncogenesis, and they are known to be altered in other metabolic disorders as well. Despite its huge potential as an attractive target in these diseases, there is an unmet need for the development of a successful inhibitor. Unlike protein kinase inhibitors, screening for lipid kinase inhibitors has been challenging. Here we report, for the first time, the development of a radioactive lipid kinase screening platform using a phosphocellulose plate that involves transfer of radiolabeled [γ-(32)P]ATP to phosphatidylinositol 4,5-phosphate forming phosphatidylinositol 3,4,5-phosphate, captured on the phosphocellulose plate. Enzyme kinetics and inhibitory properties were established in the plate format using standard inhibitors, such as LY294002, TGX-221, and wortmannin, having different potencies toward PI3K isoforms. ATP and lipid apparent Km for both were determined and IC50 values generated that matched the historical data. Here we report the use of a phosphocellulose plate for a lipid kinase assay (PI3Kß as the target) as an excellent platform for the identification of novel chemical entities in PI3K drug discovery.

Biochanin-A, an isoflavon, showed anti-proliferative and anti-inflammatory activities through the inhibition of iNOS expression, p38-MAPK and ATF-2 phosphorylation and blocking NFκB nuclear translocation.

Biochanin-A, an isoflavone, existing in red clover, cabbage and alfalfa, has an inhibitory and apoptogenic effect on certain cancer cells. However, the actual mechanism by which this compound inhibits proliferation and induces apoptosis in cancer cells and the mechanism of its anti-inflammatory activities have not been well characterized. In this study, we have investigated the anti-inflammatory and anti-proliferative activity of Biochanin-A. The effects of Biochanin-A on RAW 264.7, HT-29 cell lines and mouse peritoneal macrophages have been investigated in vitro. Cell proliferation and anti-inflammatory effects were analyzed by 3-(4-5-dimethylthiozol-2-yl)2-5-diphenyl-tetrazolium bromide (MTT) assay, (3)H-thymidine incorporation assay, Western blot, cytokines estimation, Luciferase assay, Electrophoretic mobility shift assay (EMSA) and Kinase assay. Present investigation demonstrated that, Biochanin-A inhibited lipopolysacharide (LPS)-induced nitric oxide(NO) production in macrophage and showed dose dependent inhibition of inducible nitric oxide synthase (iNOS) expression. The induction of NF-κB binding activity by LPS was inhibited markedly by co-incubation with different doses of Biochanin-A. Biochanin-A inhibited the LPS-induced IkB kinase (IKK) activity and nuclear factor kappa beta (NF-κB) activation associated with the inhibition of iNOS expression. LPS-induced phosphorylation of IκBα and p38 MAPK was blocked by Biochanin-A and it inhibited IL-6, IL-1ß and TNF-α production in RAW264.7 cells indicating its anti-inflammatory activity in association with anti-proliferation. Biochanin-A is important for the prevention of phosphorylation and degradation of IκBα, thereby blocking NF-κB activation, which in turn leads to decreased expression of the iNOS, thus preventing proliferation and inflammation.

The major chemical-detoxifying system of UDP-glucuronosyltransferases requires regulated phosphorylation supported by protein kinase C.

Finding rapid, reversible down-regulation of human UDP-glucuronosyltransferases (UGTs) in LS180 cells following curcumin treatment led to the discovery that UGTs require phosphorylation. UGTs, distributed primarily in liver, kidney, and gastrointestinal tract, inactivate aromatic-like metabolites and a vast number of dietary and environmental chemicals, which reduces the risk of toxicities, mutagenesis, and carcinogenesis. Our aim here is to determine relevant kinases and mechanism(s) regulating phosphorylation of constitutive UGTs in LS180 cells and 10 different human UGT cDNA-transfected COS-1 systems. Time- and concentration-dependent inhibition of immunodetectable [(33)P]orthophosphate in UGTs and protein kinase Cepsilon (PKCepsilon), following treatment of LS180 cells with curcumin or the PKC inhibitor calphostin-C, suggested UGT phosphorylation is supported by active PKC(s). Immunofluorescent and co-immunoprecipitation studies with UGT-transfected cells showed co-localization of UGT1A7His and PKCepsilon and of UGT1A10His and PKCalpha or PKCdelta. Inhibition of UGT activity by PKCepsilon-specific antagonist peptide or by PKCepsilon-targeted destruction with PKCepsilon-specific small interference RNA and activation of curcumin-down-regulated UGTs with typical PKC agonists verified a central PKC role in glucuronidation. Moreover, in vitro phosphorylation of nascent UGT1A7His by PKCepsilon confirms it is a bona fide PKC substrate. Finally, catalase or herbimycin-A inhibition of constitutive or hydrogen peroxide-activated-UGTs demonstrated that reactive oxygen species-related oxidants act as second messengers in maintaining constitutive PKC-dependent signaling evidently sustaining UGT phosphorylation and activity. Because cells use signal transduction collectively to detect and respond appropriately to environmental changes, this report, combined with our earlier demonstration that specific phospho-groups in UGT1A7 determined substrate selections, suggests regulated phosphorylation allows adaptations regarding differential phosphate utilization by UGTs to function efficiently.

