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CELLULAR AND MOLECULAR
Departments of Pathology (N.B., K.C.N., A.H., M.N.A., K.J., J.V.), Obstetrics and Gynecology (A.W.O.), Dermatology (S.K.), and Chemistry (T.B.S., G.D.G.), University of Michigan, Ann Arbor, Michigan
Received September 5, 2007; accepted November 28, 2007.
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Abstract |
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; Boitano et al., 2003a; Varani et al., 2005). Bz-423 defines a structural class of benzodiazepines that differs from those currently in clinical use as anxiolytics and hypnotics by the presence of a hydrophobic substituent at the C-3 position, which blocks binding to the central benzodiazepine receptor and renders binding to the peripheral benzodiazepine receptor weak (Boitano et al., 2003b; Cleary et al., 2007). In vivo, this compound reduces pathologically expanded populations of B and T lymphocytes in autoimmune-prone (NZB x NZW)F1 and MRL-lpr strains of mice, respectively (Blatt et al., 2002; Bednarski et al., 2003). In association with its effects on lymphocytes, Bz-423 treatment results in less immune-mediated kidney damage (glomerulonephritis) and reduced autoimmune arthritis.
The antiproliferative and proapoptotic response to Bz-423 depends on its binding to the oligomycin sensitivity-conferring protein (OSCP) in mitochondria, a component of the F1F0-ATPase. Its binding to the OSCP increases mitochondria-derived intracellular superoxide that is coupled to a signaling response in a cell type-specific manner (Johnson et al., 2006). Indeed, the antiproliferative response of Burkitt's lymphoma cells to Bz-423 seems to result from the reactive oxygen species (ROS) response triggering c-myc protein degradation (Sundberg et al., 2006). In addition, Bz-423 has also been shown to limit the proliferation of rapidly dividing skin cells. Specifically, Bz-423 was found to suppress hyperplastic changes induced by all-trans retinoic acid (RA) in the epidermis of human skin in organ culture without affecting the structure of the dermis (Varani et al., 2005). In the same study, dermal fibroblasts in monolayer culture were less sensitive to the growth-modulating activity of this compound than keratinocytes.
Given that epidermal hyperplasia in retinoid-treated skin and psoriatic epidermal hyperplasia both involve increased keratinocyte proliferation driven by aberrant signaling through the epidermal growth factor receptor (EGF) receptor pathway (Gottlieb et al., 1988; Elder et al., 1989; Cook et al., 1992; Varani et al., 1998, 2001; Piepkorn et al., 2003; Rittié et al., 2006), it was of interest to determine whether Bz-423 would reduce psoriatic keratinocyte hyperproliferation. To begin addressing this issue, we compared the effects of a topical formulation of Bz-423 with a potent topical steroid on human psoriatic skin transplanted onto severe, combined immunodeficient (scid) mice. Our data show that Bz-423 has efficacy in this disease model and points to a mechanism of action consistent with Bz-423-induced ROS mediating these effects. Finally, our studies identify additional analogs of Bz-423 with keratinocyte growth-inhibiting activity in cell and organ culture models. These results support continued clinical development of this class of compounds for psoriasis and, perhaps, other hyperproliferative skin disorders.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). The nuclear magnetic resonance and mass spectroscopy data confirming the structure of Bz-423 was published in the initial report (Bunin et al., 1994), and both the nuclear magnetic resonance and mass spectral analysis of the new compounds are consistent with the proposed structures. Each of these benzodiazepine derivatives was dissolved in dimethyl sulfoxide at 20 mg/ml and diluted in culture medium at the time of use. Clobetasol propionate cream (0.05%; Temovate; GlaxoSmithKline Healthcare, Pittsburgh, PA) was obtained through the University of Michigan Hospital Pharmacy. RA was obtained from Sigma-Aldrich (St. Louis, MO). The retinoid was diluted in DMSO at 20 mg/ml and stored frozen. At the time of use, the RA stock solution was diluted in culture medium and used in organ culture studies at a final concentration of 1.0 µg/ml. DMSO was present at a final concentration of 0.5% (v/v) or less in all experiments. This concentration of DMSO is below the concentration at which biological effects are observed in monolayer cultures of human skin cells or organ cultures of human skin under the conditions used here (Varani et al., 1994). Mouse monoclonal antibodies to human Ki-67 CD3 were obtained from Dako North America, Inc. (Carpinteria, CA). Anti-β-catenin was obtained from Millipore Bioscience Research Reagents (Temecula, CA). Anti-cyclin D1 and anti-c-myc were obtained from Cell Signaling Technology Inc. (Danvers, MA). The superoxide dismutase mimetic manganese (III)meso-tetrakis (4-benzoic acid) porphyrin (MnTBAP) was obtained from Alexis Laboratories (San Diego, CA), and vitamin E was from Sigma-Aldrich. Human Skin. Replicate 6-mm punch biopsies of full-thickness psoriatic plaque skin were obtained from human skin donors with psoriasis. These biopsies were used in the transplant studies. Six-millimeter punch biopsies of sun-protected (hip) skin from nonpsoriatic donors were obtained as controls. In addition, replicate 2-mm full-thickness punch biopsies of sun-protected hip skin were obtained from normal donors for use in organ culture studies. Four to six 6-mm biopsies or up to 12 2-mm biopsies were obtained from each tissue donor. This project was approved by the University of Michigan Institutional Review Board. All subjects provided written informed consent before biopsy.
