Isosilybin B causes androgen receptor degradation in human prostate carcinoma cells via PI3K-Akt-Mdm2-mediated pathway.

The identification and development of novel nontoxic phytochemicals that target androgen and androgen receptor (AR) signaling remains a priority for prostate cancer (PCA) control. In the present study, we assessed the antiandrogenic efficacy of isosilybin B employing human PCA LNCaP (mutated AR), 22Rv1 (mutated AR) and LAPC4 (wild-type AR) cells. Isosilybin B (10-90 microM) treatment decreased the AR and prostate specific antigen (PSA) levels in LNCaP, 22Rv1 and LAPC4 cells, but not in non-neoplastic human prostate epithelial PWR-1E cells. Isosilybin B treatment also inhibited synthetic androgen R1881-induced nuclear localization of AR, PSA expression and cell growth, and caused G(1) arrest. In mechanistic studies identifying AR degradation, isosilybin B caused increased phosphorylation of Akt (Ser-473 and Thr-308) and Mdm2 (Ser-166), which was linked with AR degradation as pretreatment with PI3K inhibitor (LY294002)-restored AR level. Further, overexpression of kinase-dead Akt largely reversed isosilybin B mediated-AR degradation suggesting a critical role of Akt in AR degradation. Antibody pull-down results also indicated that isosilybin B treatment enhances the formation of complex between Akt, Mdm2 and AR, which promotes phosphorylation-dependent AR ubiquitination and its degradation by proteasome. Together, present findings identify a novel mechanism for isosilybin B-mediated anticancer effects in human PCA cells.

Silymarin and silibinin cause G1 and G2-M cell cycle arrest via distinct circuitries in human prostate cancer PC3 cells: a comparison of flavanone silibinin with flavanolignan mixture silymarin.

Here, we assessed and compared the anticancer efficacy and associated mechanisms of silymarin and silibinin in human prostate cancer (PCA) PC3 cells; silymarin is comprised of silibinin and its other stereoisomers, including isosilybin A, isosilybin B, silydianin, silychristin and isosilychristin. Silymarin and silibinin (50-100 microg/ml) inhibited cell proliferation, induced cell death, and caused G1 and G2-M cell cycle arrest in a dose/time-dependent manner. Molecular studies showed that G1 arrest was associated with a decrease in cyclin D1, cyclin D3, cyclin E, cyclin-dependent kinase (CDK)4, CDK6 and CDK2 protein levels, and CDK2 and CDK4 kinase activity, together with an increase in CDK inhibitors (CDKIs) Kip1/p27 and Cip1/p21. Further, both agents caused cytoplasmic sequestration of cyclin D1 and CDK2, contributing to G1 arrest. The G2-M arrest by silibinin and silymarin was associated with decreased levels of cyclin B1, cyclin A, pCdc2 (Tyr15), Cdc2, and an inhibition of Cdc2 kinase activity. Both agents also decreased the levels of Cdc25B and cell division cycle 25C (Cdc25C) phosphatases with an increased phosphorylation of Cdc25C at Ser216 and its translocation from nucleus to the cytoplasm, which was accompanied by an increased binding with 14-3-3beta. Both agents also increased checkpoint kinase (Chk)2 phosphorylation at Thr68 and Ser19 sites, which is known to phosphorylate Cdc25C at Ser216 site. Chk2-specific small interfering RNA largely attenuated the silymarin and silibinin-induced G2-M arrest. An increase in the phosphorylation of histone 2AX and ataxia telangiectasia mutated was also observed. These findings indicate that silymarin and silibinin modulate G1 phase cyclins-CDKs-CDKIs for G1 arrest, and the Chk2-Cdc25C-Cdc2/cyclin B1 pathway for G2-M arrest, together with an altered subcellular localization of critical cell cycle regulators. Overall, we observed comparable effects for both silymarin and silibinin at equal concentrations by weight, suggesting that silibinin could be a major cell cycle-inhibitory component in silymarin. However, other silibinin stereoisomers present in silymarin also contribute to its efficacy, and could be of interest for future investigation.

Catalytic inhibition of human DNA topoisomerase IIalpha by hypericin, a naphthodianthrone from St. John's wort (Hypericum perforatum).

