Primary NHOK and NHOF cultures were established from discarded oral mucosa without patient identification or medical information, under exemption from the Institutional Review Board of UCLA (Los Angeles, CA, USA) as described previously (21). Briefly, the discarded oral mucosal tissues of approximately 25 mm² were collected from healthy patients who were undergoing routine dental procedures at the UCLA Dental Center between 2010 and 2016. The oral mucosal tissues were minced into 2 mm² × 0.5 mm sections and incubated in 2.5 mg/ml dispase solution (cat. no. 17105; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in 37°C for 1 h, then the epithelial layers and connective layers were separated. The separated epithelial layers were minced and subjected to trypsin digestion (cat. no. 25200056; Gibco; Thermo Fisher Scientific, Inc.) in 37°C for 5 min to harvest the individual epithelial cells to establish NHOK. The separated connective tissue layers were minced and digested with collagenase (cat. no. 17100017; Gibco; Thermo Fisher Scientific, Inc.) in 37°C for 1 h to harvest the individual mesenchymal cells to establish NHOF. NHOKs were cultured in EpiLife supplemented with human keratinocyte growth supplement (HKGS; Cascade Biologics; Thermo Fisher Scientific, Inc.), and NHOFs were cultured in DMEM supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.). For radiation exposure, NHOKs were exposed to varying doses of IR using a Mark I-30 Cesium-137 irradiator (JL Shepherd & Associates, San Fernando, CA, USA) with the delivery rate of 4.86 Gy min⁻¹. IR exposure led to the induction of a senescent phenotype, described as stress-induced premature senescence (SIPS) (22). To confirm SIPS, NHOKs were stained for senescence-associated β-galactosidase (SA β-Gal) activity, which was viewed/imaged under an Olympus phase-contrast microscope (Olympus Corporation, Tokyo, Japan) (21). Squamous cell carcinomas (SCC) 4 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) and 0.4 µg/ml hydrocortisol (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany).

The following primary antibodies were used in the present study: GAPDH (cat. no. sc-47724), zinc finger E-box binding homeobox 1 (ZEB1; cat. no. sc-25388), E-Cadherin (E-Cad; cat. no. sc-21791), P21/WAF1 (cat. no. sc-397), 53BP1 (cat. no. sc-22760), P16^INK4A (cat. no. sc-468), phosphorylated (p-)Akt (Ser473; cat. no. sc-7985), collagen type I α1 chain (Col1a1; cat. no. sc-8784) and Col3a1 (cat. no. sc-28888) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); GRHL2 (cat. no. H00079977) from Abnova (Taipei City, Taiwan); N-Cadherin (N-Cad; cat. no. 610920) from BD Biosciences (San Jose, CA, USA); fibronectin (FN; cat. no. F3648), α-smooth muscle actin (α-SMA; cat. no. A5228) and Snail (cat. no. SAB1406456) from Sigma-Aldrich; Merck KGaA; p-small mothers against decapentaplegic (Smad)3 (ser423/425; cat. no. 9520), p-Smad2 (ser465/467; cat. no. 3108), Smad4 (cat. no. 38454) and p-p53 (Ser15; cat. no. 9284) from Cell Signaling Technology, Inc. (Danvers, MA, USA); and γ-H2AX (phospho S139; cat. no. ab2893) from Abcam (Cambridge, MA, USA). Alexa Fluor^® anti-rabbit or anti-mouse secondary antibodies were from Thermo Fisher Scientific, Inc. TGF-β1 (cat. no. 100-21) was purchased from PeproTech, Inc. (Grand Island, NY, USA); TGF-β receptor I kinase inhibitor (TRI; cat. no. 616451) was obtained from Calbiochem; EMD Millipore (Billerica, MA, USA). BLM was purchased from Sigma-Aldrich; Merck KGaA; GV1001 was provided by Gemvax & Kael Co. Ltd. (Seoul, Korea).

Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific, Inc.). DNA-free total RNA (5 µg) was used for the RT reaction, followed by qPCR for each 1 µl cDNA sample with 1 µl primer pair mix (5 pmol/µl each primer) and LC480 SYBR Green I master using universal cycling conditions in a LightCycler^® 480 (Roche Diagnostics, South San Francisco, CA, USA). The primer sequences were obtained from the Universal Probe Library (Roche Diagnostics). The PCR cycling conditions were as follows: 45 cycles of 10 sec at 95°C, 45 sec at 55°C, and 20 sec at 72°C. The second derivative Cq value determination method was used to compare the fold-differences (23). Cq was the cycle at which the threshold was crossed. The experiments were performed in triplicate. The primer sequences used for the PCR to determine gene expression were as follows: Col1a1, forward 5′-GGGATTCCCTGGACCTAAAG-3′ and reverse 5′-GGAACACCTCGCTCTCCA-3′; Col3a1, forward 5′-CTGGACCCCAGGGTCTTC-3′ and reverse 5′-CATCTGATCCAGGGTTTCCA-3′; FN, forward 5′-GGGAGAATAAGCTGTACCATCG-3′ and reverse 5′-TCCATTACCAAGACACACACACT-3′; N-Cad, forward 5′-CTCCATGTGCCGGATAGC-3′ and reverse 5′-CGATTTCACCAGAAGCCTCTAC-3′; ZEB1, forward 5′-AACTGCTGGGAGGATGACAC-3′ and reverse 5′-TCCTGCTTCATCTGCCTGA-3′; ZEB2, forward 5′-CCAGACCGCAATTAACAATG-3′ and reverse 5′-ATGCTGACTGCATGACCATC-3′; glyceraldehyde 3-phosphate dehy drogenase (GAPDH), forward 5′-AGCCACATCGCTCAGACAC-3′ and reverse 5′-GCCCAATACGACCAAATCC-3′.

Whole cell extracts (WCEs) from the cultured cells were isolated using lysis buffer [1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 µM β-glycerophosphate, 1 mM sodium orthovanadate and 1 mg/ml PMSF]. The protein concentration of the lysates was measured using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of protein (20 µg/lane) were separated by 8 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto an immobilon membrane (EMD Millipore), which was incubated in blocking solution containing 5% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) in Tris-buffered saline (TBS) for 30 min at room temperature. The membrane was then incubated with primary antibodies (e.g., GRHL2 and N-Cad) diluted in blocking solution (1:1,000) at 4°C overnight. Following washing with TBST three times, the membrane was incubated with anti-mouse or rabbit secondary antibodies (1:2,000) for 1 h at room temperature and then exposed to the chemiluminescence reagent (GE Healthcare Life Sciences, Piscataway, NJ, USA) for signal detection.

The cells were cultured in an Nunc™ Lab-Tek™ II Chamber Slide™ system (Thermo Fisher Scientific, Inc.) to reach 70–80% confluence, and then fixed in 2% paraformaldehyde for 20 min. The cells were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 10 min, and then blocked for 1 h in PBS containing 2% BSA (Sigma-Aldrich; Merck KGaA) at room temperature and incubated overnight at 4°C with primary antibodies (ZEB1 and α-SMA; 1:100). Following three washes with PBS, the cells were incubated with the secondary antibodies, Alexa Fluor^® 594 goat anti-rabbit IgG or Alexa Fluor^® 488 goat anti-mouse IgG (1:400; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Slides were mounted in Prolong Gold w/DAPI (Invitrogen; Thermo Fisher Scientific, Inc.). Images were captured with an Olympus epifluorescence inverted microscope (Olympus Corporation).

Paraffin-embedded histological sections were stained with hematoxylin and eosin (H&E) for determination of the histological changes in the dermis. The mouse skin specimens were fixed in 4% (wt/vol) paraformaldehyde at 4°C for 24 h. The samples were embedded in paraffin, sectioned at 4 µm thickness, and stained as described previously (24). Briefly, the tissue slides were dewaxed, and antigen retrieval was performed using an unmasking solution (Vector Laboratories, Inc., Burlingame, CA, USA) boiled in a microwave for 20 min. The slides were then incubated in 3% hydrogen peroxide in PBS for 10 min to quench the ZEB endogenous peroxidase. The slides were sequentially washed with distilled water and PBS, and incubated in blocking solution (2.5% BSA in PBS) for 30 min at room temperature. The slides were incubated with Col3a1 primary antibody diluted in blocking solution (1:100) at 4°C overnight. Following washing with PBS, the slides were incubated with the secondary antibody, Alexa Fluor^® 594 goat anti-rabbit IgG (1:400; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. The slides were mounted in Prolong Gold w/DAPI (Invitrogen; Thermo Fisher Scientific, Inc.).

Following irradiation, the cells were fixed in 2% paraformaldehyde for 20 min at room temperature. The cells were permeabilized with 0.5% TritonX-100 and blocked with 5% FBS in PBS. The cells were then incubated with 53BP1 or γ-H2A.X primary antibody overnight at 4°C in 1% BSA. The coverslips were mounted with ProLong^® Gold antifade reagent with DAPI to counterstain the nuclei. The foci were quantified using Cell Profiler to count cells with >3 foci per nucleus in ≥10 different fields from each experiment under confocal microscopy (Olympus Corporation). Experimental data were determined as the average of three independent experiments.