Skin Transplantation and Treatment Protocol. Scid mice (CB-17 strain; Taconic Farms, Germantown, NY) were used as tissue recipients in a human skin-scid mouse transplant model previously used to evaluate potential antipsoriatic agents (Dam et al., 1999; Ellis et al., 2000; Zeigler et al., 2001; Zollner et al., 2002; Villadsen et al., 2003; Bhagavathula et al., 2005a,b). One biopsy from each normal or psoriatic volunteer was transplanted onto the dorsal surface of a recipient mouse as follows. After the animal was anesthetized, the dorsal surface of the mouse was shaved. Mouse skin was surgically removed to size and replaced with the human tissue. This tissue was secured to the back of the mouse with absorbable sutures (4-0 Dexon "S"; Davis-Geck, Manati, Puerto Rico). The transplant was then bandaged with Xeroform petrolatum dressing (Kendall Company, Mansfield, MA) for 5 days. The animals were maintained in a pathogen-free environment throughout the preparation and treatment phases. Treatment was initiated 1 to 2 weeks post-transplantation. Animals with the human skin transplants were divided into treatment groups (vehicle plus test reagent, vehicle alone, or clobetasol propionate). Animals were treated twice daily for 14 days. All procedures involving animals were approved by the University Committee on Use and Care of Animals.
At the end of the treatment phase, animals were photographed, and then they were euthanized. The transplanted human tissue along with the surrounding mouse skin was surgically removed and fixed in 10% formalin. After embedding tissue in paraffin, multiple 5-µm sections were cut from each tissue piece (approximately 50 µm between sections), mounted onto microscope slides, and stained with hematoxylin and eosin. Epidermal thickness (distance from the dermo-epidermal juncture to the uppermost layer of viable cells) was measured at several sites in each tissue section at 200x magnification. The relationship between epidermal thickness and treatment was determined by ANOVA, making comparisons between paired groups. In addition to assessing epidermal thickness in hematoxylin- and eosin-stained sections, tissue sections from the same specimens were stained with an antibody to the proliferation-associated antigen Ki-67 and with an anti-human CD3+ monoclonal antibody to detect human T lymphocytes in the transplanted tissue. Selected sections were also probed with antibodies to c-myc and β-catenin.
Human Skin Organ Cultures. Immediately upon biopsy, replicate 2-mm punch biopsies (nonpsoriatic skin only) were immersed in keratinocyte basal medium (KBM) (Lonza Walkerville, Inc., Walkersville, MD). KBM is a low-Ca2+, serum-free modification of MCDB-153 medium. It was supplemented with CaCl2 to bring the final Ca2+ concentration to 1.4 mM. Biopsies were incubated in wells of a 24-well dish containing 400 µl of Ca2+-supplemented KBM with or without additional treatments (RA and/or one of the analogs of Bz-423) as described under Results. Cultures were incubated at 37°C in an atmosphere of 95% air and 5% CO2. Other than to maintain the tissue in a minimal volume of medium, nothing further was done to ensure a strict air-liquid interface. Incubation was for 8 days, with change of medium and fresh treatments provided every second day. At the end of the incubation period, tissue was fixed in 10% buffered formalin and examined histologically after staining with hematoxylin and eosin. Routinely, three to six tissue sections were prepared from each block. Epidermal thickness measurements were made at several sites in each tissue section (distance from the dermal-epidermal juncture to the top of the viable portion of the epithelium). The organ culture procedure used here has been described in the past (Varani et al., 1993, 1994).