A crystalline compound (BM-ANF1) from the Indian toad (Bufo melanostictus, Schneider) skin extract, induced antiproliferation and apoptosis in leukemic and hepatoma cell line involving cell cycle proteins.

In our earlier communication, it was reported that Indian toad (Bufo melanostictus) skin extract (TSE) possesses antiproliferative and apoptogenic activity in U937 and K562 cells [Giri et al., 2006. Antiproliferative, cytotoxic and apoptogenic activity of Indian toad (Bufo melanostictus, Schneider) skin extract in U937 and K562 cells. Toxicon 48 (4), 388-400]. In the present study, a compound (BM-ANF1) has been isolated from the TSE by alumina gel column chromatography, crystallized and evaluated for its antiproliferative and apoptogenic activity in U937, K562 and HepG2 cells. BM-ANF1 produced dose-dependent inhibition of U937, K562 and HepG2 cell growth. The antiproliferative activity was reflected by the MTT assay and demonstrated by the reduced expression of proliferative cell nuclear antigen (PCNA). Flow-cytometric analysis showed that BM-ANF1 arrested the cell cycle at G1 phase and enhanced annexin-V binding in U937 and K562 cells. Scanning electron microscopic and fluorescent microscopic analysis of U937 and K562 cells revealed the apoptogenic nature of the compound. Alkaline comet assay showed that BM-ANF1 produced DNA fragmentation. The dose-dependent expression of caspase 3 indicated that the apoptogenic properties of BM-ANF1 were mediated through the activation of downstream effector nucleases in the cancer cells. The increased expression of p53 and moderate expression of p21(Cip1)/p27(Kip1) due to BM-ANF1 treatment in HepG2 cells supported that the apoptogenic activity of BM-ANF1 was mediated through p53 tumor-suppressor gene expression followed by the expression of p21(Cip1) and p27(Kip1) and it was likely to be linked with cell cycle arrest at G1 phase in cancer cells. From the present study, it may be suggested that the crystalline compound, BM-ANF1, was antiproliferative and apoptogenic in human leukemic and hepatoma cells.

NRH:quinone oxidoreductase 2 and NAD(P)H:quinone oxidoreductase 1 protect tumor suppressor p53 against 20s proteasomal degradation leading to stabilization and activation of p53.

Tumor suppressor p53 is either lost or mutated in several types of cancer. MDM2 interaction with p53 results in ubiquitination and 26S proteasomal degradation of p53. Chronic DNA damage leads to inactivation of MDM2, stabilization of p53, and apoptotic cell death. Here, we present a novel MDM2/ubiquitination-independent mechanism of stabilization and transient activation of p53. The present studies show that 20S proteasomes degrade p53. The 20S degradation of p53 was observed in ubiquitin-efficient and -deficient cells, indicating that this pathway of degradation did not require ubiquitination of p53. The cytosolic quinone oxidoreductases [NRH:quinone oxidoreductase 2 (NQO2) and NAD(P)H:quinone oxidoreductase 1 (NQO1)] interacted with p53 and protected p53 against 20S proteasomal degradation. Further studies revealed that acute exposure to radiation or chemical leads to induction of NQO1 and NQO2 that stabilizes and transiently activates p53 and downstream genes. These results suggest that stress-induced NQO1 and NQO2 transiently stabilize p53, which leads to protection against adverse effects of stressors.

