Effect of Luteolin and Apigenin on the Expression of Oct-4, Sox2, and c-Myc in Dental Pulp Cells with In Vitro Culture

Introduction. Dental pulp cells (DPCs) are promising cell source for dental tissue regeneration. Recently, small molecules which optimize microenvironment or activate the reprogramming network provide a new way to enhance the pluripotency. Two promising bioflavonoids luteolin and apigenin were reported to enhance reprogramming efficiency in mouse embryonic fibroblast (MEF). However, their effect and underlying mechanism in cell fate determination of human DPCs remain unclear. Methods. To elucidate the effect of luteolin and apigenin on the cell fate determination of DPCs, we explored the cell proliferation, cell cycle, senescence, apoptosis, expression of pluripotency markers Oct-4, Sox2, and c-Myc, and multilineage differentiation capability of DPCs with luteolin or apigenin treatment. Results. We demonstrated that luteolin and apigenin inhibited cell proliferation, arrested DPCs in G2/M and S phase, and upregulated PI value and apoptosis. Moreover, luteolin and apigenin increased telomerase activity, maintained DPCs in a presenescent state, and activated the expression of Oct-4, Sox2, and c-Myc at a dose- and time-dependent pattern in DPCs even at late passages, albeit repressed lineage-specific differentiation. Conclusions. Addition of luteolin and apigenin in the culture medium might provide an effective way to maintain DPCs in an undifferentiated stage and inhibit lineage-specific differentiation.


Introduction
Dental pulp cells (DPCs) with self-renewal, colony forming efficiency, and multilineage differentiation capability are promising cell source for dental tissue regeneration [1,2]. It is verified that DPCs were capable of differentiating into odontoblasts, adipocytes, chondrocytes, and so forth [1]. Since DPCs are easily obtained from extracted teeth, they may be ideal cell resource to repair injured tooth structures. However, the potential application of DPCs in dental regeneration is limited by loss of stem cell characteristics in in vitro culture conditions [3].
Mesenchymal stem cells from dental tissues are able to be reprogrammed to induced pluripotent stem cells (iPSCs) with embryonic stem cells (ESCs) like characteristics by introducing transcription factors Oct-4/Sox2/Klf4/c-Myc or Oct-4/Sox2/Nanog/Lin28. Thus, the transcription factors Oct-4, Sox2, and c-Myc are closely correlated with pluripotency and reprogramming [4]. Recent studies reported that small molecules increased the expression of STRO-1, Nanog, Oct-4, and Sox2 and decreased cell proliferation and odonto/osteogenic, adipogenic, and neurogenic lineages differentiation through Ras-GAP-, ERK1/2-, and mTORsignaling pathways. Small molecules might provide a new way to enhance the immature state and maintain the potential capability of dental derived cells in tissue engineering through optimizing microenvironment [5]. Therefore, improving culture condition through adding growth factors to culture solution might be a simple and effective way of optimizing microenvironment and delivering signals to cells. Luteolin and apigenin, two important flavonoids, possess antiproliferation, proapoptosis, antiangiogenesis, antitumor, and anti-inflammatory properties [6,7]. They are able to counteract oxidative mechanisms and suppress cell growth of prostate cancer through inhibition of insulin-like growth factor-I receptor, Akt signaling, cell cycle arrest, and induction of cell apoptosis [6,7]. Notably, luteolin and apigenin are reported to enhance reprogramming efficiency through the upregulation of E-cadherin, which could replace Oct-4 during iPSC generation [8]. However, to date, the specific role of luteolin and apigenin in regulating pluripotency and multilineage differentiation capability of DPCs remains unknown.
Our previous study revealed that Sox2, and c-Myc maintained nucleus location and relatively high mRNA expression even at late passages in periodontal ligament cells (PDLCs) with rhBMP4 induction, indicating that small molecules may provide a suitable microenvironment to maintain PDLCs in an undifferentiated stage [9]. These results implied that chemical approaches may play essential roles in the regulation of cell fate determination and pluripotency, which will shed light on the potential application of DPCs in dental regeneration. Therefore, in the present study, we investigated the effect of luteolin and apigenin on cell proliferation, apoptosis, cell cycle, senescence, expression of pluripotency markers (Oct-4, Sox2, and c-Myc), and multilineage differentiation capability of DPCs, to demonstrate the essential role luteolin and apigenin played in cell fate determination of dental derived cells.

