PPAR-γ Agonists for the Treatment of Major Depression: A Review.

Introduction: Selective agonists of the nuclear transcription factor peroxisome proliferator-activated receptor-gamma (PPAR-γ) are used for the treatment of type 2 diabetes. We reviewed their efficacy and safety for the treatment of major depression and the association of their potential antidepressant effects with changes in biomarkers of metabolism and inflammation. Methods: From 8 studies, 4 open-label trials, and 4 randomized controlled trials (RCT) (3 vs. placebo and 1 vs. metformin), 448 patients with major depression were included, of which 209 patients received PPAR-γ agonists (pioglitazone or rosiglitazone) for 6-12 weeks, either alone or in add-on therapy to conventional treatments. Results: PPAR-γ agonists have antidepressant effects in the 4 open-label studies and in 3 out of 4 RCT. No major adverse event was reported. Improvement in depression scores was associated with improvement in 3 biomarkers of insulin resistance (homeostatic model assessment [HOMA-IR], oral glucose tolerance test, and fasting plasma glucose) and 1 biomarker of inflammation (interleukin-6) among 21 biomarkers studied. Conclusion: PPAR-γ agonists may have antidepressant properties, which need to be assessed in further studies of major depressive episodes.

In vitro cytotoxicity of dental composites based on new and traditional polymerization chemistries.

UNLABELLED: The biological response to dental restorative polymer composites is mediated by the release of unpolymerized residual monomers. Several new composite formulations claim to reduce unpolymerized residual mass. The current study assessed the cytotoxic responses to several of these new formations and compared them with more traditional formulations. Our hypothesis predicted that if these new polymerization chemistries reduce unpolymerized residual mass, the cytotoxicity of these materials also should be reduced relative to traditional formulations. METHODS: Materials (HerculiteXRV, Premise, Filtek Supreme, CeramxDuo, Hermes, and Quixfil) were tested in vitro in direct contact with Balb mouse fibroblasts, initially, then after aging in artificial saliva for 0, 1, 3, 5, or 8 weeks. The toxicity was determined by using the MTT assay to the estimate SDH activity. Knoop hardness of the materials also was measured at 0 and 8 weeks to determine whether surface breakdown of the materials in artificial saliva contributed to cytotoxic responses. RESULTS: Materials with traditional methacrylate chemistries (Herculite, Premise, Filtek Supreme) were severely (>50%) cytotoxic throughout the 8-week interval, but materials with newer chemistries or filling strategies (Hermes, CeramXDuo, and Quixfil) improved over time of aging in artificial saliva. Hermes showed the least cytotoxicity at 8 weeks, and was statistically equivalent to Teflon negative controls. Hardness of the materials was unaffected by exposure to artificial saliva. CONCLUSIONS: Newer polymerization and filling strategies for dental composites show promise for reducing the release of unpolymerized components and cytotoxicity.

Intra- and extracellular reactive oxygen species generated by blue light.

Blue light from dental photopolymerization devices has significant biological effects on cells. These effects may alter normal cell function of tissues exposed during placement of oral restorations, but recent data suggest that some light-induced effects may also be therapeutically useful, for example in the treatment of epithelial cancers. Reactive oxygen species (ROS) appear to mediate blue light effects in cells, but the sources of ROS (intra- versus extracellular) and their respective roles in the cellular response to blue light are not known. In the current study, we tested the hypothesis that intra- and extracellular sources of blue light-generated ROS synergize to depress mitochondrial function. Normal human epidermal keratinocytes (NHEK) and oral squamous cell carcinoma (OSC2) cells were exposed to blue light (380-500 nm; 5-60 J/cm(2)) from a dental photopolymerization source (quartz-tungsten-halogen, 550 mW/cm(2)). Light was applied in cell-culture media or balanced salt solutions with or without cells present. Intracellular ROS levels were estimated using the dihydrofluorescein diacetate (DFDA) assay; extracellular ROS levels were estimated using the leucocrystal violet assay. Cell response was estimated using the MTT mitochondrial activity assay. Blue light increased intracellular ROS equally in both NHEK and OSC2. Blue light also increased ROS levels in cell-free MEM or salt solutions, and riboflavin supplements increased ROS formation. Extracellularly applied ROS rapidly (50-400 muM, <1 min) increased intracellular ROS levels, which were higher and longer-lived in NHEK than OSC2. The type of cell-culture medium significantly affected the ability of blue light to suppress cellular mitochondrial activity; the greatest suppression was observed in DMEM-containing or NHEK media. Collectively, the data support our hypothesis that intra- and extracellularly generated ROS synergize to affect cellular mitochondrial suppression of tumor cells in response to blue light. However, the identity of blue light targets that mediate these changes remain unclear. These data support additional investigations into the risks of coincident exposure of tissues to blue light during material polymerization of restorative materials, and possible therapeutic benefits.