Targeted inhibition of glucuronidation markedly improves drug efficacy in mice - a model.

Finding UDP-glucuronosyltransferases (UGT) require protein kinase C-mediated phosphorylation is important information that allows manipulation of this critical system. UGTs glucuronidate numerous aromatic-like chemicals derived from metabolites, diet, environment and, inadvertently, therapeutics to reduce toxicities. As UGTs are inactivated by downregulating PKCs with reversibly-acting dietary curcumin, we determined the impact of gastro-intestinal glucuronidation on free-drug uptake and efficacy using immunosuppressant, mycophenolic acid (MPA), in mice. Expressed in COS-1 cells, mouse GI-distributed Ugt1a1 glucuronidates curcumin and MPA and undergoes irreversibly and reversibly dephosphorylation by PKC-specific inhibitor calphostin-C and general-kinase inhibitor curcumin, respectively, with parallel effects on activity. Moreover, oral curcumin-administration to mice reversibly inhibited glucuronidation in GI-tissues. Finally, successive oral administration of curcumin and MPA to antigen-treated mice increased serum free MPA and immunosuppression up to 9-fold. Results indicate targeted inhibition of GI glucuronidation in mice markedly improved free-chemical uptake and efficacy using MPA as a model.

BALT development and augmentation of hyperoxic lung injury in mice deficient in NQO1 and NQO2.

NAD(P)H/NRH:quinone oxidoreductases (NQO1 and NQO2) protect against oxidative stress and neoplasia. Cross-breeding of NQO1-/- with NQO2-/- mice generated double-knockout (DKO) mice. DKO mice were born normal yet showed myelogenous hyperplasia as observed in single-knockout mice. DKO mice also showed bronchial-associated lymphoid tissue (BALT) that increased in number and size with age. BALT was absent in wild-type and single-knockout mice. Further analysis demonstrated infiltration of neutrophils and macrophages in BALT and significant increases in the serum cytokines TNFalpha, IL-6, and IL-1beta and increased expression of iNOS and higher nitric oxide in lung macrophages. The development of BALT in DKO mice presumably led to the release of cytokines and higher lung macrophage activation, because histologically spleen, thymus, and blood cultures and urine analysis showed absence of infection. Additionally, the DKO mice upon exposure to hyperoxia demonstrated severe intra-alveolar edema and perivascular inflammation and massive infiltration with neutrophils, compared with wild-type mice. These results suggest that NQO1 and NQO2 combined protect mice against lung inflammation, BALT, and hyperoxic lung injury.

Human UDP-glucuronosyltransferases show atypical metabolism of mycophenolic acid and inhibition by curcumin.

Although the promising immunosuppressant, mycophenolic acid (MPA), has many desirable properties and is widely prescribed for organ transplant recipients, its high oral dosage requirement is not understood. Whereas previous Northern blot analysis by Basu and colleagues (2004) located the mRNAs encoding MPA primary metabolizers, UDP-glucuronosyltransferases (UGTs) 1A7, 1A8, 1A9, and 1A10, in human gastrointestinal (GI) tissues, in situ hybridization analysis of mRNAs found that the isozymes were restricted to the mucosal layer of various GI organs. Concomitantly, MPA was glucuronidated by microsomes isolated from normal adjoining specimens. Microsomal studies showed the highest relative rates of metabolism in esophagus, ileum, duodenum, colon, and stomach at pH 6.4; only esophagus showed high pH 7.6 activity. Properties of the recombinant UGTs indicate that MPA is metabolized with pH 6.4 or 7.6 optimum. Activity for 1A7 and 1A9 increased with increasing concentrations up to 2.4 mM, with parallel production of both ether- and acylglucuronides; similarly, 1A8 and 1A10 reached plateaus at 1.6 mM, producing both glucuronides. K(m) values were 250 to 550 microM. Between 400 and 1600 microM MPA, isozymes generated between 15 and 42% of the acylglucuronides. In effect, high K(m) values (MPA) are associated with high concentrations to achieve saturation kinetics. Finally, transient inhibition of UGTs in human LS180 colon cells and mouse duodenum by the dietary agent, curcumin, has implications for in vivo pretreatment to reduce MPA glucuronidation to increase the therapeutic index.