A ChIP assay was performed based on the protocols described in our previous study (22). The sequences for the PCR primers of gene promoters were as follows, Col1a1 (-1,750 bp), forward 5′-TCTCCCAGGTGTCTGTCTCC-3′ and reverse 5′-AGGAGAGGGGTCTGCTGAG-3′; Col1a1 (-1,000 bp), forward 5′-AGGGGGAAAAACTGCTTTGT-3′ and reverse 5′-TCCGACCTCTCTCCTCTGAA-3′; Col1a1 (−200 bp), forward 5′-CCACTTGGGTGTTTGAGCA-3′ and reverse 5′-CCTCCCCTCCACTCCTTC-3′; Col3a1 promoter, forward 5′-TCTCCATTGCCAGATATAGCC-3′ and reverse 5′-TCCAGATCCTCATCCACAAA-3′. The promoter regions analyzed in the present study for p-Smad2 enrichment included the Smad-binding element (SBE), AGACA sequence.

SCC4 cells (2×10⁵ cells per well) were seeded in 6-well plate and cultured for 48 h in culture medium. A scratch (wound) was then introduced in the confluent cell layer using a P200 yellow tip. The cells were washed three times with medium to remove detached cells. The cells were then incubated with or without TGF-β (10 ng/ml) or GV1001 (1 µM) for 48 h and images of a defined wound spot were captured with a computer-aided phase contrast microscope (Olympus). Experiments were performed in triplicate.

Pathogen-free, female C57BL/6 mice (6 week-old; Jackson Laboratory, Ben Harbor, ME, USA) were used for BLM-induced dermal fibrosis by subcutaneous injection of BLM to the shaved upper back skin for 4 weeks. The animals were housed in a pathogen-free environment with a 12 h light/dark cycle in the Division of Laboratory and Animal Medicine at UCLA (Los Angeles, CA, USA). The experiments were performed based on the protocol approved by the Institutional Animal Care and Use Committee. A total of 20 mice were divided into four groups: Control group, injected subcutaneously (s.q.) with 100 µl vehicle (PBS); BLM group, injected with 100 µl BLM solution (100 µg/ml); BLM and Low GV1001 group, injected with 1 mg/kg GV1001 and 100 µg/ml BLM; BLM and high GV1001 group, injected with 5 mg/kg GV1001 and 100 µg/ml BLM. Each group included five mice. The s.q. injection continued daily for 4 weeks and the shaved upper back skin tissues were harvested for histological assessment of the lesions.

Statistical evaluations were performed using SPSS version 11.0 (SPSS, Inc., Chicago, IL, USA). All numerical data were expressed as the mean ± standard deviation. Statistical analysis was performed using Student's t-test (two-tailed) for quantitative experiments. P<0.05 was considered to indicate a statistically significant difference.

Our previous study showed that exposure of primary human keratinocytes to IR triggered a senescence response (22). To examine the effect of GV1001 on the cellular response to IR, NHOKs were exposed to 6 Gy IR and the cells were cultured in the presence or absence of GV1001. The rapidly proliferating NHOKs underwent cell proliferation arrest upon irradiation and exhibited cellular morphology consistent with senescence, including a flattened cytosolic region and perinuclear vacuolization (21), whereas these senescing effects of IR were reduced in the cells treated with GV1001 (Fig. 1A). While irradiation inhibited cell proliferation, treatment of the NHOKs with GV1001 mitigated the growth suppressive effects of IR (Fig. 1B). With increased dose of IR, the percentage of cells expressing SA β-Gal was increased in the NHOKs; however, GV1001 treatment significantly reduced the percentage of cells stained positively for SA β-Gal (Fig. 1C). Similarly, the colony forming ability of the cultured NHOKs was suppressed by exposure to IR, whereas this effect was mitigated by GV1001 treatment (Fig. 1D). These results demonstrated that GV1001 treatment ameliorated IR-induced premature senescence in cultured NHOKs.

Keratinocyte proliferation is regulated in part through Grainyhead-like 2 (GRHL2), which is a novel transregulator of the hTERT gene and determines epithelial phenotype through the suppression of EMT regulators (25–27). The western blot analysis showed that the endogenous level of GRHL2 was induced by GV1001, even following exposure to 6 Gy IR (Fig. 2A). GV1001 treatment led to reduction in the level of N-Cad, encoded by the CDH2 gene, which is elevated during EMT (28), consistent with the increased expression of GRHL2. As EMT is induced by TGF-β signaling, these data indicated the possible activation of the TGF-β pathway in NHOKs following IR exposure. IR also led to p53 phosphorylation (Ser15) and an increased level of p21/WAF1 cyclin-dependent kinase inhibitor, however, these protein levels were reduced in the cells treated with GV1001. p-Akt (Ser473), which is necessary for radioresistance in cells by enhancing DNA double strand break (DSB) repair (29), was elevated in the NHOKs treated with GV1001. The level of DNA DSB in the irradiated cells was measured directly by staining for 53BP1 and γ-H2AX, which form fluorescent foci at the sites of DSBs (30,31), as shown in Fig. 2B. The NHOKs were irradiated with 6 Gy IR in the presence or absence of GV1001, and the percentages of cells with intranuclear 53BP1 and γ-H2AX foci were determined at 10 days post-IR by IFS using confocal microscopy. There was elevation of DSBs in the irradiated cells, compared with the control cells without IR exposure, and the level of DSBs was decreased in cells cultured with GV1001, indicating that GV1001 enhanced the removal of DSBs in the cells (Fig. 2C). Taken together, these data indicated that GV1001 suppressed IR-induced premature senescence, in part via mitigating the genotoxic effects.