Atypical protein kinase Czeta suppresses migration of mouse melanoma cells.

Mouse melanoma B16 F1 cells cultured in RPMI 1640 supplemented with the melanin precursors tyrosine and phenylalanine display increased melanin levels and elevated migration while down-regulating protein kinase C (PKC)zeta to low levels. Although control experiments rule out a direct role by melanin, PKCzeta down-regulation is shown to be a critical determinant of cell migration. Transfection of high-motility cells with either wild-type PKCzeta or its regulatory domain suppresses migration. Known to bind to the regulatory domain of PKCzeta, the proapoptotic protein prostate apoptosis response-4 (Par-4) coimmunoprecipitates with PKCzeta as a 47-kDa protein. Transfection of Par-4 (or its leucine zipper element) further suppresses migration of low-motility cells (which express high levels of PKCzeta), whereas high-motility cells (which express low levels of PKCzeta) are unaffected by Par-4 overexpression. It is proposed that in nonmetastatic cells, the PKCzeta Par-4 complex provides a brake on migration that is released by melanin precursors that initiate PKCzeta down-regulation. Elevation of PKCzeta in melanoma cells, or preventing its down-regulation through the dietary restriction of tyrosine and phenylalanine, may therefore control metastatic behavior.

Photo-induced inactivation of protein kinase calpha by dequalinium inhibits motility of murine melanoma cells.

Dequalinium (DECA) is a potent antitumor agent and inhibitor of protein kinase C (PKC). Previously it was shown that PKCalpha activity in vitro could be irreversibly inhibited when treated with DECA at low micromolar concentrations and irradiated with 366 nm of light. This approach was used to probe the role of intracellular PKC activity in the motility of metastatic murine melanoma B16 F10 cells and as a target for DECA analogs with increasing PKC inhibitory potencies. Pretreatment of a monolayer of B16 F10 cells with 250 nM of a DECA analog in the presence of UV irradiation for 5 min resulted in 1) complete inhibition of cell motility for up to 4 h in a time-lapse motility assay and 40 to 60% inhibition of cell migration in a Boyden chamber, and 2) inhibition by 40 to 60% of intracellular phosphatidylserine/Ca(2+)-dependent PKC catalytic activity, signifying inactivation of a conventional PKC isoform. Because PKCalpha is the only conventional PKC isoform detected in B16 F10 cells, a stably transfected clone expressing a kinase-defective mutant of PKCalpha was developed that exhibited a substantial loss of adhesion and motility and was refractory to further inhibition by DECA. These findings identify PKCalpha catalytic activity both as a mechanistic component of cell motility and adhesion and as a critical intracellular target of DECA. These studies further suggest that the combined use of UV with nanomolar concentrations of DECA offers an effective chemotherapeutic approach to inhibit metastatic behavior of melanoma cells.

Scanning electrochemical microscopy of living cells: different redox activities of nonmetastatic and metastatic human breast cells.

Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Calpha), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators.

Inhibition of protein kinase C(alpha) by dequalinium analogues: dependence on linker length and geometry.

