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Plastic and Reconstructive Surgery Research, Manchester Institute of Biotechnology, University of Manchester, Manchester, United KingdomInstitute of Inflammation and Repair, University of Manchester, Manchester, United Kingdom
Institute of Inflammation and Repair, University of Manchester, Manchester, United KingdomDepartment of Dermatology, University of Luebeck, Luebeck, Germany
Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts
Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts
Address reprint requests to Ardeshir Bayat, M.B.B.S., Ph.D., Plastic and Reconstructive Surgery Research, Manchester Institute of Biotechnology, University of Manchester, 131 Princess St, Manchester, M1 7ND UK
Plastic and Reconstructive Surgery Research, Manchester Institute of Biotechnology, University of Manchester, Manchester, United KingdomInstitute of Inflammation and Repair, University of Manchester, Manchester, United KingdomDepartment of Plastic and Reconstructive Surgery, University Hospital South Manchester NHS Foundation Trust, Wythenshawe Hospital, Manchester, United KingdomUniversity of Manchester, Manchester Academic Health Science Centre, University Hospital South Manchester NHS Foundation Trust, Wythenshawe Hospital, Manchester, United Kingdom
Keloid disease (KD) is a fibroproliferative lesion of unknown etiopathogenesis that possibly targets the PI3K/Akt/mTOR pathway. We investigated whether PI3K/Akt/mTOR inhibitor, Palomid 529 (P529), which targets both mammalian target of rapamycin complex 1 (mTORC-1) and mTORC-2 signaling, could exert anti-KD effects in a novel KD organ culture assay and in keloid fibroblasts (KF). Treatment of KF with P529 significantly (P < 0.05) inhibited cell spreading, attachment, proliferation, migration, and invasive properties at a low concentration (5 ng/mL) and induced substantial KF apoptosis when compared with normal dermal fibroblasts. P529 also inhibited hypoxia-inducible factor-1α expression and completely suppressed Akt, GSK3β, mTOR, eukaryotic initiation factor 4E-binding protein 1, and S6 phosphorylation. P529 significantly (P < 0.05) inhibited proliferating cell nuclear antigen and cyclin D and caused considerable apoptosis. Compared with rapamycin and wortmannin, P529 also significantly (P < 0.05) reduced keloid-associated phenotypic markers in KF. P529 caused tissue shrinkage, growth arrest, and apoptosis in keloid organ cultures and substantially inhibited angiogenesis. pS6, pAkt-Ser473, and mTOR phosphorylation were also suppressed in situ. P529 reduced cellularity and expression of collagen, fibronectin, and α-smooth muscle actin (substantially more than rapamycin). These pre-clinical in vitro and ex vivo observations are evidence that the mTOR pathway is a promising target for future KD therapy and that the dual PI3K/Akt/mTOR inhibitor P529 deserves systematic exploration as a candidate agent for the future treatment of KD.
The mammalian target of rapamycin (mTOR) is a 289-kDa serine-threonine kinase that regulates cell mobility, survival, proliferation, transcription, and protein synthesis
However, keloids are not truly malignant neoplasms because they never metastasize. In KD, there is excessive angiogenesis and inflammatory cell infiltration,
Increased vascular endothelial growth factor may account for elevated level of plasminogen activator inhibitor-1 via activating ERK1/2 in keloid fibroblasts.
Notch signaling pathway in keloid disease: enhanced fibroblast activity in a Jagged-1 peptide dependent manner in lesional versus extra-lesional fibroblasts.
and, therefore, the mTOR pathway may be a plausible target pathway in management of KD.
mTOR kinases form two distinct multiprotein complexes: mTORC1 and mTORC2. mTORC1 consists of regulatory associated protein of mTOR (Raptor), mLST8 (also known as G-protein β-subunit–like protein, a yeast homolog of LST8), and PRAS40 (proline-rich Akt substrate of 40 kDa). mTORC2 contains mTOR and mLST8, but instead of Raptor, mTORC2 contains rapamycin-insensitive companion of mTOR (Rictor) and mammalian stress-activated protein kinase-interacting protein-1 (mSin1).
The TORC1/TORC2 inhibitor, Palomid 529, reduces tumor growth and sensitizes to docetaxel and cisplatin in aggressive and hormone-refractory prostate cancer cells.
mTORC1, the molecular target of rapamycin, phosphorylates downstream proteins S6K (p70S6 kinase) and 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1), both involved in translation. However, inhibition of mTORC1 alone by rapalogs could lead to enhanced activation of the PI3K axis because of the mTOR-S6K-IRS1 negative feedback loop.
AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity.
AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity.
Considering the importance of Akt signaling and the critical role of mTORC2 in Akt activation, mTORC2, has become an attractive drug target for many cancers.
Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/Akt signaling pathways.
Notch signaling pathway in keloid disease: enhanced fibroblast activity in a Jagged-1 peptide dependent manner in lesional versus extra-lesional fibroblasts.
Notch signaling pathway in keloid disease: enhanced fibroblast activity in a Jagged-1 peptide dependent manner in lesional versus extra-lesional fibroblasts.
Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/Akt signaling pathways.
Angiotensin II regulates phosphoinositide 3 kinase/Akt cascade via a negative crosstalk between AT1 and AT2 receptors in skin fibroblasts of human hypertrophic scars.
Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/Akt signaling pathways.
The challenge, therefore, is to generate convincing pre-clinical evidence in support of this novel KD treatment strategy, ideally using the best pre-clinical KD research assay that is currently available, ie, KD organ culture (OC),
and to identify well-tolerated mTOR inhibitors that effectively antagonize KD growth and ECM deposition in vitro and in situ.
To meet this challenge, we hypothesized that dual mTORC-1 and mTORC-2 inhibition would provide superior inhibition of Akt signaling and angiogenesis and would produce a greater effect on KF and KD vasculature than does inhibition of mTORC-1 alone. Unlike rapamycin, which inhibits only mTORC-1,
The TORC1/TORC2 inhibitor, Palomid 529, reduces tumor growth and sensitizes to docetaxel and cisplatin in aggressive and hormone-refractory prostate cancer cells.
High-performance liquid chromatography analysis of a novel small-molecule, anti-cancer drug, Palomid 529, in human and mouse plasma and in mouse tissue homogenates.
J Chromatogr B Analyt Technol Biomed Life Sci.2011; 879: 3823-3831
Therefore, we investigated the following areas in the present study: whether the baseline cellular levels of mTOR and p70S6K and their activated forms differ between primary KF and normal dermal fibroblasts (NF) isolated from extralesional and normal healthy skin; whether P529 antagonizes KD growth and ECM deposition in vitro and in situ in a manner that renders this novel dual mTORC1/2 inhibitor an ideal candidate for clinical testing in the future management of KD; and whether and how P529 differs in this respect from standard PI3K/Akt/mTOR inhibitors (rapamycin and wortmannin).
Materials and Methods
Patient Selection and Recruitment
Keloid tissue was harvested during excisional surgery in patients confirmed to have clinical and pathologic evidence of KD as described previously
Notch signaling pathway in keloid disease: enhanced fibroblast activity in a Jagged-1 peptide dependent manner in lesional versus extra-lesional fibroblasts.
Fibroblasts from the growing margin of keloid scars produce higher levels of collagen I and III compared with intralesional and extralesional sites: clinical implications for lesional site-directed therapy.
(Figure 1A). Included in the study were 31 patients (16 men and 15 women; age range, 17–74 years) with KF, including 8 with extralesional fibroblasts (normal skin adjacent to a keloid lesion), and 11 patients with NF (4 men and 7 women; age range, 23–65 years). Of 31 keloid tissue samples, 11 were used for ex vivo KD OC study (see Supplemental Figure S1 at http://ajp.amjpathol.org). Tissue samples were selected carefully from patients with no previous treatment history. In most of the in vitro experiments, samples were matched for sex and age (patient demographic data are given in Supplemental Tables S1 and S2 at http://ajp.amjpathol.org). Keloid samples were obtained from individuals of different races/ethnicities, all of whom had given approval (obtained from the local hospital, university, and regional NHS Ethics Committee) before the surgical procedure.
