Quercitrin Stimulates Hair Growth with Enhanced Expression of Growth Factors via Activation of MAPK/CREB Signaling Pathway

The present study aimed to investigate the molecular mechanism of quercitrin, a major constituent of Hottuynia cordata extract, for its hair growth stimulating activities in cultured human dermal papilla cells (hDPCs). Quercitrin enhanced the cell viability and cellular energy metabolism in cultured hDPCs by stimulating the production of NAD(P)H and mitochondrial membrane potential (ΔΨ). The expression of Bcl2, an essential marker for anagen hair follicle and cell survival, was increased by quercitrin treatment. Quercitrin also increased the cell proliferation marker Ki67. The expression of growth factors—such as bFGF, KGF, PDGF-AA, and VEGF—were increased by quercitrin both in mRNA and protein levels. In addition, quercitrin was found to increase the phosphorylation of Akt, Erk, and CREB in cultured hDPCs, while inhibitors of MAPKs reversed the effects of quercitrin. Finally, quercitrin stimulated hair shaft growth in cultured human hair follicles. Our data obtained from present study are in line with those previously reported and demonstrate that quercitrin is (one of) the active compound(s) of Hottuynia cordata extract which showed hair growth promoting effects. It is strongly suggested that the hair growth stimulating activity of quercitrin was exerted by enhancing the cellular energy metabolism, increasing the production of growth factors via activation of MAPK/CREB signaling pathway.


Introduction
Androgenetic alopecia (AGA) is the most common type of hair loss in men and is a naturally progressive disorder [1]. In the process of AGA, large terminal scalp hairs are gradually replaced by thinner and smaller vellus hairs, which is called hair follicle miniaturization. The pathogenesis and underlying molecular mechanisms of AGA are not yet fully understood but androgen hormone, dihydrotestosterone (DHT) is suspected to be the most potent causative agent in genetically predisposed individuals [2].
Although AGA is not a life-threatening disease it has great psychosocial impact on affected patients, resulting in a significant impairment of quality of life [3]. Despite tremendous efforts for

Quercitrin Increased Cell Viability in Cultured Human DPCs
The treatment of quercitrin increased the viability of DPCs in a concentration dependent manner. NAD(P)H generation, presented as cell viability, was increased by 9.6%, 24.4%, and 39.0% in the presence of 0.1, 1, and 10 nM quercitrin, respectively. Above 10 nM concentrations, the cell viability was reached to a plateau (Figure 1b). The increments were comparable with that of 100 nM of minoxidil, approved topical hair growth stimulating medicine, a positive control.
Because NAD(P)H production was markedly increased by quercitrin treatment, the changes in mitochondrial membrane potential (∆Ψ) were investigated. The mitochondrial membrane potential indicates the capacity for energy generation in mitochondria. As shown in Figure 2, the membrane potential was increased in a concentration dependent manner. The treatment of quercitrin at concentration of 10 and 100 nM significantly increased the membrane potential by 34.0% and 63.2%, respectively. Combining the data in Figures 1 and 2, the treatment of quercitrin resulted in potentiation of energy metabolism in cultured hDPCs. Because NAD(P)H production was markedly increased by quercitrin treatment, the changes in mitochondrial membrane potential (∆Ψ) were investigated. The mitochondrial membrane potential indicates the capacity for energy generation in mitochondria. As shown in Figure 2, the membrane potential was increased in a concentration dependent manner. The treatment of quercitrin at concentration of 10 and 100 nM significantly increased the membrane potential by 34.0% and 63.2%, respectively. Combining the data in Figure 1 and Figure 2, the treatment of quercitrin resulted in potentiation of energy metabolism in cultured hDPCs.   Cell viability was assessed using CCK-8 assay kit after quercitrin treatment (0.1, 1, 10, 100 nM and 1 µM) for 24 h. The value of non-treated control was taken to be 100%. N.T, non-treated control; MNX, minoxidil. Significantly different compared with N.T (* p < 0.05, ** p < 0.01, *** p < 0.001).
Because NAD(P)H production was markedly increased by quercitrin treatment, the changes in mitochondrial membrane potential (∆Ψ) were investigated. The mitochondrial membrane potential indicates the capacity for energy generation in mitochondria. As shown in Figure 2, the membrane potential was increased in a concentration dependent manner. The treatment of quercitrin at concentration of 10 and 100 nM significantly increased the membrane potential by 34.0% and 63.2%, respectively. Combining the data in Figure 1 and Figure 2, the treatment of quercitrin resulted in potentiation of energy metabolism in cultured hDPCs.