Analogues of a bipartite compound, dequalinium (DECA) (quinolinium, 1,1'-(1,10-decanediyl)bis(4-amino-2-methyl diiodide)), were tested for inhibition of protein kinase C(alpha) (PKC(alpha)). In vitro assays of monomeric and dimeric analogues support a model in which DECA inhibits PKC(alpha) by an obligatory two-point contact, a unique mechanism among PKC inhibitors. The presence of unsaturation in the center of the C(10)-alkyl linker produced geometric isomers with different inhibitory potencies: cis IC(50) = 52 +/- 12 microM and trans IC(50) = 12 +/- 3 microM, where the trans isomer was equipotent to that of the saturated C(10)-DECA. DECA analogues with longer, saturated linkers (C(12), C(14), or C(16)) exhibited enhanced inhibitory potencies which reached a plateau with the C(14)-linker (IC(50) = 2.6 +/- 0.2 microM). Metastatic melanoma cells treated with 250 nM C(12)-, C(14)-, or C(16)-DECA and irradiated with long-wave UV light (which causes irreversible inhibition of PKC(alpha) by DECA) confirmed the linker-dependent inhibition of intracellular PKC(alpha) activity.

Overexpression of protein kinase Calpha in MCF-10A human breast cells engenders dramatic alterations in morphology, proliferation, and motility.

A nonmetastatic human mammary epithelial cell line (MCF-10A) was engineered to overproduce protein kinase Calpha (PKCalpha) so as to investigate a role for this isoform in the metastatic phenotype. PKCalpha transfectants (clone 26alpha) expressed an 8-fold higher level of PKCalpha protein without compensatory alterations in other isoforms. Clone 26alpha proliferated slowly (accumulating in G1 of the cell cycle) but exhibited pronounced increases in motility and adhesion. Elevated expression of cell cycle inhibitor p27 and focal adhesion proteins was observed, whereas E-cadherin expression decreased to undetectable levels. These observations were consistent with the morphology of PKCalpha transfectants (large, disaggregated, and flat, with lamellipodia and extensive actin fibers) and control cells (small, aggregated, and refractile). Treatment with PKC inhibitors or transfection of a dominant negative (dn) mutant of Rac1, but neither dn RhoA nor dn cdc42, reduced the motility of clone 26alpha, implicating PKCalpha catalytic activity and endogenous Rac1, respectively, in the PKCalpha-induced phenotype. Overall, PKCalpha overexpression suppresses proliferation while endowing MCF-10A cells with properties consistent with the metastatic phenotype.

Deletion analysis of protein kinase Calpha reveals a novel regulatory segment.

Using a combined pharmacological and genetic approach, we have identified aa 260-280 in the C2 region as a critical factor in the catalytic function of protein kinase Calpha (PKCalpha). Progressive truncations from the N-terminus as well as selected internal deletion mutants were expressed in Saccharomyces cerevisiae and tested for altered sensitivity to dequalinium, a PKC inhibitor whose target site was previously mapped to the catalytic domain. PKC mutants representing truncations of up to 158 amino acid residues (aa) from the N-terminus (ND84 and ND158) displayed 60-63% inhibition of kinase activity by 50 microM dequalinium, somewhat more sensitive than the wild-type PKCalpha enzyme (45% inhibition). Mutant ND262, lacking N-terminal aa 1-262, was inhibited by almost 72% with 50 microM dequalinium, but mutant ND278, which lacked an additional 16 aa, was inhibited by only 9% of total activity. This result suggests that a C-terminal segment of the C2 region (aa 263-278) influences inhibition by dequalinium at low micromolar concentrations. An internal deletion mutant (D260-280) which retains the entire primary structure of PKCalpha except for aa 260-280, was similarly inhibited by only 4% with 50 microM dequalinium. In the absence of dequalinium and despite the presence of a nearly complete regulatory domain, this mutant exhibited constitutive activity (both in vitro and in a phenotypic assay with S. cerevisiae) that could not be further stimulated even by the potent activator TPA. Taken together, our findings suggest that, in the native structure of PKCalpha, the segment described by aa 260-280 regulates PKCalpha activity and influences the sensitivity of PKCalpha to dequalinium.

Photoinduced inactivation of protein kinase C by dequalinium identifies the RACK-1-binding domain as a recognition site.