St. John's wort (Hypericum perforatum) is the most widely used herbal medicine for the treatment of depression. However, concerns have arisen about the potential of its interaction with other drugs due to the induction of cytochrome P450 isozymes 1A2 and 3A4 by the components hypericin and hyperforin, respectively. Structurally similar natural products are often employed as antitumor agents due to their action as inhibitors of DNA topoisomerases, nuclear enzymes that modify DNA during cellular proliferation. Preliminary findings that hypericin inhibited the DNA relaxation activity of topoisomerase IIalpha (topo II; EC 5.99.1.3) led us to investigate the mechanism of enzyme inhibition. Rather than stabilizing the enzyme in covalent complexes with DNA (cleavage complexes), hypericin inhibited the enzyme prior to DNA cleavage. In vitro assays indicate that hypericin is a potent antagonist of cleavage complex stabilization by the chemotherapeutics etoposide and amsacrine. This antagonism appears to be due to the ability of hypericin to intercalate or distort DNA structure, thereby precluding topo II binding and/or DNA cleavage. Supporting its non-DNA damaging, catalytic inhibition of topo II, hypericin was shown to be equitoxic to both wild-type and amsacrine-resistant HL-60 leukemia cell lines. Moreover, hypericin was incapable of stimulating DNA damage-responsive gene promoters that are activated by etoposide. As with the in vitro topo II assay, antagonism of DNA damage stimulated by 30 microM etoposide was evident in leukemia cells pretreated with 5 microM hypericin. Since many cancer patients experience clinical depression and concomitantly self-medicate with herbal remedies, extracts of St. John's wort should be investigated further for their potential to antagonize topo II-directed chemotherapy regimens.

Benzene metabolites antagonize etoposide-stabilized cleavable complexes of DNA topoisomerase IIalpha.

Chronic exposure to benzene is associated with hematotoxicity and acute myelogenous leukemia. Inhibition of topoisomerase IIalpha (topo II) has been implicated in the development of benzene-induced cytogenetic aberrations. The purpose of this study was to determine the mechanism of topo II inhibition by benzene metabolites. In a DNA cleavage/relaxation assay, topo II was inhibited by p-benzoquinone and hydroquinone at 10 microM and 10 mM, respectively. On peroxidase activation, inhibition was seen with 4,4'-biphenol, hydroquinone, and catechol at 10 microM, 10 microM, and 30 microM, respectively. But, in no case was cleavable complex stabilization observed and the metabolites appeared to act at an earlier step of the enzyme cycle. In support of this conclusion, several metabolites antagonized etoposide-stabilized cleavable complex formation and inhibited topo II-DNA binding. It is therefore unlikely that benzene-induced acute myelogenous leukemia stems from events invoked for leukemogenic topo II cleavable complex-stabilizing antitumor agents. (Blood. 2001;98:830-833)

The histone deacetylase inhibitor sodium butyrate induces DNA topoisomerase II alpha expression and confers hypersensitivity to etoposide in human leukemic cell lines.

The differentiating agent and histone deacetylase inhibitor, sodium butyrate (NaB), was shown previously to cause a transient, 3-17-fold induction of human DNA topoisomerase II alpha (topo II alpha) gene promoter activity and a 2-fold increase in topo II alpha protein early in monocytic differentiation of HL-60 cells. This observation has now been extended to other short chain fatty acids and aromatic butyrate analogues, and evidence is presented that human topo II alpha promoter induction correlates closely with histone H4 acetylation status. Because increased topo II alpha expression is associated with enhanced efficacy of topo II-poisoning antitumor drugs such as etoposide, the hypothesis tested in this report was whether NaB pretreatment could sensitize HL-60 myeloid leukemia and K562 erythroleukemia cells to etoposide-triggered DNA damage and cell death. A 24-72 h NaB treatment (0.4-0.5 mM) induced topo II alpha 2-2.5-fold in both HL-60 and K562 cells and caused a dose-dependent enhancement of etoposidestimulated, protein-linked DNA complexes in both cell lines. At concentrations with minimal effects on cell cycle kinetics (0.4 mM in HL-60; 0.5 mM in K562), NaB pretreatment also modestly enhanced etoposidetriggered apoptosis in HL-60 cells, as determined morphologically after acridine orange/ethidium bromide staining, and substantially increased K562 growth inhibition and poly(ADP-ribose)polymerase cleavage after etoposide exposure. Therefore, a temporal window may exist whereby a differentiating agent may sensitize experimental leukemias to a cytotoxic antitumor agent. These results indicate that histone deacetylase inhibitors should be investigated for etoposide sensitization of other butyrate-responsive hematopoietic and nonhematopoietic tumor lines in vitro and in vivo.