Quercitrin Enhanced Bcl2 Expression
The mRNA expression level of cell viability related genes was assessed in cultured hDPCs. The expression of Bad, an apoptotic marker in mitochondria, was significantly decreased by quercitrin treatment. On the contrary, an anti-apoptotic gene Bcl2 level was significantly increased by 47% and 54% with 10 and 100 nM quercitrin, respectively. In addition, cell proliferation marker, Ki67 was also increased by quercitrin in a concentration dependent manner (Figure 3a). Western blot analysis revealed that quercitrin also significantly increased the amount of Bcl2 protein by 66.2% with 100 nM treatment (Figure 3b,c). treatment. On the contrary, an anti-apoptotic gene Bcl2 level was significantly increased by 47% and 54% with 10 and 100 nM quercitrin, respectively. In addition, cell proliferation marker, Ki67 was also increased by quercitrin in a concentration dependent manner (Figure 3a). Western blot analysis revealed that quercitrin also significantly increased the amount of Bcl2 protein by 66.2% with 100 nM treatment (Figure 3b

Quercitrin Increased mRNA Expression of Growth Factors and Their Receptors
The DPCs secrete myriad of growth factors to regulate hair follicle growth. To specify the factors affecting metabolic stimulation and cell viability, the mRNA expression of growth factors and growth factor receptors was evaluated. The expression levels of 19 growth factor genes and 15 growth factor receptor genes were measured. Among 34 genes tested, the expression of 8 genes (bFGF, KGF, IGFBP2, TGFb1, VEGFA, FGFR1, PDGFRa, and PDGFRb) was increased by quercitrin treatment. Among growth factors, mRNA expression of bFGF, KGF and VEGFA was significantly increased by 50.1%, 79.5%, and 32.7% at 100 nM quercitrin treatment, respectively. For growth factor receptors, the expression level of FGFR1 and PDGFRa was significantly increased (Figure 4).

Quercitrin Increased mRNA Expression of Growth Factors and Their Receptors
The DPCs secrete myriad of growth factors to regulate hair follicle growth. To specify the factors affecting metabolic stimulation and cell viability, the mRNA expression of growth factors and growth factor receptors was evaluated. The expression levels of 19 growth factor genes and 15 growth factor receptor genes were measured. Among 34 genes tested, the expression of 8 genes (bFGF, KGF, IGFBP2, TGFb1, VEGFA, FGFR1, PDGFRa, and PDGFRb) was increased by quercitrin treatment. Among growth factors, mRNA expression of bFGF, KGF and VEGFA was significantly increased by 50.1%, 79.5%, and 32.7% at 100 nM quercitrin treatment, respectively. For growth factor receptors, the expression level of FGFR1 and PDGFRa was significantly increased ( Figure 4).

Quercitrin Increased the Protein Expression of Growth Factors
To elucidate whether quercitrin affect the gene expression of growth factors and their receptors to protein level, dot blot analysis was performed. The amount of bFGF, KGF, PDGF-AA, and VEGF, which were reported to enhance the cell viability of DPCs [12], was significantly increased by quercitrin treatment ( Figure 5).

Quercitrin Increased the Protein Expression of Growth Factors
To elucidate whether quercitrin affect the gene expression of growth factors and their receptors to protein level, dot blot analysis was performed. The amount of bFGF, KGF, PDGF-AA, and VEGF, which were reported to enhance the cell viability of DPCs [12], was significantly increased by quercitrin treatment ( Figure 5).

Quercitrin Increased the Protein Expression of Growth Factors
To elucidate whether quercitrin affect the gene expression of growth factors and their receptors to protein level, dot blot analysis was performed. The amount of bFGF, KGF, PDGF-AA, and VEGF, which were reported to enhance the cell viability of DPCs [12], was significantly increased by quercitrin treatment ( Figure 5). Protein levels of bFGF and KGF both in culture medium and cell lysate were further investigated using ELISA to differentially assess the intracellular and secreted form. The intracellular bFGF and KGF were significantly increased by quercitrin treatment (Figure 6a,c). Furthermore, quercitrin at concentration of 100 nM also increased the secretion of bFGF and KGF by 36.5% and 27.3%, Protein levels of bFGF and KGF both in culture medium and cell lysate were further investigated using ELISA to differentially assess the intracellular and secreted form. The intracellular bFGF and KGF were significantly increased by quercitrin treatment (Figure 6a,c). Furthermore, quercitrin at concentration of 100 nM also increased the secretion of bFGF and KGF by 36.5% and 27.3%, respectively (Figure 6b,d). Taken together, quercitrin enhanced not only the production of bFGF and KGF but also the secretion of them.