1,1'-Decamethylenebis-4-aminoquinaldinium diiodide (DECA; dequalinium) is an anti-tumor agent and protein kinase C (PKC) inhibitor whose mechanism of action with PKC is unknown. This study reports that with human PKC alpha, DECA exhibited competitive inhibition (Ki = 11.5 +/- 5 microM) with respect to RACK-1 (receptor for activated C kinase-1), an adaptor protein that has been proposed to bind activated PKC following translocation (Ron, D., Luo, J., and Mochly-Rosen, D. (1995) J. Biol. Chem. 270, 24180-24187). When exposed to UV light, DECA covalently modified and irreversibly inhibited PKC (alpha or beta), with IC50 = 7-18 microM. UV/DECA treatment of synthetic peptides modeled after the RACK-1-binding site in the C2 region of PKC beta induced modification of Ser218-Leu-Asn-Pro-Glu-Trp-Asn-Glu-Thr226, but not of a control peptide. This modification occurred at a tryptophan residue (Trp223) that is conserved in all conventional PKC isoforms. In overlay assays with native RACK-1 that had been immobilized on nitrocellulose, UV-treated control PKC alpha bound well to RACK-1, whereas UV/DECA-inactivated PKC alpha had reduced binding activity. The significance of these findings is shown with adenocarcinoma cells, which, when pretreated with 10 microM DECA and UV light, exhibited diminished 12-O-tetradecanoylphorbol-13-acetate-induced PKC alpha translocation. Overall, this work identifies DECA as a tool that prevents PKC translocation by inhibiting formation of the PKC.RACK-1 complex.

Deletion analysis of protein kinase C inactivation by calphostin C.

Protein kinase C (PKC) undergoes specific inactivation by nanomolar concentrations of calphostin C. Both PKC-alpha (a Ca(2+)-dependent conventional isoform) and PKC-epsilon (a Ca(2+)-independent novel isoform) are similarly inactivated by calphostin C (75-100 nM produced 50% inhibition), suggesting that inactivation requires a site common to both classes of PKC. We therefore performed studies to identify a critical region in the regulatory domain of PKC-alpha required for inactivation by calphostin C. A series of N-terminal-truncation mutants of bovine PKC-alpha expressed in Saccharomyces cerevisiae was tested with 500 nM calphostin C, a concentration sufficient to inactivate wild-type PKC-alpha by 80-90%. This concentration was as effective with mutant proteins containing deletions of up to 91 amino acid (aa) residues from the amino terminus (ND91), whereas a mutant protein truncated by 140 aa (ND140) was inactivated by only 20%. These findings imply that the aa sequence 92-140 is a structural determinant of PKC-alpha inactivation by calphostin C. This sequence contains one of the phorbol ester-binding sites (aa 102-144), which is highly conserved among most PKC isoforms including PKC-epsilon. In addition to aa 92-140, PKC-stimulating cofactors (phosphatidylserine, phorbol ester, and Ca2+) are required for inactivation by calphostin C even in the case of PKC mutants that do not require these cofactors for enzymatic activity. These results suggest that cofactors provide a template that is required for productive interaction of PKC and the inhibitor. The significance of the proposed proximity effect to calphostin C action is discussed.

Effects of suramin-related and other clinically therapeutic polyanions on protein kinase C activity.

The mechanism of the antineoplastic effects of suramin may involve interference with signal transduction, but in general is not well understood. We examined several polyanions to determine their effects on the kinase activity of the protein kinase C (PKC) beta1 and other PKC isoforms. Similar to suramin, a phosphorothioate oligodeoxynucleotide 28-mer homopolymer of cytidine (SdC28) inhibited the phosphatidylserine and Ca2+-dependent phosphorylation of an epidermal growth factor receptor octapeptide substrate. The inhibition by suramin was mixed competitive/noncompetitive with respect to ATP, but uncompetitive with respect to substrate. In contrast, the inhibition by SdC28 was competitive with respect to substrate (Ki = 5.4 microM) and not competitive with respect to ATP. The PKC alpha and beta1 isoforms were inhibited to the same extent with SdC28, while PKC epsilon was not inhibited. SdC28, in the absence of lipid cofactor, stimulated substrate phosphorylation, and in the absence of substrate induced PKC beta1 autophosphorylation. Similar behavior was seen with another polyanion, the polysulfated carbohydrate pentosan polysulfate (polyxylyl hydrogen sulfate). H4, a bis-naphthalene disulfonate tetraanion structurally related to suramin, also inhibited kinase activity but was not competitive with respect to ATP. Dianions closely related to H4 failed to inhibit PKC beta1, suggesting that multiple (>2) negative charges are required. The interactions of polyanions with PKC are complex, and are dependent on the molecular structure of the polyanion, the presence of cofactors, and the PKC isoform.