Figure 1A: Schematic representation of a keloid tissue sample and extralesional keloid tissue sample (internal control tissue harvested from the same patient). B: mTOR and p-mTOR expression pattern in keloid and extralesional tissue samples. Original magnification ×200. C: Representative infrared images from ICWB analysis of total mTOR, p- mTOR, total p70S6K, and p-p70S6K in primary KF compared with primary extralesional fibroblasts (ELF) and NF from healthy control subjects. Bar graph indicates mean fluorescence intensity of ICWB from three independent experiments. *P < 0.02 indicates significant overexpression in KF compared with ELF and NF. ELF, extra-lesional fibroblasts; KF, keloid fibroblasts; NF, normal fibroblasts.
Notch signaling pathway in keloid disease: enhanced fibroblast activity in a Jagged-1 peptide dependent manner in lesional versus extra-lesional fibroblasts.
Fibroblasts from the growing margin of keloid scars produce higher levels of collagen I and III compared with intralesional and extralesional sites: clinical implications for lesional site-directed therapy.
In brief, tissue was washed in PBS (PAA Laboratories, Ltd., Yeovil, Somerset, UK) and incubated in 10 mg/mL dispase II (Roche Diagnostics, Ltd., Burgess Hill, West Sussex, UK) for 3 hours at 37°C, and epidermis and fat were removed. Next, the dermis was minced and incubated in 10 mg/mL collagenase A (Roche Diagnostics). The tissue/cell suspension was passed through a 100-μm filter (cell strainer; BD Biosciences, Oxford, UK) to remove any remaining tissue residuals. Cells were then washed once with PBS and plated in T25 CellBind flasks (Nunc, Life Technologies Ltd., Wiesbaden, Germany) as passage 0 (p0) cells. Cells were maintained in DMEM (Dulbecco's modified Eagle's medium). Passage 1 (p1) to passage 4 (p4) cell cultures were used in the present study.
Notch signaling pathway in keloid disease: enhanced fibroblast activity in a Jagged-1 peptide dependent manner in lesional versus extra-lesional fibroblasts.
Fibroblasts from the growing margin of keloid scars produce higher levels of collagen I and III compared with intralesional and extralesional sites: clinical implications for lesional site-directed therapy.
P529, Rapamycin, Wortmannin, and Camptothecin Regimen
P529 was provided by Paloma Pharmaceuticals, Inc. (Boston, Massachusetts). Rapamycin and wortmannin were purchased from Merck Chemicals, Ltd. (Beeston, Nottingham, UK) to assess and compare the effect of P529. The concentration of rapamycin was determined on the basis of similar studies of primary fibroblasts.
The following concentrations were compared: P529 (5, 10, 20, and 40 ng/mL), rapamycin (50 and 100 ng/mL), and wortmannin (5 and 10 ng/mL). Camptothecin (Sigma-Aldrich Corp., St. Louis, MO) (250 ng/mL) was used as a positive control for real-time cell analysis (RTCA), cytotoxicity assay via detection of lactate dehydrogenase (LDH), viability/metabolic activity assay via water-soluble tetrazolium salts-1 (WST-1), and apoptotic assay (see Supplemental Figure S1 at http://ajp.amjpathol.org).
Label-Free RTCA
To determine the effect of various drug concentrations on KF compared with NF in vitro, label-free RTCA was used, as described previously.
Notch signaling pathway in keloid disease: enhanced fibroblast activity in a Jagged-1 peptide dependent manner in lesional versus extra-lesional fibroblasts.
Cytotoxicity (LDH) and Cell Viability/Metabolic Activity (WST-1) Assay
LDH and WST-1 (Roche Diagnostics) assays were performed according to the manufacturer's instructions after various drug therapies, as indicated above.
High Throughput In-Cell Western Blotting and Quantification
Primary KF and NF were subjected to various concentrations of mTOR drugs, as indicated above, for 24 hours at 37°C in 5% CO2. Then, In-Cell Western Blotting (ICWB) was performed using the similar protocol as described previously.
Notch signaling pathway in keloid disease: enhanced fibroblast activity in a Jagged-1 peptide dependent manner in lesional versus extra-lesional fibroblasts.
Fibroblasts from the growing margin of keloid scars produce higher levels of collagen I and III compared with intralesional and extralesional sites: clinical implications for lesional site-directed therapy.
In brief, KF and NF were grown to 80% to 85% confluence in T75 flasks. Before inoculating the cells in 96-well plates, the cells were starved in 0.2% serum DMEM for 24 hours. Cells were trypsinized and counted using FACS (fluorescence-activated cell sorting), and 10,000 cells were inoculated in each well of the 96-well plates (Dow Corning, Ltd., Barry, Vale of Glamorgan, UK) in triplicate and grown in DMEM for up to 7 to 8 hours at 37°C in 5% CO2. The cells were then treated for 24 hours using various drugs. Subsequent to this, the cells were fixed in 4% formaldehyde for 30 minutes at room temperature. The wells were washed three times with PBS (200 μL/well), permeabilized with PBS/0.1% Triton X-100 (150 μL/well, three times for 5 minutes each), and blocked in Odyssey blocking buffer (LI-COR Biosciences UK, Ltd., Cambridge, UK) (200 μL/well) for 2 hours at room temperature. The 96-well plates were then incubated using various primary antibodies (Table 1) overnight at 4°C (50 μL/well). They were subsequently washed three times with PBS/0.1% Tween-20 (150 μL/well). Secondary antibody (1:500) in PBS/0.5% Tween-20 was then added (50 μL/well) (Table 2). The plates were then incubated for 1 hour at room temperature, and the wells were washed with PBS/0.1% Tween-20 three times. The plates were covered with aluminum foil and imaged on an Odyssey infrared scanner (LI-COR Biosciences) using microplate 2 setting with sensitivity of 7 in the 800-nm wavelength channel. Data were retrieved using Odyssey software, which were then exported and analyzed using Excel software (Microsoft Corp., Redmond, WA). β-Actin was used as loading control.
Table 1Primary Antibodies Used in the Present Study
Antibody
Species raised
Isotype
Clone
In-cell Western dilution
IHC-dilution
Product code
Source
S6 ribosomal protein
Mouse monoclonal
IgG1
54D2
1:1000
1:100
2317
CST
pS6 ribosomal protein (Ser235/236)
Rabbit monoclonal
IgG
91B2
1:1000
1:75
4857
CST
p70S6K
Rabbit monoclonal
IgG
NA
1:500
NA
9202
CST
p-p70S6K
Rabbit monoclonal
IgG
NA
1:500
NA
9204
CST
AKT
Rabbit
NA
NA
1:1000
1:200
9272
CST
p-AKT (Thr308)
Rabbit monoclonal
IgG
244F9
1:1000
NA
4056
CST
p-AKT (Ser473)
Rabbit monoclonal
IgG
D9E
1:1000
1:50
4060
CST
mTOR
Rabbit
NA
NA
1:1000
1:100
2972
CST
p-mTOR (Ser2448)
Rabbit
NA
NA
1:1000
1:100
2971
CST
GSK-3-α/β
Rabbit monoclonal
IgG
D75D3
1:1000
NA
5676
CST
p-GSK-3-α/β (Ser9)
Rabbit monoclonal
IgG
5B3
1:1000
1:400
9323
CST
p38 MAP kinase
Rabbit
NA
NA
1:1000
1:50
9212
CST
p-p38 MAP kinase (Thr180/Tyr182)
Rabbit
NA
NA
1:1000
NA
9211
CST
PCNA
Mouse monoclonal
IgG2a
PC10
1:2000
1:4000
2586
CST
Cyclin D1
Rabbit monoclonal
IgG
92G2
1:1000
1:25
2978
CST
Anti-HIF-1α
Mouse monoclonal
IgG1
ESEE122
1:500
NA
Ab8366
Abcam
Collagen I
Mouse monoclonal
IgG1
COL-1
1:500
1:200
Ab6308
Abcam
CD34
Mouse monoclonal
IgG1
581
NA
1:100
Ab45524
Abcam
CD31
Mouse monoclonal
IgG1
1A10
NA
1:50
ncl-cd31-1a10
Leica
α-SMA
Mouse monoclonal
IgG2a
1A4
1:500
1:250
A5691
Sigma-Aldrich
Fibronectin
Rabbit polyclonal
IgG
NA
1:500
1:250
ab2413
Abcam
β-Actin
Mouse monoclonal
IgG1
NA
1:1000
NA
Ab8226
Abcam
Abcam PLC, Cambridge, UK; CST, Cell Signaling Technology, Inc., Danvers, MA; Leica Microsystems (UK), Ltd., Milton Keynes, Buckinghamshire, UK; NA, not available or not applicable; Sigma-Aldrich Corp., St. Louis, MO.
