BMS-863233 | PDK1 signal

Experimental Cell Research

XL413, a cell division cycle 7 kinase inhibitor enhanced the anti-ﬁbrotic effect of pirfenidone on TGF-β1-stimulated C3H10T1/2 cells via Smad2/4

Shu-fang Jin 1, Hai-long Ma 1, Zhong-long Liu, Shui-ting Fu, Chen-ping Zhang, Yue Hen
Department of Oral Maxillofacial-Head and Neck Oncology, Faculty of Oral and Maxillofacial Surgery, Shanghai Ninth People’s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai, China
A R T I C L E I N F O

Article history:
Received 18 July 2015 Received in revised form 9 October 2015
Accepted 12 November 2015
Available online 14 November 2015
Keywords:
Pirfenidone
Cell division cycle 7 Smads
Proliferation Differentiation

A B S T R A C T

Pirfenidone is an orally bioavailable synthetic compound with therapeutic potential for idiopathic pul- monary ﬁbrosis. It is thought to act through antioxidant and anti-ﬁbrotic pathways. Pirfenidone inhibits proliferation and/or myoﬁbroblast differentiation of a wide range of cell types, however, little studies have analyzed the effect of pirfenidone on the mesenchymal stem cells, which play an important role on the origin of myoﬁbroblasts. We recently found that pirfenidone had anti-proliferative activity via G1
phase arrest and cell division cycle 7 (Cdc7) kinase expression decrease in transforming growth factor-β1 (TGF-β1)-stimulated murine mesenchymal stem C3H10T1/2 cells. Pirfenidone also had inhibiting effect on the migration and α-SMA expression. Moreover, in this study we showed for the ﬁrst time that Cdc7
inhibitor XL413 enhanced the anti-ﬁbrotic activity of pirfenidone via depressed the expression of Smad2/ 4 proteins, and also prevented the nuclear accumulation and translocation of Smad2 protein. In con- clusion, we demonstrated that pirfenidone inhibited proliferation, migration and differentiation of TGF-
β1-stimulated C3H10T1/2 cells, which could be enhanced by Cdc7 inhibitor XL413, via Smad2/4. Com- bination with pirfenidone and XL413 might provide a potential candidate for the treatment of TGF-β1
associated ﬁbrosis. It needs in vivo studies to further validate its therapeutic function and safety in the future.& 2015 Elsevier Inc. All rights reserved.
1. Introduction

Fibrosis is the end result of chronic inﬂammatory reaction in- duced by a variety of stimuli including tissue injury, persistent infections, autoimmune reactions, allergic responses, chemical insults and radiation. The key cellular mediator of ﬁbrosis is the myoﬁbroblast, which when activated serves as the primary col- lagen-producing cell [1], yet the cell types of origin and the me- chanisms that regulate proliferation and differentiation are un- known. Studies have shown that mesenchymal stem cells are an important source of myoﬁbroblasts in the ﬁbrotic organ. Kramann
et al. [2] showed that perivascular Gli1 þ mesenchymal stem cell-
like cells generated a large fraction of myoﬁbroblasts in disease conditions. The migrated bone marrow derived-mesenchymal

n Correspondence to. Department of Oral Maxillofacial-Head and Neck Oncology, Faculty of Oral and Maxillofacial Surgery, Shanghai Ninth People’s Hospital, Shanghai JiaoTong University School of Medicine, No 639, Zhi Zao Ju Road, Shanghai 200011, China.E-mail address: [email protected] (Y. He).
1 These authors contributed equally to the work.

stem cells also contributed signiﬁcantly to α-smooth muscle actin (α-SMA)-positive myoﬁbroblasts in the bleomycin-induced mouse lung-ﬁbrosis model [3] and carbon tetrachloride-induced micecirrhotic liver model [4].Pirfenidone is an orally bioavailable synthetic compound with therapeutic potential for idiopathic pulmonary ﬁbrosis. Pirfeni- done inhibits proliferation and/or myoﬁbroblast differentiation of a wide range of cell types including primary human lung ﬁbroblast [5], rat hepatic stellate cells [6], and rat cardiac ﬁbroblasts [7] as well as in animal models of lung [8], kidney [9], hepatic [10] and cardiac ﬁbrosis [11]. Some double blind, placebo-controlled phaseⅡ and Ⅲ studies showed that pirfenidone had clinically meaningful effects and a favorable safety proﬁle in patients with idiopathic pulmonary ﬁbrosis [12–14].Although its mechanism of action has not been fully estab-
lished, existing data suggest that it has anti-inﬂammatory, anti-
ﬁbrotic, and antioxidant properties, with antagonism of activities mediated by transforming growth factor β1 (TGF-β1) and tumour necrosis factor α (TNF-α) [15]. An important aspect of the anti-
ﬁbrotic mechanism of pirfenidone is associated with its inhibition of both production and activity of TGF [16,17]. To the best of our