Dynamics of the internalization of phosphodiester oligodeoxynucleotides in HL60 cells.

We have examined the cellular association and internalization of phosphodiester (PO) oligodeoxynucleotides (oligos) with HL60 cells. At 4 degrees C, a 15-mer PO homopolymer of thymidine (FOdT15) exhibits apparent saturation binding (Km = 22 +/- 1 nM) that is competitive with the binding of phosphorothioate (PS) oligos. The value of Kc for SdC28, a PS 28-mer homopolymer of cytidine, is 5 +/- 2 nM. SdC28 was used to strip cell surface fluorescence: Internalized fluorescence accumulated in a (concentration)(time)-dependent fashion, consistent with a pinocytotic mechanism. PS, and to a lesser extent, PO oligos inhibited the rate of internalization of fluorescent albumin, also a marker of pinocytosis. This was correlated with direct in vitro inhibition of protein kinase C (PKC) beta 1 by the PS and PO oligos. Furthermore, other PKC inhibitors (H7, staurosporine, DMSO, PKC pseudosubstrate polypeptide) also inhibited intracellular accumulation of pinocytosed materials, perhaps by stimulating the exocytosis rate. In HL60 cells, the pinocytotic internalization of charged oligos appears to be dependent on intact PKC kinase activity, which is inhibited in vitro by PS and PO oligos.

A self-assembling protein kinase C inhibitor.

Previous studies have described a dicationic anticarcinoma agent that can chemically assemble in situ from monocationic phosphonium salts. The chemical combination of these monocationic precursors in the micromolar concentration range, occurring after their uptake by cells, was probably responsible for their synergistic inhibition of cell growth and for their selective cytotoxicity to Ehrlich ascites murine carcinoma cells relative to untransformed epithelial cells. Here, we report that the dicationic product that forms in this assembly reaction is an in vitro inhibitor of protein kinase C (PKC) alpha and beta 1 isoforms, exhibiting IC50 values of 20.4 microM and 35 microM, respectively. The monocationic precursors proved to be much weaker inhibitors of PKC (IC50 values greater than 200 microM). When PKC is exposed to combinations of the two precursors, the enzymatic activity decreases steadily as a function of time. Using dose-response data and HPLC kinetic studies, we show that when the two precursor compounds are added as a combination to PKC under these conditions, the rate of formation of the inhibitory product follows the observed time course of decline in PKC activity under identical conditions. We discuss the possibility that antiproliferative effects against carcinoma cells of the preformed dication and of the combined monocationic precursors involve inhibition of PKC.

Two polychlorinated hydrocarbons cause phospholipid-dependent protein kinase C activation in vitro in the absence of calcium.

Polychlorinated hydrocarbons known to be nongenotoxic carcinogens were screened as activators of protein kinase C (PKC)-beta 1 either at high concentrations of Ca2+ or in the absence of Ca2+ (i.e., with 1 mM ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N',-tetraacetic acid). Of those compounds tested, kepone and dicofol significantly stimulated PKC activity in the absence, but not the presence, of Ca2+. PKC activation was most pronounced in the presence of phosphatidylserine. Kepone and dicofol stimulated PKC activity 26% and 13%, respectively, as compared with the PKC activity (100%) stimulated by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Northern blot analysis of expression of TPA-inducible genes by kepone showed slight expression of phorbin and ornithine decarboxylase in murine embryo fibroblasts. Future studies are required to determine the relevance of PKC activation by kepone and dicofol to the known carcinogenicity of these compounds.