Measurement of Early Apoptosis via Annexin V Staining Followed by FACS
Primary KF and NF were treated using various concentrations of P529, rapamycin, and wortmannin, as described above. Apoptosis was detected using an annexin V–FITC (fluorescein isothiocyanate)–labeled apoptosis detection kit (Abcam PLC, Cambridge, UK) as described previously.
Each well of a 96-well Oris plate (Oris migration assay kit; Cambridge Bioscience, Ltd., Cambridge, UK) was coated with 9 μg/mL collagen (BD Biosciences) and incubated for 30 to 45 minutes at 37°C in 5% CO2. After incubation, wells were rinsed with 1X PBS once, and Oris cell seeding stoppers were inserted according to the manufacturer's instructions. Serum-starved KF and NF were prelabeled with PKH26 (Sigma-Aldrich) according to the manufacturer's instructions, and 2.5 × 104 cells per well were seeded in triplicate. For each treatment, one well without a cell-seeding stopper served as the positive control. The plate was then incubated overnight at 37°C in 5%CO2. The next day, the cell-seeding stoppers were removed from the test wells, and cells were washed once with PBS 1X (PAA Laboratories). Then 100 μL fresh medium was added with or without various drugs, and the cells were allowed to migrate for ∼30 hours in the migration zone created by cell-seeding stoppers. After completion of the experiment, micrographs were captured using an inverted IX71 microscope (Olympus Corp., Tokyo, Japan) at ×4 magnification. Migrated cells in the migration zone were counted from four independent experiments, and the mean number of migrated cells was plotted on the graphs.
In Vitro Three-Dimensional Invasion Assay
Inhibition of the invasive capacity of primary KF was tested using an in vitro three-dimensional (3D) invasion assay (Oris invasion and detection assay kit; Cambridge Bioscience) as described previously.
Notch signaling pathway in keloid disease: enhanced fibroblast activity in a Jagged-1 peptide dependent manner in lesional versus extra-lesional fibroblasts.
In brief, 96-well plates were coated with basement membrane extract (BME) per the manufacturer's instructions. After BME coating, Oris cell-seeding stoppers were inserted in each well. Serum-starved cells, 2.5 × 104 cells/well, were seeded. At 8 to 12 hours after cell attachment, the stoppers were removed, the cells were washed once with 1X PBS, and 40 μL BME was added on top of the cells. The plates were reincubated at 37°C in 5% CO2 for 45 to 60 minutes to enable polymerization of BME. After BME polymerization, various mTOR drug treatments were given for ∼48 hours, and the cells were allowed to invade (in the presence of mTOR drugs) the 2-mm invasion zone created by cell-seeding stoppers. After completion of the experiment, cells were stained with calcein AM. Micrographs were obtained using an inverted IX71 microscope (Olympus) at ×4 magnification. Invaded cells in the invasion zone were counted from four independent experiments, and the mean number of invaded cells were plotted on graphs.
Actin Reorganization
Serum-starved, low-density (1.5 × 104 cells/well), primary KF (passages p1 to p4) were grown on glass coverslips in 24-well plates, were pre-treated with or without P529, rapamycin, and wortmannin for ∼8 hours, then stimulated with or without 10 ng/mL insulin-like growth factor-1 (IGF-1) (R&D Systems Europe, Ltd., Abingdon, Oxfordshire, UK) for ∼3.5 hours. Cells were then fixed in 4% paraformaldehyde buffered solution for 20 minutes at 4°C and permeabilized with 0.1% Triton X-100 in 1× PBS (PAA Laboratories) for 15 minutes. Cells were stained with 1 mg/mL TRITC-conjugated phalloidin (Sigma-Aldrich) for 30 minutes at room temperature. Actin filaments (F-actin) were visualized and photographed using a BX53 digital microscope (Olympus). The percentage of cells with lamellipodia formation was determined by randomly counting at least 250 cells under the fluorescence microscope at ×600 magnification.
Ex Vivo Keloid OC Model
For the serum-free long-term OC of keloid tissue, a recently developed KD OC system was used.
In brief, 4-mm punch biopsy fragments were prepared from keloid tissue. These were embedded in rat tail collagen gel matrix at the air-liquid interface. Serum-free William's E medium, supplemented with 10 μg/mL insulin, 10 ng/mL hydrocortisone, 2 mmol/L l-glutamine, 100 IU/mL penicillin, and 10 μg/mL streptomycin,
Tissue specimens were fixed in formaldehyde, embedded in paraffin blocks, and sectioned to 4- to 5-μm thickness. Paraffinized tissue sections (4 to 5 μm) collected at various times (day 0, day 3, week 1, week 2, and week 4) were deparaffinized and stained using standard H&E (Surgipath; Leica Microsystems (UK), Ltd., Milton Keynes, Buckinghamshire, UK). Micrographs were obtained using an upright fluorescence microscope (BX51; Olympus).
Notch signaling pathway in keloid disease: enhanced fibroblast activity in a Jagged-1 peptide dependent manner in lesional versus extra-lesional fibroblasts.
Fibroblasts from the growing margin of keloid scars produce higher levels of collagen I and III compared with intralesional and extralesional sites: clinical implications for lesional site-directed therapy.
Tissues were immersed in freshly prepared 4% paraformaldehyde in 0.02 mol/L phosphate buffer (pH 7.4) overnight at 4°C, washed in 1 × PBS, and embedded in paraffin. For immunostaining, 4- to 5-μm tissue sections were hydrated and incubated in 3% H2O2 in methanol for 20 minutes, washed in water, and boiled in 1 mmol/L EDTA (pH 8.0) for 15 to 20 minutes. Sections were blocked with 3% to 6% normal goat serum (Vector Laboratories, Ltd., Orton Southgate, Peterborough, UK) and incubated with various primary antibodies (Table 1) at specific dilutions overnight at 4°C. After two washes with 1× Tris-buffered saline solution, tissue sections were incubated with respective secondary antibodies (Table 2) for 30 to 45 minutes at room temperature and detected using an avidin-biotin complex method kit (Vector Laboratories).
Notch signaling pathway in keloid disease: enhanced fibroblast activity in a Jagged-1 peptide dependent manner in lesional versus extra-lesional fibroblasts.
To evaluate apoptosis caused by the various concentrations of drugs, double immunofluorescence was used. Terminal dUTP nick-end labeling [Dead-End TUNEL assay; Promega (UK), Ltd., Southampton, Hampshire, UK) was used as a marker for apoptosis, and DAPI was used to localize the DNA as described previously.
The micrographs were obtained using an upright fluorescence microscope (BX51; Olympus) at ×200 magnification. The mean number of apoptotic cells was quantified in five random fields from each stained tissue section.
OC Fluorescence-Associated Immunohistochemistry
Cell-specific antigen (CD34 expression) was localized on 4- to 5-μm paraffin sections using a similar technique as described previously.
Each experiment was performed independently at least three or four times. Data are given as mean ± SEM. Statistical evaluation of the continuous data was performed using one-way analysis of variance, followed by Dunnett's t-test for between-group comparisons. Statistical analyses were performed using commercially available software (SPSS version 13.0; SPSS, Inc., Chicago, IL). P < 0.05 was considered statistically significant.
Results
Elevated Levels of Total and Phosphorylated Forms of mTOR and p70S6K in KF
mTOR regulates metabolic processes and translation rates in response to intracellular ATP levels, as well as extracellular signals by phosphorylating two major translational components: ribosomal p70KDa S6 kinase (p70S6K) and eIF4E.
Hence, we initially investigated whether there was overexpression of mTOR, p70S6K, and their phosphorylated forms in the keloid samples.