Ligand-dependent interaction of hepatic fatty acid-binding protein with the nucleus.

Our studies were conducted to explore the role of hepatic fatty acid-binding protein (L-FABP) in fatty acid transport to the nucleus. Purified rat L-FABP facilitated the specific interaction of [(3)H]oleic acid with the nuclei. L-FABP complexed with unlabeled oleic acid decreased the nuclear association of [(3)H]oleic acid:L-FABP; however, oleic acid-saturated bovine serum albumin (BSA) or fatty acid-free L-FABP did not. The peroxisome-proliferating agents LY171883, bezafibrate, and WY-14,643 were also effective competitors when complexed to L-FABP. Nuclease treatment did not affect the nuclear association of [(3)H]oleic acid:L-FABP; however, proteinase treatment of the nuclei abolished the binding. Nuclei incubated with fluorescein-conjugated L-FABP in the presence of oleic acid were highly fluorescent whereas no fluorescence was observed in reactions lacking oleic acid, suggesting that L-FABP itself was binding to the nuclei. The nuclear binding of FABP was concentration dependent, saturable, and competitive. LY189585, a ligand for L-FABP, also facilitated the nuclear binding of fluorescein-conjugated L-FABP, although it was less potent than oleic acid. A structural analog that does not bind L-FABP, LY163443, was relatively inactive in stimulating the nuclear binding. Potential interactions between L-FABP and nuclear proteins were analyzed by Far-Western blotting and identified a 33-kDa protein in the 500 mm NaCl extract of rat hepatocyte nuclei that bound strongly to biotinylated L-FABP. Oleic acid enhanced the interaction of L-FABP with the 33-kDa protein as well as other nuclear proteins. We propose that L-FABP is involved in communicating the state of fatty acid metabolism from the cytosol to the nucleus through an interaction with lipid mediators that are involved in nuclear signal transduction.

Modulation of human DNA topoisomerase IIalpha function by interaction with 14-3-3epsilon.

Human DNA topoisomerase IIalpha (topo II), a ubiquitous nuclear enzyme, is essential for normal and neoplastic cellular proliferation and survival. Several common anticancer drugs exert their cytotoxic effects through interaction with topo II. In experimental systems, altered topo II expression has been associated with the appearance of drug resistance. This mechanism, however, does not adequately account for clinical cases of resistance to topo II-directed drugs. Modulation by protein-protein interactions represents one mechanism of topo II regulation that has not been extensively defined. Our laboratory has identified 14-3-3epsilon as a topo II-interacting protein. In this study, glutathione S-transferase co-precipitation, affinity column chromatography, and immunoprecipitations confirm the authenticity of these interactions. Three assays evaluate the impact of 14-3-3epsilon on distinct topo II functional properties. Using both a modified alkaline comet assay and a DNA cleavage assay, we demonstrate that 14-3-3epsilon negatively affects the ability of the chemotherapeutic, etoposide, to trap topo II in cleavable complexes with DNA, thereby preventing DNA strand breaks. By electrophoretic mobility shift assay, this appears to be due to reduced DNA binding activity. The association of topo II with 14-3-3 proteins does not extend to all 14-3-3 isoforms. No protein interaction or disruption of topo II function was observed with 14-3-3final sigma.

Long chain arginine esters: a new class of cationic detergents for preparation of hydrophobic ion-paired complexes.