Human Epidermal Keratinocytes and Dermal Fibroblasts in Monolayer Culture. Epidermal keratinocytes were isolated from fresh tissue biopsies as described previously (Varani et al., 1994). Primary and early passage cells were maintained in keratinocyte growth medium (KGM) (Lonza Walkerville, Inc.). KGM contains the same basal medium as KBM, but it is further supplemented with a mixture of growth factors, including 0.1 ng/ml EGF, 0.5 µg/ml insulin, and 0.4% (v/v) bovine pituitary extract. In addition to using low-passage keratinocytes, we also used the HaCat line of immortalized human epidermal keratinocytes in some experiments (Boukamp et al., 1988). The immortalized keratinocytes were handled exactly as low-passage keratinocytes.
Fibroblasts obtained from the same tissue as keratinocytes were grown in monolayer culture using Dulbecco's modified minimal essential medium supplemented with nonessential amino acids and 10% fetal bovine serum as culture medium. Both keratinocytes and fibroblasts were maintained at 37°C in an atmosphere of 95% air and 5% CO2. Cells were subcultured by exposure to EDTA and used at passages 2 to 4.
Proliferation Assay. Keratinocyte proliferation was assessed by seeding 4 x 104 cells per well in a 24-well plate using KGM as culture medium. After the cells had attached (overnight), they were washed, and triplicate samples were harvested for zero time counts. The remaining cells were then incubated in KGM with different concentrations of test reagents or DMSO control as indicated under Results Section. Proliferation was measured on day 2 by releasing the cells with trypsin/EDTA and enumerating them using a particle counter (Beckman Coulter, Inc., Fullerton, CA). Fibroblast proliferation studies were conducted in a similar manner except that KBM supplemented with 1.4 mM Ca2+ was used as culture medium.
Cytotoxicity and Apoptosis Assays. Cytotoxicity and apoptosis analysis was done by staining the cells with Annexin V-FITC and propidium iodide and by analyzing them via flow cytometry. In brief, keratinocytes were exposed to different concentrations of Bz-423 (0.1–4 µM) for 48 h. After 48 h, cells were washed twice with ice-cold phosphate-buffered saline and then resuspended in 1x binding buffer (BD Biosciences PharMingen, San Diego, CA) at a concentration of 1 x 106 cells/ml. Then, 200 µl of the above-mentioned cell suspension was transferred to 96-well V-bottom plates, and 10 µl of Annexin V-FITC (BD Biosciences PharMingen) and 5 µl of propidium iodide (Invitrogen, Carlsbad, CA) were added to the wells, and the plats were incubated for 15 min in the dark. Samples were then analyzed by flow cytometry (LSR II; BD Biosciences, San Jose, CA). Data acquisition and analysis were done using BD FACSDiva software.
Preparation of Cell Lysates and Immunoblot Analysis. Keratinocytes were plated at 3 x 105 cells per well in six-well tissue culture dish in KGM as culture medium, and cells were allowed to attach overnight. The next day, cultures were washed, and then they were incubated for 2 days under the desired conditions as described under Results. At the end of the incubation period, cells were lysed in 1x cell lysis buffer consisting of 20 mM Tris-HCl, pH 7.4, 2 mM sodium vanadate, 1.0 mM sodium fluoride, 100 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 25 µg/ml each of aprotinin, leupeptin, and pepstatin, and 2 mM EDTA and EGTA. Lysis was performed by adding 200 µl of lysis buffer to each well and incubating the plate on ice for 5 min. After the incubation, cells were scraped, and samples were sonicated. Then, the extracts were cleared by microcentrifugation at 14,000g for 15 min. Supernatants were collected, and protein concentrations were estimated using the Bio-Rad DC Protein Assay kit (Bio-Rad, Hercules, CA).