http://dx.doi.org/10.1016/j.yexcr.2015.11.013

0014-4827/& 2015 Elsevier Inc. All rights reserved.
knowledge, however, there was little studies had analyzed the effect of pirfenidone on the mesenchymal stem cells, which play an important role on the origin of myoﬁbroblasts.
Cell division cycle 7 (Cdc7) is a serine threonine kinase that is of critical importance in the initiation of eukaryotic DNA replica- tion and checkpoint response. Shi et al. [18] identiﬁed Cdc7 as one
of the factors mediating both the proliferation and smooth muscle cell (SMC) differentiation of the TGF-β-induced murine me- senchymal stem C3H10T1/2 (10T1/2) cells. C3H/10T1/2, Clone
8 was isolated by Reznikoff and Brankow in 1973 from a line of C3H mice embryo cells [19] and can differentiate into osteoblast [20,21], adipocytes [22], chondrocytes [23,24], myoﬁbroblasts [25], and tenocytes [26], which was considered as mesenchymal stem cells.
Considering the important effect of TGF-β1 signaling pathway,
therefore, we ﬁrst evaluated the effect of pirfenidone on pro- liferation, migration, expression of α-SMA and Smad2/4 proteins, and the nuclear translocation of Smad2 of 10T1/2 cells in the presence of TGF-β1. We also investigated whether Cdc7 inhibitor XL413 would enhanced the anti-ﬁbrotic activity of pirfenidone.
2. Material and methods

2.1. Reagents and chemicals

Unless otherwise stated, all compounds were purchased from Sigma-Aldrich (St. Louise, MO, USA). Pirfenidone (5-methyl-1- phenyl-2-[1 H]-pyridone) with a purity greater than 98% was dissolved in culture medium to make a stoke solution and sub- sequently diluted by cell culture medium before the experiments. Cell Counting Kit (CCK8) and Cytotoxicity LDH Assay Kit-WST (CK12) were from DOJINDO Laboratories (Kumamoto, Japan). An- tibodies against smad2, p-Smad2, Smad4 were purchased from Cell Signaling Technology (Danvers, MA, USA), monoclonal anti-
body against Cdc7 and α-SMA were from Abcam (Cambridge, MA, USA). Recombinant human TGF-β1 was from PeproTech (Rocky
Hill, NJ, USA). Pirfenidone was from TCI Shanghai (CAS No: 53179- 13-8, Shanghai, China). XL413 (BMS-863233) was from Selleck Chemicals (Shanghai, China).

2.2. Cell culture

C3H10T1/2 cells were obtained from the ATCC (Manassas, VA, USA), and cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS) in a 5% CO2 humidiﬁed atmosphere at 37 °C. Prior to various concentrations of pirfenidone treatment, cells were grown in serum-free medium for 24 h. In the combined experiments, cells were treated with 1 mg/ml pirfenidone and/or
10 μM Cdc7 inhibitor XL413. TGF-β1 (5 ng/ml) was added at the
same time as pirfenidone or XL413 treatment. The dose and con-
centration of XL413 were used according to the previous study
[27] and the manufacture’s protocol.

2.3. Cell proliferation (CCK8) and Cytotoxicity (LDH) assay

Cells were seeded in the 96-well plates at a density of 2 103 cells per well and starved for 24 h. They were then treated with
the indicated concentration of drugs in 10% FBS DMEM containing 5 ng/ml TGF-β1 for 0, 24, 48 or 72 h. The total cell numbers were assessed after incubation with 10 μl of CCK8 for other 3 h. The
optical density (OD) of each well was measured using a microplate reader at 450 nm, and the OD values are reported as the means 7SD. The Lactate dehydrogenase (LDH) cytotoxicity was performed according to the manufacturer’s guidelines. The

experiments were both repeated three times.

2.4. Observation of morphologic changes

Cultured 10T1/2 cells were exposed to pirfenidone, XL413 or pirfenidone plus XL413 at the same concentration for 24 h, and the cellular morphology was observed using inverted microscopy (Olympus, Tokyo, Japan).

2.5. Flow cytometry

Cells were seeded in the 6-well plates and incubated with 0, 0.5, 1.0 or 1.5 mg/ml pirfenidone for 24 h. The cells were digested, harvested, and ﬁxed with 70% ethanol for at least 12 h in 4 °C. After ﬁxation, the cells were stained with propidium iodide (PI) using a PI/RNase staining buffer (BD Biosciences, San Diego, CA, USA) according to the manufacture’s protocol. Cells were analyzed for DNA content by ﬂow cytometry (FACSCaliburTM, BD Bios- ciences, San Jose, CA, USA) and the cycle distribution was analyzed by ModFit LTTM software (BD Biosciences).