Characterization of a specific form of protein kinase C overproduced by a C3H 10T1/2 cell line.

Murine embryo fibroblasts (C3H 10T1/2) which were genetically engineered to overproduce the beta 1 isoform of protein kinase C (PKC-beta 1) were used to obtain homogeneous preparations of PKC-beta 1 for the purpose of characterizing the specific structural and functional properties of this isoform. Fractionation of PKC activity from these cells by hydroxyapatite chromatography produced one major peak, which represented 93% of the total cellular PKC activity and was not detected in control cells. This major peak of activity was shown by Western-blotting analysis with a beta 1-specific antiserum to be the overproduced beta 1-isoform, and exhibited a band at 77 kDa. The functional properties of the overproduced PKC-beta 1 were established with regard to phospholipid-dependence, Ca2(+)-dependence, responsiveness to a phorbol ester tumour promoter, activation by arachidonic acid (plus Ca2+), and inhibition by known PKC inhibitors. From these studies we conclude that PKC-beta 1 overproduced by C3H 10T1/2 cells exhibits the structural and functional properties previously ascribed to native PKC. Furthermore, these data provide the first definitive biochemical characteristics of this isoform of PKC.

Inhibition of rodent protein kinase C by the anticarcinoma agent dequalinium.

Dequalinium has previously been shown to be an anticarcinoma agent (M. J. Weiss et al., Proc. Natl. Acad. Sci. USA, 84: 5444-5448, 1987). The present study demonstrates that it can inhibit protein kinase C-beta 1 isolated from an overproducing cell line with a 50% inhibitory concentration of 8-15 microM. Further examination of the inhibition by using structural analogues of dequalinium reveals that the length of the methylene bridge between the two quinaldinium moieties, the presence of the ring substituents, and the bipartite character of the compound each contributes to the inhibitory potency. Related studies show that the analogues display the same rank order of inhibitory potency when tested with the trypsin-generated catalytic fragment of the enzyme, indicating that dequalinium inhibits kinase activity through an interaction with the catalytic subunit. Further studies argue that the ability of a given analogue to inhibit phosphotransferase activity correlates with its ability to compete with [3H]phorbol-12,13-dibutyrate binding on the intact enzyme (50% inhibitory concentration of 2-5 microM). This suggests that the inhibitor is either binding directly to the regulatory subunit as well, or that due to its interaction with the catalytic subunit, dequalinium produces an indirect effect on sites defined by phorbol ester binding. Kinetic analysis revealed that inhibition is noncompetitive with respect to ATP or phosphatidylserine. Studies conducted with types I, II, and III rat brain isozymes, resolved by hydroxylapatite chromatography, demonstrate that dequalinium inhibits each of them with similar potency (50% inhibitory concentration of 11 microM) and imply that the site of contact on the enzyme is a highly conserved region. Morphology studies with dequalinium in intact cells demonstrate that the inhibitor can protect control cells against phorbol ester-induced morphology changes but cannot protect protein kinase C-overproducing cells, suggesting that an elevation in protein kinase C levels alone is sufficient to overturn the protection conferred by dequalinium. On the basis of these results, we propose that protein kinase C could be a critical in vivo target of dequalinium.

Inactivation of soybean lipoxygenase 1 by 12-iodo-cis-9-octadecenoic acid.

12-Iodo-cis-9-octadecenoic acid (12-IODE) is a time-dependent, irreversible inactivator of soybean lipoxygenase 1. The rate of inactivation is independent of 12-IODE concentration above 20 microM and is half-maximal at about 4 microM. Inactivation by 12-IODE requires lipid hydroperoxide, which must be present even after the initial oxidation of the iron in the enzyme from ferrous to ferric. Inactivation by 12-IODE is also dependent on O2. These findings suggest that 12-IODE is converted by the enzyme into a more reactive species, which is responsible for inactivation. No inactivation has been detected with 12-iodooctadecanoic acid, 12-bromo-cis-9-octadecenoic acid, 12-iodo-trans-9-octadecenoic acid, or a mixture of stereoisomers of 9,11-octadecadienoic acid.