The ability of stoichiometric amounts (based on charged groups) of ionic detergents to bind to oppositely charged ionic compounds has been recently reviewed. These hydrophobic ion-paired (HIP) complexes display altered solubility properties. Most of the work to date on HIP compelxes has focused on basic drugs and anionic detergents. It would be extremely useful to extend this approach to acidic compounds, including DNA and RNA. However, most cationic detergents are relatively toxic. It is hypothesized that detergents constructed from naturally occurring or well tolerated components, coupled by labile linkages, will be less toxic and still able to form strong HIP complexes. This study describes the synthesis and characterization of long chain alkyl esters of arginine. This class of cationic detergents, which have not been reported previously, are less cytotoxic than alkyltrimethylammonium detergents, possibly making them more acceptable in drug delivery applications. These arginine esters exhibit detergent-like properties. For example, the dodecyl ester of arginine has a critical micelle concentration of 0.07 mM, while being approximately 5-10 fold less toxic than tetradecyltrimethylammonium bromide. The arginine dodecyl ester forms stable HIP complexes with plasmid DNA. The complex is sufficiently stable to allow some modest level of transfection with Cos-7 cells in a time- and concentration-dependent fashion. This work demonstrates that arginine-based cationic detergents are effective ion-pairing agents, appear to be less toxic than alkyltrimethylammonium compounds, and form stable complexes with DNA.

Herbs as medicines.

Herbs and related products are commonly used by patients who also seek conventional health care. All physicians, regardless of specialty or interest, care for patients who use products that are neither prescribed nor recommended. Some herbs have been extensively studied, but little is known about others. When a patient asks for advice regarding the use of a particular herb, how should a physician respond? Similarly, how does a physician determine if a patient's symptoms are caused by a "remedy"? This review attempts to answer these questions by investigating pertinent definitions, the history of herbs in medicine, epidemiology and prevalence of herbal use, and relevant psychosocial issues.

Alpha,beta-unsaturated aldehydes increase glutathione S-transferase mRNA and protein: correlation with activation of the antioxidant response element.

A series of alpha,beta-unsaturated aldehydes was evaluated to determine if these compounds could mediate inducible expression of glutathione S-transferase (GST) through the 5'-flanking antioxidant response element (ARE). The ARE from rGST A1 was subcloned into a luciferase reporter construct and used to transiently transfect rat Clone 9 hepatoma cells. Transfected cells were treated with 4-hydroxy-trans-2-nonenal (4-HNE), trans-2-hexenal (t-2-HE), 2-propenal (acrolein, 2-PE), and ethacrynic acid (EA), a control compound also containing an alpha,beta-unsaturated carbonyl moiety. Each compound was evaluated for cytotoxicity to construct dosing regimens in transfection studies. IC50 values for growth inhibition were measured using 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide. IC50 values in Clone 9 cells were: 4-HNE, 6.3 +/- 0.7 microM; t-2-HE, 16.0 +/- 0.7 microM; 2-PE, 2.2 +/- 0.4 microM; and EA, 38.0 +/- 1.6 microM. A dose-dependent increase in luciferase activity was observed in transfected cells with all four compounds tested, indicating that alpha, beta-unsaturated aldehydes function as direct activators of the ARE. To determine whether or not the observed promoter activation led to increased transcriptional and translational induction of GST, cells were treated with the various compounds and assayed for increases in GST mRNA, protein, and enzyme activity. Studies in Clone 9 cells revealed increased steady-state message for GST A1 and A4, increased GST A4-4 protein by Western blotting, and increased GST activity toward 1-chloro-2,4-dinitrobenzene in response to treatment with all four compounds evaluated. Collectively, these studies demonstrate that EA and certain alpha,beta-unsaturated aldehydes produced as a result of cellular membrane lipid peroxidation are activators of the ARE and efficient inducers of GST A1-1 and A4-4.

Stability of lipid/DNA complexes during agitation and freeze-thawing.

It is well established that cationic liposomes facilitate the delivery of DNA and offer substantial advantages over viral-based delivery systems. However, these synthetic vectors readily aggregate in liquid formulations which in clinical trials requires preparation of lipid/DNA complexes at the bedside immediately before injection. This temporal requirement could be eliminated if complexes were formulated as stable preparations that could be shipped, stored, and administered as needed. To this end, our study investigates the stability of lipid/DNA complexes during physical stresses that might be encountered during shipping and storage, i.e., agitation and freeze-thawing. Our data show that agitation significantly reduces transfection rates in complexes prepared with three different commercially available lipid formulations. Additional experiments indicate that slow freezing is more damaging than rapid freezing, and that sucrose is able to preserve transfection and complex size during freeze-thawing. These results are consistent with previous reports and demonstrate that frozen formulations may be suitable for maintaining transfection rates of lipid/DNA complexes. Under certain conditions, we observe a reproducible 3-fold increase in transfection after freeze-thawing that is prevented by high concentrations of sucrose. Together, these data suggest that physical stresses can alter structural characteristics of lipid/DNA complexes that can markedly affect rates of DNA delivery.