2.6. Migration assay and scratch test

Cells migration assays were performed using a Transwell
technique. Cells were digested with 0.25% trypsin–EDTA and re- suspended in serum-free DMEM containing 5 ng/ml TGF-β1. Then, 200 μl aliquots of cells (a density of 1 105/ml) were loaded into
the upper portion of a Transwell migration chamber. In addition,
pirfenidone at different concentrations (0, 0.5, 1.0 or 1.5 mg/ml) were added to the upper chambers. 500 μl of DMEM containing 10% FBS was introduced into the lower chamber as chemotactic
stimulus. After incubations for 20 h, inserts were collected and stained with 0.1% crystal violet for 20 min and then rinsed with distilled water. Adherent non-migratory cells on the upper side of the membranes were swabbed using moist cotton buds. Migrated cells on the underside of the membrane were photographed using an inverted light microscope. Migration was quantiﬁed by count- ing the invaded cells in 6 random ﬁelds ( 40 magniﬁcation) for each membranes.
Cell motility was measured in a scratch test in which the cells move from a conﬂuent area to an area that has been mechanically denuded of cells. The 10T1/2 cells were grown to a conﬂuent monolayer and then serum deprived for 24 h. After the medium was discarded, a scratch wound was inﬂicted in a straight line across the cells with a p10 pipette tip. The plates were then rinsed with phosphate-buffered saline (PBS; pH7.4) to remove the sus- pended cells and incubated with DMEM supplemented with 0, 0.5,1.0 or 1.5 mg/ml pirfenidone. Wound closure was monitored and photographed after 24 h under a light microscope and analyzed. The distances between the edges of the cells moving from both sides were measured.

2.7. Real-time reverse transcription-polymerase chain reaction (real- time PCR) analysis

Total RNA was extracted by TRIzol reagent (Invitrogen, Life Technologies) according to the manufacturer’s instructions, and cDNA was generated using an AMV Reverse Transcriptase Kit. Real- time PCR was performed in triplicate with Power PCR SYBR Green Master Mix (Takara Biotechnology, Dalian, China) using the ABI PRIAM Step-One Real-time PCR System (Applied Biosystems, Carlsbad, CA, USA) with results normalized to GAPDH expression.
The relative expression was calculated using the ΔΔCT method.
Primer sequences used were as follows: α-SMA, 5′-GGGAG- TAATGGTTGGAATGG-3′ and 3′-GGTGATGATGCCGTGTTCTA-5′; Cy- clin D1, 5′-AAAATGCCAGAGGCGGATGA-3′ and 3′

2.8. Western blot analysis

After pirfenidone, XL413 or other factors treatment, cells were washed three times with ice-cold PBS (pH7.4) and then lysed on ice in SDS lysis buffer (50 Mm Tris, 1% SDS, protease inhibitors and phosphatase inhibitors). Protein concentration was determined with a BCA detection kit (Thermo Scientiﬁc, Waltham, MA, USA).
Total 25 μg of protein lysates were separated by 10% sodium do-
decyl sulfate-polyacrylamide (SDS-PAGE) gel and transferred onto
polyvinylidene diﬂuoride (PVDF) membranes (Millipore, Burling- ton, MA, USA) using a wet transfer system (Bio-Rad, Shanghai, China). Membranes were blocked with 5% w/v skim milk, in- cubated overnight in primary antibody with gentle shaking at 4 °C, and then incubated with secondary antibody (IRDye 800CW) for 1 h at room temperature. Detection was performed with Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Quantiﬁcation of the proteins was done using Image J software.
Protein α-tubulin (Epitomics, Burlingame, CA, USA) was used as
the loading control.

2.9. Immunoﬂuorescence

Cells were seeded in the confocal dishes and cultured in medium for 24 h before being exposed to a series of concentra- tions of agents for 24 h, ﬁxed with 4% paraformaldehyde, and permeabilized with 0.2% TritonX-100. The cells were incubated

with primary antibodies to α-SMA (1:100) and Smad2 (1:100), respectively. After incubation with the primary antibodies, cells
were rinsed in PBS and incubated in ﬂuorescein-conjugated goat anti-rabbit secondary antibodies. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Labeled cells were visualized on a LMS710 laser scanning confocal microscope (Carl Zeiss, Germany).

2.10. Statistical analysis

Statistical analysis was performed using the software of GraphPad Prism version 5 Demo for Windows (GraphPad Soft- ware, San Diego, CA, USA). All values are expressed as mean 7-
standard deviation (SD), and Student’s t test (two tailed) was
performed to assess the statistical signiﬁcance of difference. P
value o0.05 was considered statistically signiﬁcant.