The senescent phenotype in cells exposed to IR is regulated through the auto/paracrine activity of TGF-β (17,32). The irradiation of NHOKs also led to the activation of TGF-β signaling molecules, inducing the phosphorylation of Smad2/3 complex and the level of Smad4 (Fig. 3), suggesting their role in premature senescence. In addition, the irradiated cells exhibited a mesenchymal phenotype and expressed higher levels of TGF-β target genes, including N-Cad, ZEB1, FN and Snail. Treatment of the cells with GV1001 decreased the expression of these TGF-β target mesenchymal markers and TGF-β signaling molecules, including p-Smad2/3 and Smad4, compared with the control cells. The expression of target proteins and signaling induced by TGF-β were also eliminated by exposure to 1 µM TRI, which was used as the positive control for GV1001 treatment. These data indicated that the mesenchymal phenotype induced by IR was prevented by GV1001 in the NHOKs, possibly through suppressing TGF-β signaling.

To examine the effects of GV1001 on TGF-β signaling, rapidly proliferating NHOKs were exposed to TGF-β in the presence or absence of GV1001. Exposure to TGF-β for up to 10 days led to induction of a mesenchymal phenotype through EMT (Fig. 4A). It was also found that GV1001 treatment reduced the expression of Snail and α-SMA in the cells exposed to TGF-β, in addition to TGF-β signaling molecules, including p-Smad2/3 and Smad 4 (Fig. 4B). TGF-β treatment also led to enhanced cell migration of the SCC4 cell line, reminiscent of the mesenchymal phenotype, and GV1001 almost completely prevented the enhanced migratory effects of TGF-β (Fig. 4C). In the NHOFs, the expression of TGF-β target molecules and mesenchymal markers, including Col1a1, Col3a1, FN, N-Cad, ZEB1 and ZEB2, was suppressed in the cells treated with GV1001 (Fig. 5A). The effect of GV1001 on binding of p-Smad to the SBE was also confirmed by a ChIP assay of NHOFs exposed to TGF-β (Fig. 5B and C). Treatment of the cells with GV1001 markedly suppressed binding of Smad2 on the Col1a1 and Col3a1 gene promoter regions, indicating that GV1001 interfered with TGF-β-mediated target gene transcription in NHOFs.

In the subsequent experiments, the effects of GV1001 in fibrotic tissue formation were determined. Whether GV1001 treatment inhibited TGF-β signaling in myofibroblast differentiation was assessed. Rapidly proliferating NHOFs were exposed to TGF-β, which triggered the induction of α-SMA, specialized actin molecules found in myofibroblasts, in addition to Col3a1, Col1a1, Snail, FN and N-Cad (Fig. 6A). These changes occurred with marked induction of p-Smad2. However, when the cells were co-treated with GV1001, there were marked reductions in the levels of α-SMA, N-Cad, FN and p-Smad in the cells. Similarly, IFS revealed induced α-SMA in the NHOFs by TGF-β treatment, in addition to ZEB1, a mesenchymal transcription factor directly regulating the expression of α-SMA (33), whereas the levels of α-SMA and ZEB1 were suppressed by GV1001 treatment (Fig. 6B). These data indicated that GV1001 inhibited TGF-β-induced myofibroblast differentiation through the suppression of ZEB.

To confirm the antifibrogenic property of GV1001 in vivo, dermal fibrosis was induced in C57BL/6 mice by s.q. injection of BLM with or without co-administration of GV1001. BLM treatment for 4 weeks led to marked dermal sclerosis in the mice, which was characterized histologically by a thickened dermis with increased deposition of collagen bundles (Fig. 6C). When GV1001 was administered to these mice, there was significant reduction in the thickness of the fibrotic lesions, and a reduction in dermal levels of Col3a1, as determined by IF staining (Fig. 6C and D). Therefore, GV1001 exerted antifibrotic effects in vivo and may directly impair the fibrogenic differentiation of dermal fibroblasts.