Preparation and in vitro characterization of gentamycin-impregnated biodegradable beads suitable for treatment of osteomyelitis.

A new method for preparing poly(L-lactide) (PLA) biodegradable beads impregnated with an ionic aminoglycoside, gentamycin, is described. The process employs hydrophobic ion pairing to solubilize gentamycin in a solvent compatible with PLA, followed by precipitation with a compressed antisolvent (supercritical carbon dioxide). The resulting precipitate is a homogeneous dispersion of the ion-paired drug in PLA microspheres. The microspheres are approximately 1 microm in diameter and can be compressed into beads (3-6 mm in diameter) strung on surgical sutures for implantation. The bead strings exhibit no significant change in release kinetics upon sterilization with a hydrogen peroxide plasma (Ster-Rad). The kinetics of gentamycin release from the PLA beads are consistent with a matrix-controlled diffusion mechanism. While nonbiodegradable poly(methyl methacrylate) (PMMA) beads initially release gentamycin in a similar manner, the drug release from PMMA ceases after 8 or 9 weeks, while the PLA beads continue to release drug for over 4 months. Moreover, only 10% of the gentamycin is released from the PMMA beads, while PLA beads release more than 60% of their load, if serum is present in the release medium. The PLA system displays improved release kinetics relative to PMMA, is biodegradable, is unaltered by gas sterilization, can be used for a range of antibiotics, and can be manipulated without disintegration. These are all desirable properties for an implantable drug delivery system for the prevention or treatment of osteomyelitis.

Maintenance of transfection rates and physical characterization of lipid/DNA complexes after freeze-drying and rehydration.

It is well established that cationic liposomes form complexes with DNA and effectively transfect cells in vivo and ex vivo. Lipid/DNA complexes have proven safe and nonimmunogenic in clinical trials; however, they are known to aggregate readily in liquid formulations. This physical instability requires clinicians to prepare lipid/DNA complexes immediately prior to injection. In order to eliminate problems associated with this temporal requirement, we investigated the feasibility of preserving complexes as a dried preparation that could be tested, stored, and rehydrated as needed. To this end, our study evaluated the ability of different stabilizers to preserve transfection rates of complexes during acute freeze-drying stress. Our data show that complexes lyophilized in 0.5 M sucrose or trehalose possessed transfection rates similar to those of fresh preparations. In addition, dried complexes that exhibited full transfection activity upon rehydration had sizes comparable to nonlyophilized controls. Our work demonstrates that lipid/DNA complexes can be stabilized as dried powders that offer significant advantages over current liquid formulations. Furthermore, the correlation of transfection rates with maintenance of complex diameter suggests that size plays a critical role in lipid-based DNA delivery.

Homologous and heterologous protein-protein interactions of human DNA topoisomerase IIalpha.

DNA topoisomerase II (topo II; EC 5.99.1.3) is a nuclear enzyme whose DNA decatenating activity on newly replicated DNA is essential to successful cell division. Topo II catalytic activity proceeds by a concerted DNA breakage-reunion reaction coordinated between two interacting, homologous subunits. Human and yeast topo II have recently been shown to enter into heterologous protein-protein interactions and some of these interactions appear necessary for successful chromosomal segregation. In the present study, the sequences mediating homologous and heterologous protein-protein interactions have been investigated biochemically using various truncated peptides from the major alpha form of human topo II. From nonreducing gel electrophoresis and solid-phase protein-protein binding (Far Western) assays, topo II homodimerization appeared to be minimally governed by the region between amino acids 951 and 1042. However, maximal homodimerization and multimerization required sequences C-terminal to position 1042. Topo II peptides were also able to interact with 10-12 nuclear proteins from HeLa cells, termed topo II-interactive proteins or TIPs. Interestingly, small topo II peptides between residues 808 and 951 that did not homodimerize with topo II (857-1447) were nonetheless capable of binding to HeLa TIPs. These interactions were confirmed by use of topo II affinity chromatography for isolation of specific TIPs from HeLa nuclear extracts. Taken together, these data confirm that human topo II is also capable of heterologous interactions with nuclear proteins and that the region governing these interactions is distinct from, but has some overlap with, sequences directing topo II homodimerization.