3. Results

3.1. Effects of pirfenidone on 10T1/2 cell proliferation and cell cycle

CCK8 assay showed that, 5 ng/ml TGF-β1 had no effect on 10T1/ 2 cell proliferation (Fig. 1A). Compared with the untreated group,
pirfenidone inhibited cell proliferation at the concentration of 0.5,
1.0 and 1.5 mg/ml in a dose- and time-dependent manner in the presence of TGF-β1 stimulation. The maximal reduction in pro- liferation was observed in 1.5 mg/ml group after 72 h treatment

Fig. 1. Pirfenidone inhibited proliferation of 10T1/2 cells. (A) CCK8 assay. Cells were treated with 0 and 5 ng/ml TGF-β1 for 24, 48 or 72 h. Data are expressed as mean 7 SD in the three independent experiment. (B) CCK8 assay. Cells with 5 ng/ml TGF-β1 were treated with 0, 0.5, 1.0 or 1.5 mg/ml pirfenidone for 24, 48 or 72 h. Data are derived from the mean 7 SD in the three independent experiment. *P o 0.05, and **P o 0.001 from comparisons between cells with treatment and untreated cells, at different time points.
(C) Effects of pirfenidone on morphological changes of cells were observed with inverted microscope. Magniﬁcation, ~ 200. (D and E) Flow cytometric analysis of cell cycle.
Cells were treated pirfenidone for 24 h. Compared with the control group, pirfenidone arrested the cells at the G1 phase. (F) Effects of pirfenidone on Cdc7 protein expression
in 10T1/2 cells were determined by western blot. (G) The relative values of densitometric scanning was performed using the software ImageJ. Data are the mean7 SD,**P o 0.001*P o0.05 versus control.
Fig. 2. Pirfenidone inhibited migration and motility of 10T1/2 cells. (A) Representative images of 10T1/2 cells migrating to the underside of membrane after 20 h. Pores in the membrane are visible as dark circles. Magniﬁcation, ~ 40. (B) Migrated cells were counted. Data represent mean 7SD, **P o 0.001 versus control. (C) Light microscope images showed decreased motility of cells at 24 h, after scratches were applied to the cells with pirfenidone treatment. (D) Migrated distance was measured. Data represent mean 7SD, **P o 0.001 versus control.

(P o0.001) (Fig. 1B). We thus decided that pirfenidone at the dose of 1 mg/ml with 24 or 48 h of observation was the optimum protocol for all experiments. Cells were seeded in six-well plates and exposed to pirfenidone for 24 h. Cells were spindle in shape with decreased cell size and increased ratio between nucleus and plasma (Fig. 1C).
These results are in agreement with the ﬂow cytometry results (Fig. 1D and E). Pirfenidone increased the percentage of G1 phase cells and markedly decreased the percentage of S phase cells. The percentage of G1 phase cells were 59.61%, 82.49%, 66.89% and 88.23% for 0, 0.5, 1.0 and 1.5 mg/ml pirfenidone, respectively. In addition, the percentage of S phase cells were 30.06%, 5.21%, 7.33% and 7.12%. Therefore, pirfenidone inhibited the cells proliferation by arresting the cells in the G1 phase.
Further assessment of cell cycle effect was performed by detection of Cdc7, a cell division cycle protein association with DNA transcription. Western blot showed that Cdc7 expression le- vel was down-regulated at the concentration of 0.5, 1.0 and

1.5 mg/ml pirfenidone treatment (Fig. 1F and G).

3.2. Effects of pirfenidone on TGF-β1-induced cell migration and motility of 10T1/2 cells

To examine the effects of pirfenidone on 10T1/2 cells migration, a transwell assay was used, to mimic the in vivo situation of cel- lular migration. After 20 h incubation, migrated cells observed on the underside of the membrane were reduced in the pirfenidone groups (0.5, 1.0 and 1.5 mg/ml) when compared with the control group (Fig. 2A and B). This indicated that pirfenidone attenuated the migration capabilities of cells.

Fig. 3. Effect of pirfenidone on α-SMA expression. 10T1/2 cells were treated with 0, 0.5, 1.0 or 1.5 mg/ml pirfenidone for 24 h. (A) Effects of pirfenidone on α-SMA mRNA expression were determined by real-time PCR. (B) Effect of pirfenidone on α- SMA protein expression were determined by western blot. (C) The relative values of densitometric scanning. (D) Cells were treated with 1.0 mg/ml pirfenidone for 0, 8, 18, 24 or 48 h. α-SMA protein expression was determined by western blot. (E) The relative values of densitometric scanning. Data are the mean 7 SD, **P o0.001 and

*P o 0.05 versus control.