Immunoperoxidase staining for total and phosphorylated forms of mTOR showed high expression in KD tissue (n = 7) compared with extralesional tissue sections (n = 5) (Figure 1B). ICWB analysis and mean total immunoreactivity (fluorescence intensity) also exhibited significant (P < 0.02) up-regulation of mTOR, p-mTOR, p70S6K, and p-p70S6K in KF (n = 12) compared with fibroblasts isolated from extralesional sites (n = 8) and normal skin (n = 5) (Figure 1C). There was also noticeable overexpression of pS6, one of the downstream targets of the mTOR signaling pathway, in KD compared with normal skin tissue (see Supplemental Figure S2 at http://ajp.amjpathol.org). However, there was no statistically significant difference in the expression of mTOR, p70S6K, and their phosphorylated forms between primary extralesional fibroblasts and NF (Figure 1C). These analyses demonstrate that mTOR is highly active in KD lesions compared with extralesional and normal skin.
Dose-Dependent Effect of P529 on PI3K/Akt/mTOR signaling in KF
We next investigated the efficacy of P529 on intracellular PI3K/Akt/mTOR signaling of primary human KF (n = 10) and NF (n = 6). This was compared with rapamycin, the reference mTOR inhibitor, and wortmannin, a specific inhibitor of PI3K signaling, using ICWB. Results showed a statistically significant (P < 0.02) concentration-dependent decrease in pAkt-S473 in primary KF at 24 hours of treatment with both P529 and wortmannin, whereas total Akt levels were unaffected by both compounds. mTORC1 downstream substrates, 4E-PB, and S6 ribosomal protein were also efficiently (P < 0.02) dephosphorylated by both compounds. Both P529 and wortmannin inhibited neither phosphorylated mitogen-activated protein kinase nor pAkt-T308 as potently as pAkt-S473. Furthermore, P529 successfully reduced (P < 0.02) phosphorylation of p-GSK3β, a critical downstream element of the PI3kinase/Akt cell survival pathway, and HIF1-α (P < 0.02). As expected, rapamycin (100 ng/mL) reduced pAkt-S473 at very low levels, compared with both P529 and wortmannin (5 ng/mL), at a higher concentration of 100 ng/mL (Figure 2, A and B). P529 did not cause any significant inhibition of PI3K/Akt/mTOR signaling in NF at a low concentration (5 ng/mL) as compared with rapamycin and wortmannin (see Supplemental Figure S3 at http://ajp.amjpathol.org). Considered together, these results demonstrate that, compared with rapamycin, P529 is a specific and highly potent inhibitor of Akt/mTOR in primary human KF.
Figure 2ICWB analysis of intracellular signaling events in KF induced by increasing concentration of P529 compared with rapamycin and wortmannin. A: Representative photographs of ICWB for KF treated with P529, rapamycin, and wortmannin for 24 hours. B: Average immunoreactivity from three independent ICWB experiments of KF normalized to β-actin. *P < 0.05, †P < 0.02, indicates significant difference compared with vehicle control group (DMSO).
Subsequently, we compared the inhibition potency of P529 on p-mTOR with rapamycin and wortmannin. In ICWB, all three compounds showed significantly (P < 0.02) reduced p-mTOR levels (Figure 3, A and B). However, rapamycin (100 ng/mL) was less effective in reducing p-mTOR than were P529 and wortmannin (5 ng/mL). Hence, at low concentrations, P529 is a highly potent p-mTOR inhibitor in KF (n = 8). Unlike rapamycin and wortmannin, P529 did not have any inhibitory effect on p-mTOR in NF (n = 5) (see Supplemental Figure S4 at http://ajp.amjpathol.org) compared with KF, which suggests that P529 has a greater p-mTOR inhibitory effect on KF.
Figure 3Expression pattern of total and p-mTOR in KF at 24 hours after treatment with P529, rapamycin, and wortmannin. A: In-Cell Western Blot was performed to assess the immunoreactivity of mTOR and p-mTOR after various drug treatments. Representative output infrared images of treated and untreated fibroblasts, stained for protein expression (visible in red) are shown. B: Bar graphs represent the quantification of mean protein expression with various treatments from three independent experiments after normalization to loading control β-actin. *P < 0.05, significant difference in the treated group vs the vehicle (DMSO) control group.
Dose-Dependent Inhibition of Cell Spreading, Attachment, and Cell Proliferation in KF
We investigated whether P529 has any substantial inhibitory effect at the cellular level on KF and NF activity. We used a label-free RTCA system. RTCA results showed that primary KF (n = 10) and NF (n = 5) had cell indices of ∼3.8 to 4.2 and ∼3.0 to 3.7, respectively, at 24 hours of cell growth in the absence of any test compounds. This confirmed that fibroblasts obtained in KD (Figure 4, A and B) were significantly (P < 0.05) more aggressive in terms of cell spreading, attachment, and proliferation
Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/Akt signaling pathways.
Green tea polyphenol epigallocatechin-3-gallate suppresses collagen production and proliferation in keloid fibroblasts via inhibition of the STAT3-signaling pathway.
Figure 4Role of mTOR inhibitors on keloid cell behavior using dynamic and real-time monitoring of cell adhesion, spreading, and proliferation on microelectronic sensor arrays. A: Effect of P529, rapamycin, and wortmannin on KF using real-time cell analysis. B: Quantitative analysis of RTCA cell index of KF and NF. Representative data from five independent experiments performed in triplicate are shown. *P < 0.05, †P < 0.02, significant difference vs DMSO control group. ‡P < 0.05, significant difference in growth of KF versus NF.
By using the RTCA assay, we also demonstrated that P529, rapamycin, and wortmannin inhibited cell spreading, attachment, and proliferation in primary KF in a dose- and time-dependent manner. Compared with rapamycin and wortmannin, P529 was highly effective even at very low concentrations (5 ng/mL). A similar dose- and time-dependent inhibition was also observed in NF, but with slightly less efficacy than in KF. However, compared with rapamycin and wortmannin, P529 was more efficient in inhibiting both primary KF and NF (Figure 4B).
Consequently, we investigated whether P529 had a sustained effect on KF and NF after drug retrieval. In both cell types, when P529 was retrieved, the cells were not able to recover within 30 hours; rather, the average cell index was further reduced, which suggests that the drug effect was sustained in this group compared with groups treated with rapamycin and wortmannin (Figure 4B). These results suggest that P529 decreased KF and NF activity more efficiently than did rapamycin and wortmannin.
Increased Dose-Dependent Cytotoxicity and Cell Viability/Metabolic Activity in KF
To better understand the inhibitory effect of KF and NF observed in the RTCA assay, we assessed the efficacy of P529 on cytotoxicity using an LDH assay, and cell viability/metabolic activity using a WST-1 assay.
Both membrane disturbance (cytotoxicity) and mitochondrial activity (viability/metabolic activity) were studied using a range of concentrations of P529 (5, 10, 20, and 40 ng/mL). All three compounds (P529, rapamycin, and wortmannin) significantly (P < 0.05) increased cytotoxicity at various concentrations in primary human KF (n = 10) and NF (n = 6). However, the P529-treated group exhibited higher cytotoxicity than did the groups treated with rapamycin or wortmannin in KF compared with NF (see Supplemental Figure S6A at http://ajp.amjpathol.org). When drug treatments were stopped, P529 sustained cytotoxicity significantly (P < 0.05) for up to 30 hours compared with the groups treated with rapamycin or wortmannin. P529 demonstrated significant toxicity in KF and NF; however, the effect was reversible within 24 hours of drug removal in NF but not in KF, and much higher dosages were required for the growth-inhibitory effects of P529 in NF compared with KF (see Supplemental Figure S6B at http://ajp.amjpathol.org).
WST-1 analysis demonstrated a significant (P < 0.05) increase in viability/metabolic activity of primary KF compared with NF. These results were in agreement with those of RTCA. P529 and wortmannin were significantly (P < 0.05) more effective in reducing cell viability/metabolic activity in a dose-dependent manner compared with the dimethyl sulfoxide (DMSO) control group (see Supplemental Figure S6C at http://ajp.amjpathol.org). However, rapamycin also reduced the viability/metabolic activity of KF at much higher concentrations. Compared with the groups treated with rapamycin, KF in the groups treated with P529 or wortmannin sustained cell viability/metabolic activity inhibition up to 24 to 30 hours after discontinuation of the drug (see Supplemental Figure S6D at http://ajp.amjpathol.org). P529 was highly selective and effective in KF and less effective in NF, compared with both rapamycin and wortmannin. From these results, it was concluded that P529 caused severe toxicity and substantially inhibited viability/metabolic activity.