Prooxidant-initiated lipid peroxidation in isolated rat hepatocytes: detection of 4-hydroxynonenal- and malondialdehyde-protein adducts.

Toxicity associated with prooxidant-mediated hepatic lipid peroxidation is postulated to originate from the interaction of the aldehydic end products of lipid peroxidation with cellular constituents. The principal alpha,beta-unsaturated aldehydic products of lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), are known to modify proteins through covalent alkylation of lysine, histidine, and cysteine amino acid residues. To detect and characterize the formation of 4-HNE- and MDA-adducted proteins during prooxidant-initiated lipid peroxidation, rabbit polyclonal antibodies were raised to 4-HNE-sulfhydryl, dinitrophenylhydrazine (DNPH)-4-HNE-sulfhydryl, and MDA-amine conjugates of keyhole limpet hemocyanin (KLH). Each antiserum displayed high antibody titers to either 4-HNE-metallothionein, DNPH-albumin, or MDA-albumin adducts when measured by ELISA. To study the formation of 4-HNE- and MDA-protein adducts during prooxidant-initiated cellular injury, isolated hepatocytes were exposed to either carbon tetrachloride or iron/ascorbate for 2 h. Indices of hepatocellular oxidative stress (i.e., cell viability and glutathione status) and lipid peroxidation (i.e., formation of 4-HNE, protein carbonyls, and MDA) were monitored continuously. Hepatocellular viability was affected moderately by carbon tetrachloride, while cellular reduced glutathione status was moderately affected by both iron/ascorbate and carbon tetrachloride. Levels of MDA and protein carbonyls increased dramatically with both prooxidants, whereas 4-HNE levels did not change significantly over the time course studied. In addition, hepatocellular proteins were immunoprecipitated with each antiserum, and aldehyde-modified immunopositive proteins were detected by immunoblotting. Prooxidant-induced increases in MDA corresponded with increases in intensity and number of MDA-adducted proteins over the time course studied. A total of 13 MDA-modified proteins (20, 25, 28, 30, 33, 38, 41, 45, 80, 82, 85, 130, and 150 kDa) were detected with the MDA-amine antiserum. Additionally, both iron/ascorbate- and carbon tetrachloride-induced formation of DNPH-derivatizable protein carbonyls corresponded quantitatively with the ability to detect specific proteins (80, 100, 130, and 150 kDa) with the DNPH-4-HNE-cysteine antiserum. Neither CCl4 nor iron/ascorbate elicited changes in 4-HNE or induced the formation of 4-HNE-modified proteins when assessed by immunoprecipitation-immunoblot analysis with the 4-HNE-sulfhydryl antiserum. In all instances detection of aldehyde-modified proteins was not associated with cell death and may be related to the function of these proteins as aldehyde-binding proteins which sequester electrophilic molecules during oxidative liver injury.

NF-M trans-activates the human DNA topoisomerase II alpha promoter independently of c-Myb in HL-60 cells.

Identifying transcriptional regulators of DNA topoisomerase II alpha (topo II alpha) is essential to decipher the mechanisms underlying leukemia cell resistance to topo II-directed antitumor drugs. We have previously reported that the proto-oncogene transcription factor c-Myb transactivates the topo II alpha promoter in several hematopoietic cell lines. Currently, we investigate whether NF-M, a C/EBP beta family member, cooperates with c-Myb in activating topo II alpha transcription. Although NF-M is the most efficacious trans-activator of topo II alpha that we have examined (approximately 38-fold over basal), NF-M does not appear to be involved in the endogenous transcriptional regulation of topo II alpha. Interestingly, we report that the sodium butyrate-dependent induction of the topo II alpha promoter observed previously appears to be mediated by c-Myb, independent of NF-M.