Western blotting for β-catenin, c-myc and cyclin-D1 was carried out as described previously (Bhagavathula et al., 2004). In brief, samples were separated in SDS-polyacrylamide gel electrophoresis under denaturing and reducing conditions and transferred to nitrocellulose membranes. After blocking with a 5% nonfat milk solution in Tris-buffered saline with 0.1% Tween at 4°C overnight, membranes were incubated for 1 h at room temperature with the desired antibody, diluted 1:1000 in blocking buffer. Thereafter, the membranes were washed with Tris-buffered saline with 0.1% Tween and bound antibody detected using the Phototope-horseradish peroxidase Western blot detection kit (Cell Signaling Technology Inc.). A Kodak-1000 X-OMAT processor was used to capture the positive images of the Western blots, and these positive images were scanned and digitized. The digitized images were quantitated using NIH Image analysis software (National Institutes of Health, Bethesda, MD).
Cell Fractionation. Keratinocytes were plated in 100-mm culture dishes at a density of 1.5 x 106 cells per dish using KGM as culture medium. Cells were allowed to attach overnight. The next day, cells were incubated in fresh KGM with or without Bz-423. After 3 days of incubation, cells were harvested and lysed. Membrane extraction was done using the Mem-PER Eukaryotic Membrane Protein Extraction Reagent kit (Pierce Chemical, Rockford, IL) according to the manufacturer's "phase-partitioning" protocol for mammalian cells. The membrane fraction and soluble fraction were used for Western blotting as described above. Before loading the gels, protein levels in each preparation were determined using the BCA Protein Determination kit (Pierce Chemical), and equal amounts of protein were loaded onto each lane. After electrophoresis and protein transfer to the nitrocellulose filters, we used the Ponceau S reversible staining solution (Pierce Chemical) to visualize the transferred proteins and to confirm that comparable amounts of total protein were transferred.
Membrane enrichment (or depletion) was confirmed by blotting for a cell surface protein (EGF receptor) and a cytosolic protein (total extracellular signal-regulated). In every case, greater than 95% of the total extracellular signal-regulated immunoreactivity was in the cytoplasmic fraction with barely detectable reactivity in the membrane fraction. At the same time, most of the EGF receptor (75–80%) was found in the membrane fraction with the remainder in the cytoplasmic fraction.
Confocal Immunofluorescence Microscopy. Keratinocytes were plated on uncoated Lab-Tek II chamber slides in KGM and allowed to attach overnight. The next day, cultures were washed and then incubated for 3 days under the desired conditions as described under Results. Cells were then fixed with 4% formaldehyde for 20 min. After fixation, cells were washed two times with wash buffer (0.05% Tween 20 in Dulbecco's phosphate-buffered saline (DPBS), followed by permeabilization with 0.1% Triton X-100 for 10 min. Cells were again washed and then exposed to a blocking solution consisting of 1% bovine serum albumin in DPBS for 30 min. Next, cells were treated with a monoclonal antibody to β-catenin in blocking solution for 1 h. After three subsequent washing steps with DPBS (5 min each), cells were treated with Alexa Fluor 488-conjugated secondary antibody (Invitrogen) in blocking solution and incubated for 45 min. After three additional washing steps, the cells were rinsed once with wash buffer, and coverslips were mounted with Prolong Antifade (Invitrogen). Cells were examined with a Zeiss LSM 510 confocal microscope using a 63x (C-Apochr) numerical aperture 1.2 water immersion objective lens.