Inhibition of KF Proliferation by Down-Regulation of Cell Cycle
P529 greatly reduced KF and NF proliferation. To further investigate these findings, expression of the cell cycle proteins PCNA and cyclin D, were evaluated at the protein level using ICWB. Significant (P < 0.02) concentration-dependent down-regulation of PCNA and cyclin D was detected in fibroblasts treated with both P529 and Wortmannin compared with DMSO. However, at a higher concentration (100 ng/mL), the rapamycin-treated group also exhibited a similar level of significant (P < 0.02) reduction in PCNA and cyclin D expression compared with the DMSO control group in both KF (n = 8) (Figure 5, A and B) and NF (n = 5) (see Supplemental Figure S7 at http://ajp.amjpathol.org). Considered together, these results show that P529 significantly inhibits primary KF activity, at least in part by inhibiting PCNA and cyclin D at the molecular level.
Figure 5Inhibition of keloid-associated fibrotic markers and cell cycle–regulated genes by P529. A: In-Cell Western Blotting of expression of ECM proteins, PCNA, and cyclin D 24 at hours after treatment of KF with various drugs. B: Mean immunoreactivity values of KF from three independent experiment of In-Cell Western Blotting plotted on the graphs after normalizing to β-actin. *P < 0.05, †P < 0.02, significant difference in treated group vs DMSO control group.
On the basis of these results, we hypothesized that P529 might achieve its inhibitory effect on cell spreading, attachment, and proliferation via apoptosis or cellular necrosis. This hypothesis was assessed via dual-labeled FACS analysis using annexin V and propidium iodide labeling (annexin V detects early apoptosis and propidium iodide identifies cell death and necrosis).
P529, rapamycin, and wortmannin all significantly (P < 0.05) and dose dependently increased the percentage of cells positive for annexin V and propidium iodide, compared with control. Compared with NF (n = 4), P529 caused more apoptosis in KF (n = 8) (Figure 6, A and B; and see Supplemental Figure S8 at http://ajp.amjpathol.org). Therefore, P529 strongly promotes apoptosis and necrosis in human KF.
Figure 6P529 induces apoptosis in KF. Apoptosis was detected with drug treatment using annexin V staining. A: Annexin V staining for FACS. After 24 hours of treatment with indicated drug concentrations, the cells were harvested, and FITC-labeled annexin V was added to a final concentration of 2.5 μg/mL. To detect dead cells, propidium iodide (PI) was added at 5 μg/mL concentration. Using flow cytometry, dot plots of annexin V on the y axis against PI on the x axis were used to differentiate viable cells (negative for both PI and annexin V), apoptotic cells (annexin V–positive cells but excluding PI, with PI therefore negative), and late apoptotic or necrotic cells (double-positive for PI uptake and annexin V staining). Nonstained cells and untreated cells were used as negative controls. B: Annexin V– and PI-positive KF after 24 hours of treatment with various drugs as indicated in the bar graphs at different concentrations. Positive cells were counted from three independent experiments and plotted on the graph as mean ± SEM, *P < 0.05, significant difference vs DMSO control group.
Inhibition of Migration and Invasion Properties of KF
These inhibitory properties of P529 were further probed under 2D and 3D conditions, using a novel in vitro migration assay. Treatment with P529, rapamycin, and wortmannin significantly (P < 0.05) reduced the migration properties of KF (n = 6) in 2D migration compared with the control DMSO-treated group in a concentration-dependent manner. As expected, P529 was more potent in inhibiting migration properties of KF than was rapamycin, even at very low concentrations (Figure 7, A and B). Like other cancer cells, KF have invasive properties.
Fibroblasts from the growing margin of keloid scars produce higher levels of collagen I and III compared with intralesional and extralesional sites: clinical implications for lesional site-directed therapy.
As anticipated, in the 3D invasion model, KF (n = 8) was highly invasive. Both P529 and wortmannin significantly (P < 0.01) reduced the invasive properties of KF at 48 hours after treatment. In addition, rapamycin also showed significant (P < 0.05) inhibition of invasion properties of KF, but with very low efficacy (Figure 7, C and D). Considered together, these results suggest that P529 has a potential anti-migration and anti-invasive property for KF.
Figure 7Effect of P529, rapamycin, and wortmannin drugs on KF migration and invasion properties in in vitro 2D and 3D models, respectively. A: KF migration response toward 2-mm migration zone created by cell seeding stoppers in the presence and absence of various drugs. Representative micrographs are shown from three independent experiments. B: Number of migrated KF in the migration zone with and without drugs. Number of cells migrated into the migration zone were counted on the basis of the 0 hour migration pattern and plotted on the graph. C: Number of invaded KF in the invasion zone with and without drugs. Number of cells invaded into the invasion zone were counted on the basis of the 0 hour invasion pattern and plotted on the graph. D: KF invasive response toward 2-mm invasion zone created by cell seeding stoppers in the presence and absence of various drugs. Representative micrographs are shown from three independent experiments. *P < 0.05, †P < 0.02, significant difference in drug-treated group vs DMSO control group.
Inhibition of IGF-1–Stimulated F-Actin Reorganization in KF
Rapid reorganization of the actin cytoskeleton, a multistep process that includes cell polarization, adhesion, and de-adhesion, is an early event in cell migration and invasion.
Therefore, we attempted to understand whether the P529-induced inhibition of cell migration and invasion is related to prevention of cell polarization or protrusion. This was assessed using an IGF-1–stimulated F-actin reorganization assay.
The results showed that, in response to stimulation with IGF-1 (10 ng/mL), the actin cytoskeleton in KF (n = 8) undergoes rapid reorganization. P529, rapamycin, and wortmannin did not alter the pattern of basal F-actin distribution, but reduced IGF-1–stimulated F-actin reorganization at the leading edge and lamellipodia formation in KF, compared with the control group. P529 reduced lamellipodia formation more effectively than did rapamycin and wortmannin (Figure 8, A and B). Considered together, these results suggest that P529 significantly inhibits F-actin reorganization, which could be a potential mechanism for inhibition of cell proliferation, migration, and invasion properties of primary KF.
Figure 8P529 inhibits IGF-I–induced F-actin reorganization. A: Representative micrographs from three independent experiments performed in triplicate. In the absence of IGF-I, F-actin was randomly distributed across the cells, as revealed by staining with TRITC-conjugated phalloidin. Some of the F-actin was condensed in dotlike structures at the margins of the cells. However, within 3.5 hours of stimulation with IGF-I, more F-actin was condensed at the leading edge within structures resembling protrusions or lamellipodia-like structures compared with nontreated control cells. Formation of lamellipodia is shown (arrows). Scale bar = 20 μm. Micrographs at ×600 magnification. B: Percentage of cells with lamellipodia formation. Results are given as mean ± SEM of four independent experiments. *P < 0.05, significant difference vs IGF-I group; †P < 0.05, significant difference vs DMSO+IGF-I group.
Dose-Dependent Down-Regulated Expression of Fibrosis-Associated Keloid Markers
KD has been associated with a number of molecular markers, and there is increased expression of several key cytokine and growth factors including vascular endothelial growth factor, transforming growth factor-β, platelet-derived growth factor, and connective tissue growth factor. In addition, there is overexpression of a number of characteristic ECM-associated proteins such as collagen, fibronectin, and α-SMA.
Green tea polyphenol epigallocatechin-3-gallate suppresses collagen production and proliferation in keloid fibroblasts via inhibition of the STAT3-signaling pathway.
Suppression of TGF-β1/SMAD pathway and extracellular matrix production in primary keloid fibroblasts by curcuminoids: its potential therapeutic use in the chemoprevention of keloid.