We further investigated the effects of pirfenidone on the 10T1/ 2 cells motility with a scratch test. Pirfenidone signiﬁcantly re- duced 10T1/2 cells motility in dose-dependent manner after 24 h treatment (Fig. 2C and D). At the concentration of 0, 0.5, 1.0 and
1.5 mg/ml, motion rates were 100%, 75.3470.46%, 60.9672.00% and 39.6771.53%, respectively (P o0.001).
3.3. Effect of pirfenidone on TGF-β1-induced α-SMA expression in 10T1/2 cells

Myoﬁbroblast differentiation is considered to a key step in the
ﬁbrosis. Since the expression of smooth muscle cells (SMC) mar- kers, such as α-SMA is a hallmark of myoﬁbroblast differentiation, the effect of pirfenidone on TGF-β1-induced α-SMA mRNA and protein expression were measured by real-time PCR and western blot, respectively. Compared with the controls, 1.5 mg/ml pirfeni- done treatment for 24 h, there was signiﬁcantly down-regulationof α-SMA mRNA expression in the presence of TGF-β1 stimulation(P o0.001) (Fig. 3A). Consistent with this result, pirfenidone re- duced α-SMA protein expression after 48 h with 1.0 and 1.5 mg/ml treatment (Fig. 3B and C). We also detected the α-SMA proteinexpression with different time treatments. With 1.0 mg/ml pirfe- nidone treatment, α-SMA protein expression was increased at 8 h, and decreased at 48 h (Fig. 3D and E).

3.4. Pirfenidone inhibited TGF-β1-induced α-SMA expression via Smad2 and smad4

The intracellular effectors of TGF-β signaling, the Smad pro- teins, are activated by receptors and translocate into the nucleus,
where they regulated transcription. To elucidate molecular me- chanisms underlying the observed phenotypic pirfenidone effects, we investigated the Smad2 and Smad4 activity. Since phosphor-ylation is the key event for activating Smad2, we evaluated its phosphorylation status in the presence of TGF-β1 stimulation. In Fig. 4A, C, E and G, we show that pirfenidone signiﬁcantly reducedSmad4, Smad2 and phosphorylation of Smad2 in a dose-depen- dent manner in TGF-β1-stimulated cells. When treated with
1.0 mg/ml pirfenidone, phosphorylation of Smad2 down-regulated
at 24 and 48 h. Smad2 expression increased at 8 and 18 h, and then decreased until 48 h. Smad4 expression increased at 8 h, and then decreased until 48 h. (Fig. 4B, D, F and H).

3.5. Effects of XL413 plus pirfenidone on 10T1/2 cells proliferation, morphology and TGF-β1-induced α-SMA expression

As results shown Cdc7 expression decreased with pirfenidone treatment in 10T1/2 cells, therefore, we further investigated whether Cdc7 inhibitor, as XL413 would enhance the effect ofpirfenidone. For cell proliferation, cells were plated in 96-well plates, and then exposed to control, XL413 (10 μM), pirfenidone (1 mg/ml) or XL413 plus pirfenidone at the same concentration for72 h. XL413 plus pirfenidone enhanced the anti-proliferative activity compared with pirfenidone alone at 72 h (P o0.05) (Fig. 5A). For cell cytotoxicity, cells were incubated with 5 or 10 μM XL413 for 72 h and measured the amount of LDH release. XL413 alonehad no cytotoxicity of cells compared with the control group by LDH assay (Fig. 5B).
10T1/2 cells were seeded in six-well plates and incubated for 24 h, and then exposed to control, XL413 (10 μM), pirfenidone (1 mg/ml) or XL413 plus pirfenidone at the same concentration for
24 h. Cells exposed to XL413, or XL413 plus pirfenidone were polygonal in shape with decreased size and increased ratio be- tween nucleus and plasma (Fig. 5C).
In addition, effects of XL413 plus pirfenidone on α-SMA were
further investigated by western blot and immunoﬂuorescence.
Fig. 4. Pirfenidone inhibited Smad2, Smad4 and p-Smad2 expression. (A) 10T1/2 cells were treated with 0, 0.5, 1.0 or 1.5 mg/ml pirfenidone for 24 h. Smad2, Smad4 and p-Smad2 were assessed by western blot analysis. (B) Cells were treated with 1.0 mg/ml pirfenidone for 0, 8, 18, 24 or 48 h. Smad2, Smad4 and p-Smad2 was assessed by western blot analysis. (C–H) The relative values of densitometric scanning. Data are the mean 7 SD, **P o 0.001 and *P o0.05 versus control.