Detection of Intracellular ROS. Intracellular ROS were detected using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA; Invitrogen). This fluorescent dye is sensitive to peroxynitrite, hypochlorous acid, and hydrogen peroxide plus peroxidase (Crow, 1997). Cells growing in 96-well plates were loaded (30 min; 37°C) with DCFH-DA (3 µM). The cells were then washed and placed in fresh media before treatment. After the indicated treatments, the fluorescence of the oxidized product 2',7'-dichlorofluorescein (DCF) was monitored by flow cytometry using a FACSCalibur (BD Biosciences). For each sample, 10,000 events were recorded, and the data were analyzed to determine median fluorescence intensity. Dihydroethidium (DHE; Invitrogen) was used to detect superoxide anion. Cells were incubated with DHE (4 µM) for 30 min at 37°C as described above, and ethidium fluorescence was measured by flow cytometry. DHE is a redox-sensitive agent that reacts specifically with superoxide anion to produce the ethidium that becomes highly fluorescent and detectable in cells after interacting with nucleic acids (Benov et al., 1998).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Skin from four healthy donors was transplanted onto scid mice to determine the response of nonpsoriatic skin to Bz-423. Consistent with past observations (Zeigler et al., 1999, 2001; Bhagavathula et al., 2005a,b), all four normal skin transplants developed epidermal hyperplasia as a consequence of transplantation onto the scid mice (average epidermal thickness of 158 ± 43 µm after transplantation compared with 90 ± 40 µm at biopsy; n = 4, p < 0.05). As described above, the transplants were treated with vehicle, Bz-423 in vehicle, or clobetasol propionate. Compared with vehicle-treated animals, Bz-423 reduced epidermal hyperplasia in nonpsoriatic skin transplants (Bz-423 thickness of 110 ± 19 µm versus vehicle thickness of 158 ± 43 µm; n = 4, p < 0.05). In comparison, the average epidermal thickness of transplants treated with clobetasol propionate was 25 ± 8 µm. It should be noted that the overall reduction in epidermal thickness of Bz-423-treated nonpsoriatic skin (approximately 30%) was not statistically different from the reduction seen with psoriatic lesion skin (approximately 43%).
In addition to evaluating transplanted human skin, the response of mouse skin adjacent to the transplant site was also evaluated (Fig. 2). Clobetasol propionate treatment was associated with significant atrophy of the mouse skin including reduced dermal cellularity and reduced dermal and epidermal thickness (Fig. 2C). There was, in contrast, no apparent difference between the exposed mouse skin from vehicle control (Fig. 2A) and Bz-423-treated (Fig. 2B) animals. Hence, unlike corticosteroids, topical application of Bz-423 did not induce atrophy in normal mouse skin. Taken together, the results of these experiments show that Bz-423 limits epidermal hyperproliferation associated with xenografted normal and psoriatic human skin without producing the atrophic changes associated with corticosteroid use.
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Immunohistochemical Analysis of Xenografted Psoriatic Skin. Human skin transplants were examined to determine expression of Ki-67 in epithelial cells. As expected, Ki-67 staining was detected in basal keratinocytes in control and treated specimens, but in general there was reduced Ki-67 expression in keratinocytes from Bz-423-treated and clobetasol propionate-treated specimens compared with control (mirroring the differences in epidermal thickness described above). Staining was variable in that there was extensive staining of basal cells in some areas and little or no staining in areas immediately adjacent. Figure 3A demonstrates Ki-67 expression in a representative vehicle-treated section, and Fig. 3B shows a section of Bz-423-treated psoriatic skin.
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Finally, sections of treated skin were also stained with antibodies against c-myc and β-catenin. c-myc was detected in the nuclei of basal layer keratinocytes in both vehicle-treated and Bz-423-treated animals and the intensity of staining (weak) was similar between these groups. Staining for c-myc in suprabasal cells was apparent in sections from vehicle-treated animals, but not in sections from animals treated with Bz-423 (Fig. 3, E and F). With anti-β-catenin, intense staining of cell junctions was the predominant finding in sections from both groups (Fig. 3, G and H). In some cells from the vehicle-treated group, diffuse cytoplasmic staining was also apparent, and occasional cells also showed intense nuclear staining. With rare exception (Fig. 3H, arrow), such staining was not observed in sections from Bz-423-treated animals.
Bz-423-Mediated Keratinocyte Growth Inhibition and Apoptosis. Keratinocytes were exposed to Bz-423 in monolayer culture. After 48 h, proliferation and viability in cells treated with different concentrations of Bz-423 were assessed. The nonviable cell population included cells that were dead (via apoptotic or other cytotoxic mechanisms) and cells for which direct evidence of apoptosis was available. The results of this study are shown in Table 1. Consistent with past findings (Varani et al., 2005), there was a dose-dependent decrease in proliferation with Bz-423 over the range of 0.1 to 4.0 µM. Of interest, the percentage on nonviable cells increased only slightly between 0.1 and 4.0 µM. Likewise, the percentage of apoptotic cells also slowly increased over the same dose range. Thus, although it is impossible to completely rule out all cytotoxicity at Bz-423 concentrations below 4 µM, these data clearly indicate that growth suppression rather than cell killing is the primary mechanism leading to reduced cell number after treatment. Also consistent with what we have reported previously (Varani et al., 2005), concentrations above 4 µM produced an increase in cytotoxicity.