Therefore, using ICWB, we compared the effect of P529, rapamycin, and wortmannin on the expression of KD-associated fibrotic markers at the protein level. This demonstrated that all compounds significantly (P < 0.04) and dose-dependently down-regulated collagen I, fibronectin, and α-SMA protein expression (Figure 5, A and B), compared with controls. Again, compared with rapamycin and wortmannin, P529 was more effective at very low concentrations (5 ng/mL) in the inhibition of these ECM proteins, which was evident in KF (n = 8) (Figure 5, A and B). Compared with rapamycin and wortmannin, P529 did not show significant inhibition of ECM in NF (n = 5) (see Supplemental Figure S7 at http://ajp.amjpathol.org). In the present study, down-regulation of collagen and fibronectin by P529 (P < 0.001) was highly specific for P529 and strongly significant compared with rapamycin (P < 0.05) and wortmannin (P < 0.01). Because we normalized the expression levels of collagen and fibronectin to β-actin (internal loading control) in ICWB, our data suggest that this effect was not related to apoptosis or necrosis.
Overall, in the present study we demonstrated that P529 significantly inhibited primary KF at very low concentrations. Rather, a significant effect of P529 was observed in primary NF only at much higher concentrations. This suggests the specificity of P529, which acts selectively on cells with active mTOR signaling.
Significant Reduction of Keloid Tissue Volume by P529
To evaluate the therapeutic potential of P529 in the management of KD, 4-mm biopsy samples of KD tissue (n = 11) were subjected to various concentrations of P529 in a serum-free, keloid, long-term OC model.
P529 and wortmannin significantly (P < 0.05) reduced keloid tissue volume at week 1 (Figure 9A; and see Supplemental Figure S9 at http://ajp.amjpathol.org), and weight (Figure 9B) at day 3, compared with the vehicle- and rapamycin-treated groups. However, rapamycin also decreased (P < 0.05) the mean volume and weight of the keloid OC after 1 week. These data suggest that P529 induced rapid shrinkage of keloid volume and weight in an ex vivo model.
Figure 9Shrinkage of keloid organ culture (OC) after various drug treatments. Four-millimeter keloid tissue explants were maintained in collagen gel matrix in the presence of various concentrations of drugs up to 4 weeks, and the tissue was collected at different times, as indicated in the bar graphs. A: Average weights of the keloid tissue OC at different times are indicated in the bar graph. B: Mean length of all layers of epidermal shrinkage except stratum corneum, given in micrometers, shows a significant reduction of epidermal thickness in the treated group vs the untreated control group.*P < 0.05, †P < 0.02, significant difference vs vehicle control group.
Reduction of Keloid Tissue Epidermal Thickness, Cellularity, Inflammation, and Angiogenesis in Situ
We further delineated the anti-keloid effect of P529 in an ex vivo model. Histologic and morphometric analyses of the KD OC (n = 11) showed that the thickness of the epidermis overlying the keloid was significantly (P = 0.01) reduced at week 1 of P529 treatment compared with rapamycin and wortmannin (Figure 9B). Histologic analysis revealed that up to day 3, the overall tissue architecture was well preserved after both wortmannin and rapamycin treatment. Instead, on day 3, thinning of the stratum granulosum and papillary dermis was observed in the P529 treatment group (see Supplemental Figure S10 at http://ajp.amjpathol.org). P529 and wortmannin caused severe epidermal damage, with picnotic nuclei and dislocation of epidermis from dermis, at weeks 1 to 4 after treatment (Figure 10A). In contrast, the rapamycin-treated group exhibited minimal tissue damage from day 3 to week 4. Both P529 and wortmannin suppressed cellularity and inflammation by 20% to 30%, and a noticeable decrease was noted in the hyalinized collagen bundles at weeks 1 to 4 after treatment, compared with the DMSO control group (Figure 10). In contrast, rapamycin caused minimal changes at day 3 to week 4 when compared with the P529 treatment group.
Figure 10Effect of P529 on keloid OCcellularity/inflammation and angiogenesis. A: Morphologic analysis of keloid OC tissue after various drug treatments using H&E staining. Tissue was collected after different drug treatments at various times as indicated. H&E staining of tissue sections was performed using standard protocols. Micrographs were obtained using an Olympus BX21 microscope at ×200 magnification. E, epidermis; K, keratin layer; PD, papillary dermis; RD, reticular dermis. Arrows indicate the dislocation of epidermis from dermis after treatment. Pyknotic nuclei are shown in the blue circle. Arrows indicate dislocation of epidermis in the treated groups. B: Immunoperoxidase analysis of CD31+ endothelial cell marker after various drug treatments in keloid OC model. Original magnification 100. Arrows shows the staining pattern for CD31+ cells in the treated and untreated groups.
Notch signaling pathway in keloid disease: enhanced fibroblast activity in a Jagged-1 peptide dependent manner in lesional versus extra-lesional fibroblasts.
Angiotensin II regulates phosphoinositide 3 kinase/Akt cascade via a negative crosstalk between AT1 and AT2 receptors in skin fibroblasts of human hypertrophic scars.
compared with normal dermis (see Supplemental Figure S2 at http://ajp.amjpathol.org), we wondered whether P529 would exert any anti-angiogenic effects in an ex vivo model of KD. P529 showed a significant reduction in vascular density, as visualized by CD31 and expression of CD34 immunostaining. In addition, CD31+ cell density (endothelial cell marker) (n = 8) (Figure 10B) and expression of CD34+ immunoreactivity (n = 6) (see Supplemental Figure S11 at http://ajp.amjpathol.org) were drastically reduced at week 1 after treatment with both P529 and wortmannin compared with rapamycin. Thus, P529 exerts strong anti-inflammatory and anti-angiogenic properties in KD tissue.
P529 Inhibits KD Proliferation and Promotes Its Apoptosis in Situ
From shrinkage of KD volume and epidermal thickness, we hypothesized that P529 may inhibit proliferation and cause apoptosis in the keloid ex vivo model. To address this, we further analyzed the effect of P529 on proliferation and induction of apoptosis in keloid OC (n = 6) up to 4 weeks. P529 (20 ng/mL) showed marked reduction in PCNA expression at week 1 compared with vehicle-treated controls (see Supplemental Figure S12 at http://ajp.amjpathol.org). Furthermore, P529 significantly (P < 0.05) increased apoptosis at week 1 in keloid OC compared with groups treated with vehicle, rapamycin, and wortmannin (Figure 11, A and B). After 4 weeks, P529 exerted the most significant apoptosis-promoting effect in KD OC.
Figure 11Effect of P529 on keloid cell apoptosis in OC model. Four-millimeter keloid OC was treated with various drug concentrations as indicated. A: P529 induces massive apoptosis in keloid OC. TUNEL staining (red nuclei and green-yellow TUNEL-positive cells). The dotted lines indicate demarcation of epidermis and dermis junction. Original magnification ×200. E, epidermis; PD, papillary dermis; RD, reticular dermis. B: Morphometric analysis of percentage of TUNEL-positive cells. *P < 0.05, †P < 0.02, significant difference vs DMSO control group. Arrows indicate TUNEL-positive cells in the treated and untreated groups.
Early Inhibition of PI3K/Akt/mTOR signaling in an ex Vivo Keloid Model
We also assessed the inhibitory effect of P529 compared with rapamycin and wortmannin on intracellular signaling in an ex vivo model. Keloid OC (n = 8) tissue was assessed via immunohistochemistry for expression of mTOR pathway–related markers after various drug treatments at different times.
In the P529 treatment group, expression of p-Akt-S473, p-mTOR, and p-S6 were already significantly reduced at week 1, compared with the rapamycin or wortmannin treatment groups. In the rapamycin and wortmannin treatment groups, p-Akt-S473, p-mTOR, and p-S6 (see Supplemental Figure S13, A–C, respectively, at http://ajp.amjpathol.org) were reduced at week 1 to week 4, compared with the control group. This suggests that the anti-angiogenic effect of P529 in KD OC could be related to inhibition of mTOR pathway signaling.