Isolation and Expansion of Human DPCs.
Normal human premolars and impact third molars were extracted from healthy young adults (12-28 years) undergoing orthodontic treatment at the Department of Oral and Maxillofacial Surgery, the Affiliate Stomatology Hospital of Sun Yat-Sen University; informed consent was obtained from each patient. The protocols were approved by the University Ethic Committee. DPCs were obtained from dental pulp tissue by explant culture as previously described [10]. DPCs were cultured in Dulbecco's modified Eagle medium with low glucose (DMEM-LG, Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, UT, USA), 10 U/mL penicillin G, and 10 mg/mL streptomycin (Invitrogen, CA, USA). DPCs were incubated at 37 ∘ C in 5% CO 2 . Luteolin (Sigma-Aldrich, MO, USA) and apigenin (Sigma-Aldrich, MO, USA) were dissolved in dimethyl sulfoxide (DMSO, Invitrogen, CA, USA) and diluted in medium for cell culture. DPCs were serum-deprived for 24 h before induction with luteolin and apigenin at the concentrations of 0, 1 mol/L, 5 mol/L, and 10 mol/L. And the incubation was maintained for 0, 3, and 5 days. The medium was changed every 3 days.

Quantitative Real-Time Reverse-Transcription Polymerase
Chain Reaction and Western Blot. To examine the dose-and time-dependent effect of luteolin/apigenin on the expression of Oct-4, Sox2, and c-Myc in DPCs, total RNA was isolated from DPCs at passage 3 treated with luteolin/apigenin at various concentrations (1, 5, and 10 mol/L) for 0, 3, and 5 d using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's protocol. The concentration and quality of RNA samples were measured with spectrophotometers and gel electrophoresis. First-strand cDNA was synthesized from 1 g of total RNA using SuperScript III (Invitrogen, CA, USA) in a total volume of 20 L. 2.5 L of the reaction mixture Table 1: Primer sequences used in quantitative real-time polymerase chain reaction.

Cell Counting Kit 8 (CCK8) Assay for Cell Proliferation.
Luteolin (Sigma-Aldrich, MO, USA) and apigenin (Sigma-Aldrich, MO, USA) at the optimum concentration of 10 g/mL, indicated by result of real-time PCR and western blot mentioned above, were added to the culture medium throughout the suspension period. Cells cultured in normal medium served as control. The culture of DPCs was serumdeprived for 24 h prior to the induction. A total of 10 4 cells per well were plated in 96-well plates and cell proliferation of DPCs was evaluated using the CCK8 (Dojindo, Tokyo, Japan) according to manufacturer's instructions. Briefly, 10 L of CCK8 solution was added to the culture medium and incubated for additional 3 h. The absorbance was determined at 450 nm wave length.

Flow Cytometry for Cell
Cycle and Apoptosis. The culture of DPCs was serum-deprived for 24 h prior to the induction. 1 × 10 5 DPCs with/without luteolin and apigenin treatment were harvested by trypsinization, washed twice in cold PBS, and fixed in 70% alcohol for 30 min on ice. After washing in cold PBS three times, cells were incubated with 0.5% propidium iodinate (PI) for 30 min at 4 ∘ C. Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Data was analyzed using FCS Express software.

Beta-Galactosidase Staining for Cell Senescence and
Telomerase Activity. Cytochemical staining for the senescence-associated b-galactosidase assay was performed by seeding DPCs from passages 1, 3, 5, and 7 with/without luteolin/apigenin treatment at the cell density of 1 × 10 3 cells/well in a 24-well plate. The cells were allowed to attach overnight, washed with PBS, fixed, and incubated overnight at 37 ∘ C with an X-gal chromogenic substrate at pH 6.0 according to the protocol provided by the b-galactosidase staining kit (Cell Signaling Technology, MA, USA). The images of cell morphology were captured under an inverted light microscope. Telomerase activity in DPCs from passages 1, 3, 5, and 7 with/without luteolin/apigenin treatment was detected by using a quantitative telomerase detection kit (Allied Biotech, MD, USA) according to the manufacturer's protocol.