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Role of ROS in Bz-423-Mediated Keratinocyte Growth Inhibition. Bz-423 inhibits proliferation of transformed B cells through an oxidant-dependent mechanism (Sundberg et al., 2006). In addition, our previous study demonstrated increased oxidant generation in keratinocytes exposed to Bz-423 (Varani et al., 2005). Therefore, as part of the present study, we sought to determine whether ROS generation was necessary for Bz-423-induced keratinocyte growth arrest. As expected treatment of keratinocytes with growth-suppressing doses of Bz-423 led to increased intracellular ROS, based on increased fluorescence of cells loaded the two oxidant-sensitive fluorescent indicators—DHE and DCF-DA (Fig. 4, top). This increase was almost entirely reversed in cells treated with vitamin E (100 µM), an antioxidant reactive with a variety of ROS. In additional studies (data not shown), 50 and 10 µM vitamin E were also effective in reversing the growth-inhibitory effects of Bz-423, but they were not as effective as 100 µM. At 1 µM, there was little protection and at 200 µM vitamin E, toxicity was observed.
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), Bz-423 at concentrations of 1 to 2 µM, there was a net decrease in the number of cells present at day-2 compared with the number present in control cultures. As also shown in Fig. 4, bottom, the same concentration (100 µM) of vitamin E that suppressed intracellular ROS levels in Bz-423-treated keratinocytes also reversed the antiproliferative effects. Taken together, these data suggest that ROS mediate Bz-423-induced growth inhibition.
Effects of Bz-423 on Intracellular Signaling Events in Keratinocytes. In lymphocytes, growth inhibition by Bz-423 is associated with c-myc protein degradation and marked changes in the expression and phosphorylation of proteins that control the G1-to-S transition within the cell cycle (Sundberg et al., 2006). To determine whether similar changes in intracellular signaling accompanied keratinocyte growth suppression, we assessed c-myc and cyclin D1 expression in keratinocytes after Bz-423 treatment in vitro (2 µM Bz-423 for 18 h). Consistent with the observations in B cells, the levels of c-myc and cyclin D1 were both significantly reduced by Bz-423 (Fig. 5). Because c-myc expression can be regulated by Wnt signaling pathways, in which β-catenin is a transcriptional activator that regulates the expression of genes including c-myc (Wong and Pignatelli, 2002), we also assessed the level of this protein in control and Bz-423-treated keratinocytes. In contrast to the decrease in c-myc and cyclin D1, there was no apparent change in the level of β-catenin protein in keratinocytes in response to Bz-423 (Fig. 5).
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Structural Analogs of Bz-423: Comparison of Effects on Keratinocytes and Fibroblasts in Monolayer Culture and Human Skin in Organ Culture. Twenty-three structural analogs of Bz-423 were tested for growth-inhibitory activity against keratinocytes and fibroblasts in mono-layer culture with the intention of identifying congeners with greater activity against keratinocytes for further testing in an organ culture model. Each of the 23 compounds inhibited proliferation of both cell types, and in every case, EC50 values for keratinocytes were lower than for fibroblasts. The EC50 values for keratinocytes ranged from 0.7 µM to greater than 10 µM. For fibroblasts, EC50 values ranged from 1.4 µMto greater than 10 µM. Structures for two of the analogs (identified as 1002, and 1118) are presented in Fig. 7 along with that of Bz-423. Their respective EC50 values for inhibition of keratinocyte and fibroblast proliferation are included.