Significant Suppression of Collagen I and Fibronectin by P529 in an ex Vivo Keloid OC Model
We evaluated the inhibitory effect of P529 on ECM-related markers in the keloid OC (n = 8) model. As expected, the immunoreactivity of collagen I was reduced significantly at weeks 1 to 4 after treatment with P529 compared with rapamycin and wortmannin (Figure 12A). All three compounds produced a significant reduction in fibronectin immunoreactivity at week 1 after treatment, compared with the control group. However, the inhibitory effect on fibronectin was noticeably higher with P529 than with rapamycin and wortmannin (Figure 12B). Thus, inhibition of the PI3K/Akt/mTOR signaling pathway by dual mTORC1 and mTORC2 inhibitor P529 significantly reduced the expression of KD-associated fibrotic markers.
Figure 12Effect of P529 on collagen and fibronectin biosynthesis in the keloid OC model. A: Reduction of collagen I biosynthesis expression by P529 treatment in keloid OC model. B: Inhibition of fibronectin expression by P529 treatment in keloid OC model. Original magnification ×200. E, epidermis; K, keratin layer; PD, papillary dermis; RD, reticular dermis. Arrows, intensity of staining with and without treatment.
Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/Akt signaling pathways.
The present study confirms this by showing overexpression of total mTOR, p70S6K, and their phosphorylated forms and provides the first evidence that p-S6, a key downstream target of mTOR, is also overexpressed in KD. mTOR inhibitors such as rapalogs have been studied in KD previously using in vitro primary cell culture models.
The TORC1/TORC2 inhibitor, Palomid 529, reduces tumor growth and sensitizes to docetaxel and cisplatin in aggressive and hormone-refractory prostate cancer cells.
The PI3k/Akt/mTOR pathway has proximal and distal feedback signaling, and although mTOR is a downstream effector of Akt, mTORC2 can phosphorylate Akt at a specific site (Ser473), which then hyperactivates Akt via a feedback mechanism.
Rapamycin and its analogs (rapalogs) or rapamycin mTOR inhibitors are limited in that they do not affect mTORC2; consequently, duration of inhibition was shortened due to feedback activation of Akt.
However, targeting proximal pathway components generally results in broad inhibition of the downstream signaling cascade and may augment undesirable effects, whereas P529, a small-molecule synthetic nonsteroidal compound with a chemical structure derived from dibenzo[c]-chromen-6-one, is an allosteric dual mTORC1/2 dissociative inhibitor that abrogates compensatory feedback loop activation. The P529 mechanism of action is unique in that it dissociates the various proteins in the mTORC1/2 complex rather than inhibiting via catalytic competitive inhibition.
In the present study, P529 and wortmannin attenuated p-Akt(Ser473), p-mTOR, and ribosomal protein pS6 phosphorylation in vitro and ex vivo. In contrast, rapamycin failed to inhibit pAkt(Ser473), p-mTOR, and pS6 as potently as did P529. Our P529 findings concur with those of previous studies that showed inhibition of the PI3K/Akt/mTOR axis by this drug.
Compared with rapamycin, inhibition with both P529 and wortmannin produced a greater reduction in downstream effectors of mTOR in in vitro and ex vivo models.
Compared with rapamycin, both P529 and wortmannin demonstrated more effective inhibition of cell attachment, spreading, and cell proliferation, caused significant toxicity, and inhibited viability/metabolic activity. Consistent with our results, P529 caused significant cell proliferation, inhibition, and cytotoxicity.
Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/Akt signaling pathways.
Green tea polyphenol epigallocatechin-3-gallate suppresses collagen production and proliferation in keloid fibroblasts via inhibition of the STAT3-signaling pathway.
also showed significant inhibition of primary KF proliferation and migration. P529 sustained a significant inhibitory effect on primary KF (5 ng/mL) and NF (20 ng/mL) activity, compared with rapamycin and wortmannin, after discontinuation of the drugs. Primary KF recovered from rapamycin mode of action within 3 days.
However, in the present study, all three drugs, P529, rapamycin, and wortmannin, had no reversibility effect after discontinuation of the drug at up to 30 hours. Thus, it seems that P529 is selective and more efficacious in inhibiting primary KF.
In 2D and 3D culture models, P529 and wortmannin inhibited migration and invasion properties of KF more effectively than did rapamycin. This might have been achieved by suppressing F-actin, PCNA, and cyclin D. Consistent with our P529 data, recently it has been shown that mTORC2 controls F-actin reorganization,
AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity.
The TORC1/TORC2 inhibitor, Palomid 529, reduces tumor growth and sensitizes to docetaxel and cisplatin in aggressive and hormone-refractory prostate cancer cells.
AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity.
The TORC1/TORC2 inhibitor, Palomid 529, reduces tumor growth and sensitizes to docetaxel and cisplatin in aggressive and hormone-refractory prostate cancer cells.
we analyzed the effect of P529 on KD apoptosis. Both, P529 and wortmannin produced marked apoptosis in vitro and ex vivo. In contrast, rapamycin produced minimal apoptosis. Consistent with our results, P529 significantly increased apoptosis in tumor cells.
In the present study, P529 strongly inhibited PCNA in vitro and ex vivo and cyclin D in vitro. These data indicated specificity of P529 in KD. Corroborating our findings, Ong et al
also found that rapamycin inhibits PCNA and cyclin D in primary KF.
Both P529 and wortmannin, compared with rapamycin, demonstrated reduced cellularity, inflammation, and hyalinized collagen bundles. We further explored the efficacy of P529 by assessing shrinkage of keloid OC tissue volume and epidermal thickness. These findings demonstrated that shrinkage of keloid OC resulted in reduction of average keloid OC volume and significant shrinkage of OC epidermis at week 1 after treatment with both P529 and wortmannin at very low concentrations (20 and 10 ng/mL, respectively), compared with rapamycin (100 ng/mL). This could be achieved by inhibition of PCNA and significant induction of apoptosis. Consistent with our results, in other tumors, P529 considerably reduced tumor growth and volume,
The TORC1/TORC2 inhibitor, Palomid 529, reduces tumor growth and sensitizes to docetaxel and cisplatin in aggressive and hormone-refractory prostate cancer cells.
Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/Akt signaling pathways.
Compared with rapamycin, the effect of both P529 and wortmannin on angiogenesis showed significant reduction in CD31+ cells and expression of CD34+ immunoreactivity. Rapamycin did inhibit CD31+ cells at a much higher concentration (100 ng/mL) than did P529 and wortmannin at weeks 1 to 4. Supporting our findings in keloid OC, P529 reduces CD31+ cells in other tumors.
Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/Akt signaling pathways.
Long-term treatment of bile duct-ligated rats with rapamycin (sirolimus) significantly attenuates liver fibrosis: analysis of the underlying mechanisms.
Compared with rapamycin, both P529 and wortmannin more significantly reduced the keloid-associated fibrotic markers collagen I, fibronectin, and α-SMA in our in vitro model, and collagen I and fibronectin in our ex vivo model. Furthermore, the overall inhibitory effect on expression levels of collagen, fibronectin, and α-SMA were higher with P529 than with rapamycin and wortmannin. In line with our results, Ong et al
also found that treatment of mTOR inhibitor (rapamycin) significantly reduced ECM and α-SMA in primary KF.
P529 is highly effective in KF compared with NF, even at low concentrations, which suggests that P529 is a highly selective inhibitor of mTOR. This could be due to a more active PI3K/Akt/mTOR axis in keloids compared with normal skin. It is unlikely that complete target inhibition could be sustained over long periods in patients with KD because PI3K signaling is critical in the maintenance of normal tissue, although, it is possible that higher doses given intermittently and intralesionally might result in a better therapeutic outcome by producing sustained PI3K/Akt/mTOR inhibition in KD, while allowing time for normal tissue recovery. An alternative strategy would be to combine PI3K/Akt/mTOR inhibitors with anti-cancer agents that target other signaling pathways, which are considered to be dysregulated in KD.
Clinically adverse events have been associated with the use of the mTORC1 inhibitor rapamycin and its analogs.
However, with P529, such toxicities have not been observed to date in ongoing human phase I trials. P529 showed no toxicity in toxicology studies in dogs and rats. It is important to note that no in vivo toxicity was observed when P529 was administered in dose-ranging studies in dogs and rabbits.