Effect of Luteolin and Apigenin on Odontogenic, Adipogenic, and Chondrogenic Differentiation of DPCs.
For odontogenic differentiation, DPCs at passage 3 with/without luteolin and apigenin induction were odontogenically inducted in medium containing 10 mM -glycerophosphate (Sigma-Aldrich, MO, USA), 50 M ascorbic acid (Sigma-Aldrich, MO, USA), and 100 nM dexamethasone (Sigma-Aldrich, MO, USA) for 21 d. The expression of DSPP was detected by immunofluorescent staining as previously described [10]. Briefly, DPCs were cultured in chamber slides (Nunc, NY, USA) and fixed with 3% paraformaldehyde for 15 min. The slides were rinsed in PBS 3 times for 5 min, respectively, then permeabilized with 0.1% Triton for 20 min, and incubated with 10% swine serum for 1 h. Slides were transferred to a humidified chamber and stained with DSPP antibody (1 : 400 dilution; Chemicon, MA, USA) overnight at 4 ∘ C. Samples were washed 3 times in PBS and incubated with a fluorochrome-labeled secondary antibody (1 : 100 dilution; Invitrogen, NY, USA) for 3 h. The sections were thoroughly washed in PBS and mounted. PBS was used instead of the primary antibody as control. The images were captured  under microscope (Axiovert, Zeiss, Germany). The gene expression of ALP and DSPP was evaluated by real-time PCR as mentioned above. Primers used for detection are listed in Table 2.

Effect of Luteolin and Apigenin on the Cell Senescence and
Telomerase Activity of DPCs at Various Passages. Senescenceassociated b-galactosidase is caused by upregulated lysosomal activities and altered cytosolic pH, which are upregulated with senescence and aging. To elucidate the effect of luteolin and apigenin on replicative senescence state of DPCs, the senescence-associated b-galactosidase activity (SA-b-gal) was evaluated. DPCs from passages 1, 3, 5, and 7 with/without luteolin/apigenin treatment were detected, albeit only the representative results of passages 3 and 7 were presented. The result revealed that DPCs at passage 3 with luteolin ( Figure 2  luteolin or apigenin induction (Figure 2(C7), > 0.05), albeit DPCs at passage 7 with luteolin or apigenin induction showed significantly higher telomerase activity than the control group at passage 7 ( * < 0.05), which agreed with the result of b-galactosidase assay mentioned above. This result implied that luteolin and apigenin treatment significantly inhibited cell senescence and increased telomerase activity of DPCs, especially at late passages. Thus, luteolin and apigenin might be able to maintain DPCs in an undifferentiated and presenescent state.

Effect of Luteolin and Apigenin on the Multilineage Differentiation Capability of DPCs.
Assay of the multilineage differentiation capability of DPCs towards odontogenic, chondrogenic, and adipogenic cell lineages showed considerable variation in culture condition treated with luteolin and apigenin. The immunofluorescent staining showed that DSPP (Figure 3(A1)) and LPL (Figure 3(B1)) were strongly expressed in DPCs without luteolin/apigenin treatment after 21 d of odontogenic and adipogenic induction, mainly located in the nucleus of DPCs. Collagen type II (Figure 3(C1)) was mainly located in the cytoplasm of DPCs without luteolin/apigenin treatment after 21 d of chondrogenic induction, whereas, for the DPCs with luteolin/apigenin treatment, only LPL (Figures 3(B2) and 3(B3)) showed weak expression in the nucleus of DPCs after adipogenic induction, albeit DSPP (Figures 3(A2) and 3(A3)) and collagen type II (Figures 3(C2) and 3(C3)) were barely found in DPCs after odontogenic and chondrogenic induction. This result was confirmed by realtime PCR, which indicated that mRNA expression of odontogenic markers (ALP, DSPP), adipogenic markers (PPAR 2, LPL), and chondrogenic markers (collagen type II) was significantly downregulated in luteolin and apigenin groups after 3w induction towards multilineages compared with control group (Figures 3(d)-3(h)). These results indicated that the multilineage differentiation capability was inhibited in DPCs with luteolin/apigenin treatment.