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, 1994, 2001), treatment with RA (1 µg/ml) led to increased keratinocyte proliferation and epidermal hyperplasia (Fig. 8B). When the tissue was concomitantly treated with RA and either of the two analogs, partial suppression of the hyperplastic changes was observed. With both 1002 and 1118, reduction in thickness was seen at 1 and 2 µM (Fig. 8C and histogram). Reduction in retinoid hyperplasia was also observed with higher concentrations. However, at concentrations above 2 µM, thinning of normal skin epidermis was observed in some of the cultures (data not shown). These results confirm the general activity of this class of benzodiazepines against hyperproliferative skin responses. Moreover, these data provide support for the concept that activity in monolayer cultures may be used to select appropriate candidate molecules out of this class to carry forward for testing in more advanced tissue models of hyperplastic skin disease.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Bednarski et al., 2003). In a recent study, Bz-423 was shown to inhibit keratinocyte proliferation in monolayer culture. Retinoid-induced hyperplastic growth in the epidermis of human skin organ culture was also inhibited by Bz-423 under conditions in which normal skin structure was not affected (Varani et al., 2005). Capacity to interfere with epidermal hyperplasia without affecting normal skin function provides a potential therapeutic opportunity. Hyperplasia in skin after topical RA treatment and hyperplasia in psoriasis share common features. Both involve up-regulation of multiple growth factors that act through the EGF receptor (e.g., amphiregulin and heparin-binding EGF in the RA response and these two as well as transforming growth factor-
in psoriasis) and generate similar receptor-coupled downstream signals (Gottlieb et al., 1988; Elder et al., 1989; Cook et al., 1992; Varani et al., 2001; Piepkorn et al., 2003; Rittié et al., 2006). Interference with EGF receptor signaling reduces hyperplasia in both instances. With this in mind, the present study was undertaken to directly assess the ability of Bz-423 to modulate epidermal hyperplasia in human psoriatic plaque skin using the human skin-scid mouse transplant model. Based on the results obtained here, we conclude that Bz-423 (formulated for topical delivery) can effectively mitigate the psoriatic phenotype.
The evidence presented here strongly suggests that Bz-423 limits epidermal hyperplasia by a direct effect on keratinocyte proliferation. Although our studies do not unambiguously rule out the possibility that the compound's action against transplanted psoriatic skin in mice involves an effect on immune cell function, a direct effect on the epidermal cell proliferation probably underlies much of the response noted here. This is based on the following evidence. First, when skin from nonpsoriatic donors was transplanted onto the scid mice and then treated, the hyperplastic changes that occurred in the normal skin were also suppressed. In addition, suppression of keratinocyte proliferation in monolayer culture was observed, indicating the capacity of Bz-423 to directly inhibit keratinocyte growth. Also consistent with a direct effect on epidermal cell growth is the inhibitory effect on RA-induced epidermal hyperplasia (Varani et al., 2005; this report). It is of interest, in this regard, that in all of the tissue models where epidermal growth suppression was observed, the epidermis was in a hyperplastic condition to begin with. We are confident, based on the failure of Bz-423 to cause thinning of normal quiescent human skin in organ culture (Varani et al., 2005) or to cause thinning of normal mouse skin in the transplant model (this report), that Bz-423 will not produce the atrophic changes in the skin that are observed with other antipsoriatic agents such as clobetasol propionate. In vitro studies showing that fibroblasts are less sensitive to Bz-423 and related structures than keratinocytes provide further evidence for cell-type selectivity that should limit the potential of Bz-423 to cause skin atrophy (Varani et al., 2005; this study). Given that we were able to select two additional related structures from a small series of Bz-423 congeners that have similar keratinocyte-selective effects in monolayer culture and organ culture models, the Bz-423 chemotype seems to have the potential for topical drug development against hyperplastic skin disorders.
How Bz-423 functions at the molecular level to suppress keratinocyte proliferation is not fully understood. Past studies have shown that Bz-423 inhibits mitochondrial F1F0-ATPase in an OSCP-dependent manner, resulting in increased superoxide anion production in a range of cell types (Johnson et al., 2005). Rapid conversion of superoxide anion to hydrogen peroxide with the superoxide dismutase mimetic MnTBAP provides substantial protection against proapoptotic activity in these cells. Using similar approaches to those used in studies with lymphoid cells, we found that in keratinocytes, a rapid increase in intracellular fluorescence (both DHE and DCF) occurred in response to Bz-423. However, in contrast to findings in lymphoid cells, MnTBAP did not protect keratinocytes against growth inhibition resulting from exposure to 2 µM Bz-423 (Varani et al., 2005). Thus, superoxide anion itself is probably not directly responsible for the growth-modulating effects of Bz-423 in keratinocytes. A downstream ROS such as hydroxyl radical may be involved in mediating cytostasis. Consistent with this interpretation, the antioxidant vitamin E, which scavenges a variety of ROS types, reduced Bz-423-induced ROS levels and provided substantial protection against Bz-423-induced growth suppression.