XL413 inhibited Cdc7 expression both in the presence and absence of pirfenidone. XL413 plus pirfenidone signiﬁcantly reduced the α- SMA expression compared with pirfenidone alone at 24 h (Fig. 5D–F).Laser confocal scanning was used for visualization of actin stress ﬁlaments and cellular morphological changes. Cells were seeded in confocal dishes, and then exposed to control, pirfeni-

done (1 mg/ml) or XL413 (10 μM) plus pirfenidone at the same
concentration for 24 h. Immunoﬂuorescence revealed that XL413
plus pirfenidone signiﬁcantly reduced formation of stress ﬁbers and brightness of α-SMA staining compared with pirfenidone alone (Fig. 5G).

3.6. Effects of XL413 plus pirfenidone on cyclin D1, TGF-β1-induced Smad2/4 expression and nuclear translocation of Smad2 Cyclin D1 is a protein required for progression through the G1
phase of the cell cycle and regulars cell proliferation, growth, and differentiation. To elucidate the probable mechanism why the combination XL413 with pirfenidone producing anti-proliferative effect, we investigated the expression of cyclin D1. In Fig. 6A, we show that XL413 alone had no effect on cyclin D1 mRNA, pirfeni- done alone decreased the cyclin D1, and the combination group signiﬁcantly reduced the level of cyclin D1 mRNA.
The expression of Smad2, Smad4 and phosphorylation of Smad2 were inhibited signiﬁcantly in the XL413 plus pirfenidone group compared with control, XL413 or pirfenidone alone by western blot analysis (Fig. 6B–E).
Furthermore, the Smad2 nuclear translocation was scanned by immunoﬂuorescence assay under confocal microscopy. In the ba-
sal state, Smad2 are predominantly localized in the cytoplasm, and staining of nuclei Smad2 was barely visible. In TGF-β1-stimulated cells, Smad2 was expressed intensively both in cytoplasm and
nucleus in control group. Concentration of 1 mg/ml pirfenidone for
Fig. 5. Effects of XL413 plus pirfenidone on 10T1/2 cells morphology, proliferation and TGF-β1-induced α-SMA expression. Cells were exposed to control, XL413 (10 μM), pirfenidone (1 mg/ml) or XL413 plus pirfenidone. (A) CCK8 assay. Cells were treated for 24, 48 or 72 h. Data are derived from the mean SD in the three independent experiments. *P o 0.05 from comparisons between cells with XL413 or not, at different time points. (B) The LDH release of cells 72 h since incubation of 5 or 10 μM XL413. The percentage of LDH was calculated by the equation: LDH release (%) ¼(Experimental LDH release – Spontaneous LDH release)/Total LDH release ~ 100. (C) Cells were treated for 24 h. Morphological changes of cells were observed with inverted microscope. Magniﬁcation, 100. (D) Effect of treatment on Cdc7 and α-SMA protein expression were determined by western blot at 24 h. (E and F) The relative values of densitometric scanning. Data are the mean7 SD, **P o 0.001 and *P o0.05 versus control. (G) Laser confocal scanning was used for visualization of α-SMA protein expression and cellular morphological changes.
Fig. 6. Effects of XL413 plus pirfenidone on cyclin D1, Smad2/4 expression and nuclear translocation of Smad2. Cells were exposed to control, XL413 (10 μM), pirfenidone (1 mg/ml) or XL413 plus pirfenidone. (A) cyclin D1 mRNA expression was detected by real-time PCR. (B) Smad2, Smad4 and p-Smad2 protein was assessed by western blot analysis. (C–E) The relative values of densitometric scanning. Data are the mean 7 SD, **P o 0.001 and *P o0.05 versus control. (F) The Smad2 nuclear translocation was scanned by immunoﬂuorescence assay under confocal microscopy. (G) % of cells with nuclear imported Smad2 were scored. Data are average of three independent ex- periments. **P o0.001 versus control.24 h reduced the nuclei expression of Smad2 compared with the control group. After treatment with XL413 plus pirfenidone for 24 h, Smad2 staining was very faint both in cytoplasm and cell nucleus (Fig. 6F and G). Therefore, pirfenidone could block the

TGF-β1-induced nuclear translocation of Smad2 and XL413 could
enhanced the effect of pirfenidone.

4. Discussion

Pirfenidone is a novel, broad spectrum anti-ﬁbrotic agent. Its anti-ﬁbrotic effect was ﬁrst described in 1995 in a hamster model of bleomycin-induced lung ﬁbrosis [28], and since then, it has been reported that pirfenidone effectively reverses experimental lung [8], liver [10] and cardiac [11] ﬁbrosis. It is thought to act through antioxidant [29] and anti-ﬁbrotic pathways [30], however,the mechanisms remain elusive. TGF-β signaling and activatedmyoﬁbroblast play a key role in the development of ﬁbrosis.
Myoﬁbroblasts are generated from a variety of sources including resident mesenchymal cells, epithelial and endothelial cells in processes termed epithelial/endothelial-mesenchymal transition, as well as from circulating ﬁbroblast-like cells called ﬁbrocytes that are derived from bone-marrow stem cells [1].