Discussion
Previous studies have shown that the overexpression of Oct-4, Sox2, Klf4, and Myc (OSKM) can convert mouse fibroblasts into iPSCs with resemble global gene expression, epigenetic state, and developmental potential of mESC [12,13]. Oct-4, Sox2, and c-Myc work cooperatively in maintaining or activating the reprogramming network during this process; thus their expression levels are assumed to be closely related to pluripotency and reprogramming capability [14]. Since the loss of pluripotency could be rejuvenated by altered extracellular microenvironment and molecules controlling endogenous signaling pathways, it is possible to enhance the pluripotency and improve cell characteristics by optimizing the culture condition with certain molecules [15][16][17]. It is reported that alternation of O 2 microenvironments may regulate hESCs survival, self-renewal, and differentiation capabilities through posttranscriptional regulation of telomerase isoforms [16]. Moreover, hypoxia played a critical role in maintaining the stemness and differentiation capacity of PDLCs and DPCs through reactivation of Oct-4, Sox2, and c-Myc [17]. Therefore, simple and effective ways to improve undifferentiated state of somatic cells without integrating gene delivery methods are highly desirable. Small molecules are able to regulate specific signaling pathways involved in chromatin remodeling, gene expression alternation, and cell biology manipulation [5,18]. Instead of activating the "master genes" by virus transfection, pluripotency and reprogramming network can be reestablished by the application of small molecules and modulation of cell  culture microenvironment. Various small molecules with the advantages of low cost, biocompatibility, and easy application have been demonstrated to enhance reprogramming efficiency and improve cell characteristics. These properties make it possible for the small molecules to provide temporal modulation of the pluripotent signals [5,18,19]. However, the complete chemical inducing approach requires further studies and the underlying mechanism remains unclear. Flavonoids, polyphenolic compounds produced by plants, contribute to the prevention of heart disease, neurodegenerative diseases, diabetes, and cancer [6,7]. Flavonoids play important roles in antioxidant effect, anti-inflammatory effect, regulation of apoptosis, and suppression of tumor related genes and DNA damage through modulation of growth factor signaling pathways [6,7]. Antioxidant effect is one of the main biological functions of flavonoids, which contributes to chelating metal ions and inactivating free radicals [20]. The flavonoids luteolin and apigenin possess antiproliferation, proapoptosis, antiangiogenesis, antitumor, and anti-inflammatory properties [6]. These two compounds strongly dose-dependently inhibited tumor necrosis factor -(TNF--) induced NF-B, IL-8, and E-selectin protein expression after stimulation with lipopolysaccharide (LPS) or TNF-in endothelial cells [20,21]. Luteolin showed synergistic antiviral effect with IFN-in modulating the immune response of peripheral blood mononuclear cells [22]. Luteolin, inhibitor of cyclin-dependent kinase 9 (CDK9), can induce apoptosis in cancer cells through blocking phosphorylation of the carboxy-terminal domain of RNA polymerase II [23]. Apigenin restrained prostate carcinogenesis via TGF-activated pathways, especially the Smad2/3 and Src/FAK/Akt pathways [24].
Luteolin regulated proliferation and cell cycle transition of PC-3 cell through EGFR-tyrosine kinase independent mechanism, which arrested cells in G2/M phase, decreased the number of cells in G0/G1, and caused significant increase in apoptotic cells [25]. It is also reported that decreasing cell proliferation by removing c-Myc or adding antiproliferative compounds enhanced iPSCs generation, implying inhibiting cell proliferation and arresting cell cycle may improve reprogramming [26]. Our present study revealed that luteolin and apigenin inhibited cell proliferation, arrested DPCs in G2/M and S phase, downregulated the number of cells in G0/G1, and upregulated apoptotic cells. Moreover, additional treatment with luteolin and apigenin dramatically enhanced the expression of pluripotency and reprogramming markers Oct-4, Sox2, and c-Myc. These results agreed with the previous studies [25,26]. Although our study did not provide direct evidence that apoptosis is occurring in the cells arrested in G2/M phase, the data indicated that luteolin and apigenin may play important roles in cell proliferation, apoptosis, and phase-specific cell cycle regulation of DPCs, albeit further studies might be required to elucidate the underlying mechanism regulating apoptosis and cell cycle.
Consistent expression of telomerase activity inhibits senescence and is responsible for the maintenance of stemness properties [27,28]. Oct-4, Sox2, and c-Myc are closely related to pluripotency and reprogramming property [12][13][14]. In this study, our results revealed that luteolin and apigenin upregulated the telomerase activity, maintained DPCs in a presenescent state, and activated the expression of Oct-4, Sox2, and c-Myc in DPCs even at late passages. These results suggested that luteolin or apigenin treated microenvironment might be effective way to trigger the expression of pluripotency markers and maintain undifferentiated state of DPCs, which agreed with previous studies that luteolin and apigenin upregulated Oct-4, Sox2, and c-Myc through E-cadherin and enhanced reprogramming efficiency [8]. However, it was shown in our study that additional luteolin and apigenin in culture condition repressed lineage-specific differentiation potential of DPCs, evidenced by the real-time PCR and immunofluorescent staining. Thus, luteolin and apigenin enhanced undifferentiated state and inhibit lineage-specific differentiation of DPCs.
This study revealed that, taken together, luteolin and apigenin could enhance the expression of pluripotency markers, maintain DPCs in an undifferentiated state, and inhibit lineage-specific differentiation, which is of critical importance for the application of DPCs in dental regeneration. However, future studies are required to investigate the underlying mechanism of small molecules in the regulation of pluripotency in DPCs with in vitro culture.