In conclusion, the present study provides the first available functional evidence that the PI3K/Akt/mTOR axis is an important target in KD treatment in situ. Moreover, we show that dual inhibition of the PI3K/Akt/mTOR kinases by P529 can inhibit cell proliferation, migration, cellular invasion, inflammation, and ECM production; reduce tissue volume (shrinkage) and epidermal thickness; and induce apoptosis. Thus, this dual mTOR inhibitor deserves to be systemically explored as an attractive therapeutic option in the future treatment of KD.
Acknowledgments
We thank the GAT Family Foundation for support of this work and sponsoring the collaboration that took place between Prof. Pandolfi and Dr. Bayat's group and the study participants and nursing staff of the University Hospital of South Manchester NHS Foundation Trust (Manchester, UK).
Increased levels of pS6 and angiogenesis in keloid tissues. Sections of normal skin tissue and keloidal tissues were immunohistochemically stained using pS6 (pink) and CD31 (brown) antibodies. A: Results from keloid scar tissues isolated from four patients (KS-1 to KS4). B: Normal skin from two patients (NS1 and NS2) is included. Arrows, wound edges. Original magnification ×200 and ×600.
In-Cell Western Blot (ICWB) analysis of intracellular signaling events in NF induced by increasing concentration of P529 compared with rapamycin and wortmannin. Mean immunoreactivity from three independent ICWB experiments of NF normalized to β-actin. *P < 0.05, significant difference vs vehicle (DMSO) control group.
Expression pattern of total and p-mTOR in NF at 24 hours after treatment with P529, rapamycin, and wortmannin using ICWB. Bar graphs represent quantification of mean protein expression in various treatments from three independent experiments after normalization to loading control β-actin. *P < 0.05, significant difference in the treated group vs the DMSO control group.
Differential effect of P529, rapamycin, and wortmannin drugs on cell membrane integrity (cytotoxicity detection), cell proliferation, and cell death measured via lactose dehydrogenase and WST-1 assay. Cells were seeded in a 96-well plate at a density of 1 × 104cells per well. Cells were allowed to attach and grow for ∼7 to 8 hours; then the drugs were added at various concentrations as indicated. The cells were further allowed to grow for 24 hours in the presence of drugs. After 24 hours, the drugs were removed, and cells were supplemented with DMEM (10% fetal calf serum) without drugs, and reversibility of cytotoxicity and cell proliferation were further monitored for 24 hours. A and B: Lactose dehydrogenase leakage assay for cell membrane integrity. Cytotoxic effect of both drugs was assessed as described in “Materials and Methods”. C and D: WST-1 (water soluble-tetrazolium salt 1) assay for cell proliferation and cell death. WST-1 assay was performed according to the manufacturer's instructions. Data are given as mean ± SEM from four independent experiments. *P < 0.05, significant difference vs DMSO control group. **P < 0.05, significant difference in growth of KF compared with NF.
Inhibition of keloid-associated fibrotic markers and cell cycle–regulated genes by P529. In-Cell Western Blotting of expression of ECM proteins (collagen I, fibronectin, and α-SMA), PCNA, and cyclin D at 24 hours after treatment of NF with various drugs. Mean immunoreactivity values of NF from three independent experiments of In-Cell Western Blotting plotted on the graphs after normalizing to β-actin. *P < 0.05, significant difference in treated vs DMSO groups.
P529 induces apoptosis in NF. Apoptosis was detected with drug treatment using annexin V staining. A: Annexin V staining for FACS. After 24 hours of treatment with indicated drug concentrations, the cells were harvested, and FITC-labeled annexin V was added to a final concentration of 2.5 μg/mL. To detect dead cells, propidium iodide (PI) was added at a concentration of 5 μg/mL. Using flow cytometry, dot plots of annexin V on the y axis against PI on the x axis were used to differentiate viable cells (negative for both PI and annexin V), apoptotic cells (annexin V–positive cells but exclude PI, with PI therefore negative), and late apoptotic or necrotic cells (double-positive for PI uptake and annexin V staining). Nonstained cells and untreated cells were used as negative controls. B: Annexin V– and PI-positive NF after 24 hours of treatment with various drugs as indicated in the bar graphs at different concentrations. Positive cells were counted from three independent experiments and plotted on the graph as mean ± SEM, *P < 0.05, significant difference vs DMSO control group.
Shrinkage of keloid OC after various mTOR inhibitor treatments. Four-millimeter keloid tissue explants were maintained in collagen gel matrix in the presence of various concentrations of drug up to 4 weeks, and the tissue was collected at different times as indicated. Representative photographs show shrinkage of keloid tissue embedded in collagen gel matrix with and without treatment.
Effect of P529 on keloid OC tissue architecture. Morphologic analysis of keloid OC tissue after various drug treatments. H&E staining of tissue sections was performed using standard protocols. Micrographs were obtained using an Olympus BX21 microscope at ×200 magnification. E, epidermis; K, keratin layer; PD, papillary dermis; RD, reticular dermis.
Efficacy of P529 on expression profile of CD34 marker–positive cells in keloid organ culture (OC). CD34 is a specific marker for microvascular endothelial cells. Original magnification ×200. Red, nuclei; green, CD34+ cells). Arrows, positive staining with and without treatment.
Effect of P529 on keloid cell proliferation in organ culture (OC) model. Four-millimeter keloid OC was treated with various drug concentrations as indicated. Tissue sections were stained using anti-PCNA to assess the expression pattern. Compared with rapamycin, P529 strongly inhibits PCNA expression in the keloid OC model. Original magnification ×200. Arrows, positive staining pattern with and without treatment.
Effect of P529 on PI3K/Akt/mTOR signaling in keloid organ culture (OC) model. A: Expression pattern of phosphor-Akt-Ser473 in keloid OC after treatment with various drugs. B: Expression pattern of p-mTOR in keloid OC after various drug treatments. C: Expression profile of p-S6 in keloid OC after various drug treatments as indicated. Original magnification ×200. Arrows, staining pattern intensity with and without treatment.
Increased vascular endothelial growth factor may account for elevated level of plasminogen activator inhibitor-1 via activating ERK1/2 in keloid fibroblasts.
Notch signaling pathway in keloid disease: enhanced fibroblast activity in a Jagged-1 peptide dependent manner in lesional versus extra-lesional fibroblasts.
The TORC1/TORC2 inhibitor, Palomid 529, reduces tumor growth and sensitizes to docetaxel and cisplatin in aggressive and hormone-refractory prostate cancer cells.
AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity.
Green tea extract and (-)-epigallocatechin-3-gallate inhibit mast cell-stimulated type I collagen expression in keloid fibroblasts via blocking PI-3K/Akt signaling pathways.
Angiotensin II regulates phosphoinositide 3 kinase/Akt cascade via a negative crosstalk between AT1 and AT2 receptors in skin fibroblasts of human hypertrophic scars.
High-performance liquid chromatography analysis of a novel small-molecule, anti-cancer drug, Palomid 529, in human and mouse plasma and in mouse tissue homogenates.
J Chromatogr B Analyt Technol Biomed Life Sci.2011; 879: 3823-3831
Fibroblasts from the growing margin of keloid scars produce higher levels of collagen I and III compared with intralesional and extralesional sites: clinical implications for lesional site-directed therapy.
Green tea polyphenol epigallocatechin-3-gallate suppresses collagen production and proliferation in keloid fibroblasts via inhibition of the STAT3-signaling pathway.
Suppression of TGF-β1/SMAD pathway and extracellular matrix production in primary keloid fibroblasts by curcuminoids: its potential therapeutic use in the chemoprevention of keloid.
Long-term treatment of bile duct-ligated rats with rapamycin (sirolimus) significantly attenuates liver fibrosis: analysis of the underlying mechanisms.
Disclosure: D.S. is CEO and president of Paloma Pharmaceuticals, Inc., which donated P529 for this project.
A guest editor acted as editor-in-chief for the manuscript. No person affiliated with the University of Manchester was involved in the final disposition of this article.
In the article entitled, “Caspase 3 Silencing Inhibits Biomechanical Overload–Induced Intervertebral Disk Degeneration” (Volume 184, pages 753–764 of the March issue of The American Journal of Pathology), errors were inadvertently introduced into the figure labeling. The unit μm in the scale bars was incorrectly changed to μmol/L within Figures 2, below panel A; Figure 6, above panels A and B; and Figure 7, above panels A–H. We sincerely regret the error.
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