The inhibitory effect of pirfenidone on proliferation has been illustrated in a variety of cell types in vitro. In the present study,we addressed the effects of pirfenidone on murine mesenchymal stem 10T1/2 cells stimulated with TGF-β1. By performing the CCK8 assay, we found that pirfenidone signiﬁcantly inhibited the pro-
liferation of 10T1/2 cells at the concentration between 0.5 mg/ml and 1.5 mg/ml in a dose- and time-dependent manner. Our results are in line with previous observations in human lung [5], human Tenon’s ﬁbroblasts [31], rat hepatic stellate cells [32] as well as rat cardiac ﬁbroblasts [7]. Furthermore, we found that pirfenidone arrested the 10T1/2 cells in the G1 phase. In human Tenon’s ﬁ- broblasts, pirfenidone also decreased the percentage of cells in the S phase and arrested the cells at the G1 phase in a concentration- dependent manner [31]. Cdc7 kinase plays an essential role in establishing replication forks by phosphorylating components of the replication complexes and in S phase cell cycle check point control [33]. In this study, we ﬁrst found that Cdc7 expression level was downregulated at the concentration of 0.5 and 1.0 mg/ml pirfenidone. This ﬁnding was in accordance with the ﬂow cyto- metry data.
The main feature of myoﬁbroblast is represented by an im-
portant contractile apparatus similar to that of smooth muscle,
and the most commonly used molecular marker of differentiated myoﬁbroblast is the expression of α-smooth muscle actin (α-SMA) [34]. Expression of α-SMA in stress ﬁbers confers to the differ-
entiated myoﬁbroblast at least a twofold stronger contractile ac- tivity compared with α-SMA-negative ﬁbroblast in culture [34]. We demonstrated here that α-SMA mRNA and protein expressions
were inhibited by pirfenidone at the concentration of 1.5 mg/ml in TGF-β1-stimulated 10T1/2 cells. This ﬁnding is in agreement with previous reports on rat cardiac ﬁbroblast [7] and human lung ﬁ-
broblasts [5].
Most clinical trials describe pirfenidone as being generally well tolerated in the doses up to 2400 mg daily. The most common adverse effects include gastrointestinal, anorexia, fatigue, sedation, and photosensitivity rash. All of these symptoms can be serve enough to cause dose reduction or termination. The adverse ef- fects of pirfenidone appear to be dose-related and typically resolve completely once the drug is withdraw [35]. So a combination of therapies targeting several pathways involved in ﬁbro-prolifera- tion may ensure the efﬁcacy and reduce the side effects in the meantime.
Koltun et al. [27] reported the discovery of XL413, a potent and

selective ATP competitive Cdc7 inhibitor in 2012. In addition, ﬂow cytometry analysis of Colo-25 cells treated with XL413 causes dose-dependent accumulation of cells in the late S and G2 phases of cell cycle, consistent with impaired DNA synthesis. In this paper, we found for the ﬁrst time that XL413 could signiﬁcantly enhanced
the anti-ﬁbrotic activity of pirfenidone in the TGF-β1-stimulated
10T1/2 cells. Although XL413 alone had no effect of cells compared
with the control group. XL413 plus pirfenidone signiﬁcantly en- hanced anti-proliferative activity compared with pirfenidone alone at 72 h. Cdc7 inhibitor alone had no effect on the 10T1/2 cell proliferation in this study. Cdc7 is important for both the G1/S phase transition and S phase progression through activation of both early and late origin ﬁring points [36]. Down-regulation of Cdc7 expression using siRNA knockdown models produces an abortive S phase progression and cell death in HeLa cell lines. However, in normal ﬁbroblasts, a p53-dependent system prevents progression through a lethal S phase and protects the cell from the effects of reduced Cdc7 [37]. There may also be several other re- dundant checkpoint pathways in normal cells that may offer protection from Cdc7 depletion [38]. According to other study [39], in normal cells, a cell-cycle checkpoint mechanism that responds to Cdc7 depletion exists. Lack of Cdc7 causes a cell-cycle arrest at the G1-S boundary with unreplicated DNA, elevated p53 protein levels, and induction of the cyclin-dependent kinase (CDK) in- hibitor p21. As a consequence, Cdc7 depletion does not induce apoptotic cell death in normal human ﬁbroblasts.
In the control of G1/S progression, three CDKs, have been
identiﬁed as key regulators (Cyclin D/CDK4, cyclin E/Cdk2 and cyclin A/CDK2) [40]. Of these three CDKs, CDK2 along with Cdc7 activates the origin replication complexes and initiates the process of DNA replication and fork progression through modulation of Mcm2 [36]. The cyclin D1 forms a complex with and functions as a regulatory subunit of CDK4 or CDK6.