Consistent with the F1F0-ATPase serving as a target for Bz-423, we have previously shown that other benzodiazepines and ligands of the peripheral benzodiazepine receptor, including PK11195, clonazepam, and 4-chlorodiazepam, also inhibit F1F0-ATPase activity, although they are significantly less potent inhibitors of the enzyme than Bz-423, requiring 6- to 40-fold greater concentrations (Cleary et al., 2007). Our earlier study (Varani et al., 2005) showing that PK1195, clonazepam, and 4-chlorodiazepam were also less effective than Bz-423 in reducing keratinocyte proliferation (i.e., EC50 values >10 µM) is in agreement with their lower potency against the mitochondrial ATPase. Binding and inhibiting F1F0-ATPase activity to generate ROS, and, in turn, selectively regulating keratinocyte growth represents a novel therapeutic target for treatment of hyperproliferative skin disorders. Of interest in this regard, a recent study showed that keratinocytes use the mitochondria respiratory chain for accumulation of superoxide anion and that they have drastically lower mitochondrial superoxide dismutase activity than other skin cells, including dermal fibroblasts (Hornig-Do et al., 2007).
Mechanistic studies with B lymphocytes indicated that Bz-423 inhibited proliferation by targeting c-myc protein for rapid degradation in a proteosome-dependent manner (Sundberg et al., 2006). Concomitantly, levels of a number of cell cycle-regulating proteins thought to be downstream of c-myc were also reduced. The present studies demonstrated decreased expression of c-myc (and decreased cyclin-D1 expression) in keratinocytes, but the mechanism leading to the reduction may be different. Our studies showed that in the presence of a growth-suppressing concentration of Bz-423, a shift in distribution of β-catenin from the cytosolic to membrane fraction occurred. Because c-myc is transcriptionally up-regulated by β-catenin, which shuttles into the nucleus from the cytoplasmic pool, it is possible that Bz-423-induced sequestration of β-catenin at the cell surface effectively reduces cytoplasmic/nuclear β-catenin, decreasing Wnt pathway signaling and consequently affecting c-myc expression at the level of transcription. Because increased surface β-catenin is a marker of keratinocyte differentiation, these results also raise the intriguing possibility that epidermal growth suppression in response to Bz-423 may be a consequence of induced differentiation. Additional experiments are underway to understand the significance of this finding.
In summary, Bz-423 (topically delivered) reduced psoriatic hyperplasia in the human skin-scid mouse transplant model. The major growth-inhibitory effects of Bz-423 seem to be targeted at the keratinocyte. Based on in vitro observations, keratinocyte growth suppression seems to be oxidant-mediated, leading to subsequent mitigation of signaling through growth-promoting signaling pathways. The capacity of Bz-423 to reduce epidermal hyperplasia while not affecting normal skin structure provides a strong rationale for further clinical development of this class of compounds.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: Bz-423, 7-chloro-5-(4-hydroxyphenyl)-1-methyl-3-(naphthalen-2-ylmethyl)-4,5-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one; OSCP, oligomycin sensitivity-conferring protein; ROS, reactive oxygen species; RA, all-trans retinoic acid; EGF, epidermal growth factor; scid, severe, combined immunodeficient; DMSO, dimethyl sulfoxide; MnTBAP, manganese (III)meso-tetrakis (4-benzoic acid) porphyrin; ANOVA, analysis of variance; KBM, keratinocyte basal medium; KGM, keratinocyte growth medium; DPBS, Dulbecco's phosphate-buffered saline; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate; DCF, 2',7'-dichlorodihydrofluorescein; ANOVA, analysis of variance; DHE, dihydroethidium.
Address correspondence to: Dr. James Varani, Department of Pathology, The University of Michigan, 1301 Catherine Rd., Box 0602, Ann Arbor, MI 48109. E-mail: varani{at}umich.edu
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