Targeting of parasite-specific immunoliposome-encapsulated doxorubicin in the treatment of experimental visceral leishmaniasis.

A parasite-specific 51-kDa protein has been isolated from the membrane of macrophages infected with Leishmania donovani, the causative agent of visceral leishmaniasis. Active targeting of doxorubicin to infected macrophages was studied by incorporating it in immunoliposomes prepared by grafting F(ab)'(2) of anti-51-kDa antibody onto the liposomal surface. In a 45-day mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by doxorubicin incorporated in immunoliposome (immunodoxosome) at a dose of 250 microg/kg/day that was given for 4 consecutive days. A similar dose of free and liposomal drug (doxosome) had 45% and 84% parasite suppressive effects, respectively. Immunodoxosome and doxosome were generally less toxic than the free drug, as determined by several clinical parameters of cardiotoxicity and liver toxicity. These results not only indicate the potential of doxorubicin as an effective chemotherapeutic agent but also establish the use of immunoliposomes as drug carrier in the therapy of leishmaniasis.

Differential and special properties of the major human UGT1-encoded gastrointestinal UDP-glucuronosyltransferases enhance potential to control chemical uptake.

UDP-glucuronosyltransferase (UGT) isozymes detoxify metabolites, drugs, toxins, and environmental chemicals via conjugation to glucuronic acid. Based on the extended UGT1 locus combined with Northern blot analysis and in situ hybridization, we determined the distribution of UGT1A1 and UGT1A7 through UGT1A10 mRNAs and found them for the first time segmentally distributed in the mucosal epithelia layer of the gastrointestinal tract. Biochemically, recombinant isozymes exhibited pH optima of 5.5, 6.4, 7.6, 8.5, and/or a broad pH range, and activities were found to be unaffected or progressively inhibited by increasing substrate concentrations after attaining Vmax for certain chemicals. Under different optimal conditions, all exhibited wide substrate selections for dietary and environmentally associated chemicals. Evidence also suggests tandem effects of isozymes in the time for completion of reactions when comparing short- and long-term incubations. Moreover, treatment of colon cells with certain diet-associated constituents, curcumin and nordihydroguaiaretic acid, reversibly targets UGTs causing inhibition without affecting protein levels; there is no direct inhibition of control UGT using curcumin as substrate in the in vitro assay. In summary, we demonstrate that UGTs are located in gastrointestinal mucosa, have vast overlapping activities under differential optimal conditions, and exhibit marked sensitivity to certain dietary substrates/constituents, representing a first comprehensive study of critical properties concerning glucuronidating isozymes in alimentary tissues. Additionally, the highly dynamic, complex, and variable properties necessarily impact absorption of ingested chemicals and therapeutic drugs.

Evidence for phosphorylation requirement for human bilirubin UDP-glucuronosyltransferase (UGT1A1) activity.

Our discovery of rapid down-regulation of human bilirubin UDP-glucuronosyltransferase (UGT) in colon cell lines that was transient and irreversible following curcumin- and calphostin-C-treatment, respectively, suggested phosphorylation event(s) were involved in activity. Likewise, bilirubin-UGT1A1 expressed in COS-1 cells was inhibited by curcumin and calphostin-C. Because calphostin-C is a highly specific protein kinase C (PKC) inhibitor, we examined and found 4 to 5 predicted PKC phosphorylation sites in 11 UGTs examined. UGT1A1 incorporated [33P]orthophosphate, which was inhibited by calphostin-C. Also triple mutant, T75A/T112A/S435G-UGT1A1, at predicted PKC sites failed to incorporate [33P]orthophosphate. Individual or double mutants exhibited dominant-negative, additive, or no effect, while the triple mutant retained 10-15% activity towards bilirubin and two xenobiotics. Compared to wild-type, S435G and T112A/S435G shifted pH-optimum for eugenol, but not for bilirubin or anthraflavic acid, toward alkaline and acid conditions, respectively. This represents the first evidence that a UGT isozyme requires phosphorylation for activity.