According to our results, XL413 alone had no effect on cyclin D1 mRNA, pirfenidone alone decreased the cyclin D1, and the com- bination group signiﬁcantly reduced the level of cyclin D1 mRNA. Therefore, the probable mechanism why the combination XL413 with pirfenidone producing anti-proliferative effect might be as follow: the component of Cyclin D1/CDK4/6 and cyclin E/CDK2/ Cdc7 are required in the control of G1/S progression. XL413 in- hibited the activity of Cdc7, but had no effect on cyclin D1, the Cyclin D1/CDK4/6 made compensation for the function of cyclin E/ CDK2/cdc7. So XL413 alone had no effect on the 10T1/2 cell pro- liferation in this study. Pirfenidone inhibited the expression of cyclin D1 and Cdc7. When combination XL413 with pirfenidone, the Cyclin D1/Cdk4/6 and cyclin E/CDK2/Cdc7 were both inhibited. So combination XL413 with pirfenidone produced anti-pro- liferative effect on 10T1/2 cells.
Treatment with 1.0 mg/ml pirfenidone for 24 h had no effect on the α-SMA expression. However, XL413 plus pirfenidone could signiﬁcantly reduce the expression of α-SMA at 24 h. Other study
[18] also demonstrated that Cdc7 played an important role in the transcriptional activation of SMC marker genes including α-SMA, SM22α, and calponin. The possible mechanism is that upon TGF-
stimulation, both Cdc7 and Smad3 are activated, then translocate into nuclei. Cdc7 regulates Smad3 binding to Smad-binding ele- ment (SBE) on SMC marker gene promoter via physically inter- acting with Smad3 and enhancing Smad nuclear retention factor tafazzin (TAZ) expression level, leading to the activation of SMC marker transcription. So Cdc7 shRNA effectively blocked TGF-in- duced SMC differentiation.
TGF-β1 regulate the tissue differentiation through their effects
on cell proliferation, differentiation and migration. The in- tracellular effectors of TGF-β signal, the Smad proteins, are acti- vated by transmembrane receptor serine/threonine kinases re-
ceptors and translocate into the nucleus, where they regulated
transcription [41]. In the basal state, Smad2/3 are predominantly localized in the cytoplasm. Smad4 is distributed in both the cy- toplasm and the nucleus. After receptor activation, the phos- phorylated Smad2/3 translocate into the nucleus. Thus we asked if pirfenidone was able to inhibit the Smad2/3 and Smad4 activation. In the current study, we clearly demonstrated that pirfenidone
inhibited TGF-β1-induced synthesis of Smad2 and Smad4 proteins levels. Choi et al[42] reported that pirfenidone blocked TGF-β1-induced nuclear translocation of active Smad2/3 in human retinal pigment epithelial cell line (ARPE-19) cells. In addition, other study [5] revealed that pirfenidone impaired the phosphorylation of Smad3 in primary human lung ﬁbroblasts. Furthermore, wefound that pirfenidone treatment reduced TGF-β1-induced Smad2phosphorylation. We also demonstrated that XL413 plus pirfeni- done signiﬁcantly depressed the TGF-β1-induced expression of Smad2 and Smad4 proteins, and also prevented the nuclear ac-cumulation and translocation of Smad2 protein in 10T1/2.Upon TGF-stimulation, both Cdc7 and Smad3 are induced and/ or activated, and translocate into nuclei where Cdc7 regulates Smad3 binding to Smad-binding element (SBE). Another Cdc7 in- hibitor (PHA767491) diminished Smad3 nuclear translocation [18]. When combinational treatment, pirfenidone blocks Smad2/p- Smad2 directly, Cdc7 inhibitor might have indirect effect on Smad2 by block Smad3 nuclear translocation. However, further research should be done.
In conclusion, our data indicated that pirfenidone inhibited proliferation, migration and differentiation of TGF-β1-stimulated 10T1/2 cells, which could be enhanced by Cdc7 inhibitor XL413, viaSmad2/4. Combination with pirfenidone and XL413 might provide a potential candidate for the treatment of TGF-β1 associated ﬁ- brosis. It needs in vivo studies to further validate its therapeutic
function and safety in the future.

Acknowledgments

This work was supported by the National Natural Science Foundation of China, China (Grant 81271112), the Development Foundation supported by Shanghai Municipal Human Resources and Social Security Bureau (Grant 201312), SMC Rising Star (2013A) Scholar supported by Shanghai Jiao Tong University.

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