Quercitrin Induced Phosphorylation of Akt, Erk, and CREB
To clarify the molecular action mechanism of quercitrin, the MAP kinase molecules, which play pivotal roles in cellular signal cascades, were investigated. Among phosphorylated MAP kinase proteins, phospho-Akt and phospho-Erk were significantly increased. The protein levels of total Akt and Erk, on the other hand, were not changed. It was revealed that the phosphorylation of Akt and Erk tended to have a time dependency with maximum increases at a certain time point (Figure 7a). The ratio of pAkt/Akt was increased by 73.3% and 63.8%, when 100 nM of quercitrin was treated for 1 and 2 min, respectively. On the other hand, pErk/Erk ratio was more prominently increased by 204.0% and 285.9%, for 2 and 5 min of quercitrin treatment, respectively ( Figure 7b). As a downstream target of Thr202-Erk phosphorylation, CREB phosphorylation at Ser133 site was examined [13]. The phospho (Ser133)-CREB/CREB level was significantly elevated by 671.4%, 680.0%, and 244.0%, when 100 nM of quercitrin was treated for 5, 10, and 20 min, respectively (Figure 7b).
Molecules 2020, 25, x 6 of 14 respectively (Figure 6b,d). Taken together, quercitrin enhanced not only the production of bFGF and KGF but also the secretion of them.

Quercitrin Induced Phosphorylation of Akt, Erk, and CREB
To clarify the molecular action mechanism of quercitrin, the MAP kinase molecules, which play pivotal roles in cellular signal cascades, were investigated. Among phosphorylated MAP kinase proteins, phospho-Akt and phospho-Erk were significantly increased. The protein levels of total Akt and Erk, on the other hand, were not changed. It was revealed that the phosphorylation of Akt and Erk tended to have a time dependency with maximum increases at a certain time point (Figure 7a). The ratio of pAkt/Akt was increased by 73.3% and 63.8%, when 100 nM of quercitrin was treated for 1 and 2 min, respectively. On the other hand, pErk/Erk ratio was more prominently increased by 204.0% and 285.9%, for 2 and 5 min of quercitrin treatment, respectively (Figure 7b). As a downstream target of Thr202-Erk phosphorylation, CREB phosphorylation at Ser133 site was examined [13]. The phospho (Ser133)-CREB/CREB level was significantly elevated by 671.4%, 680.0%, and 244.0%, when 100 nM of quercitrin was treated for 5, 10, and 20 min, respectively (Figure 7b). We scrutinized whether the inhibitors of MAP kinases could inhibit the quercitrin induced gene expressions to find out the relationship between enhanced gene expression and MAPKs. As shown in Figure 8, Erk inhibitor U0126 (20 µM) significantly diminished the quercitrin stimulated expression of bFGF, KGF and Bcl2 while Akt inhibitor API-2 did not. The quercitrin-induced Ki67 expression, on the other hand, was inhibited by both U0125 and API-2. Our data suggest that quercitrin-stimulated expression of growth factors (bFGF and KGF) and Bcl2 was mediated by Erk activity and that both Erk and Akt were involved in enhanced cell proliferative potential by quercitrin. We scrutinized whether the inhibitors of MAP kinases could inhibit the quercitrin induced gene expressions to find out the relationship between enhanced gene expression and MAPKs. As shown in Figure 8, Erk inhibitor U0126 (20 µ M) significantly diminished the quercitrin stimulated expression of bFGF, KGF and Bcl2 while Akt inhibitor API-2 did not. The quercitrin-induced Ki67 expression, on the other hand, was inhibited by both U0125 and API-2. Our data suggest that quercitrinstimulated expression of growth factors (bFGF and KGF) and Bcl2 was mediated by Erk activity and that both Erk and Akt were involved in enhanced cell proliferative potential by quercitrin. Figure 7. Effect of quercitrin on Akt, Erk, and CREB phosphorylation in cultured hDPCs. Quercitrin was treated for different times (0, 1, 2, 5, 10, and 20 min). (a) Whole cell lysates were analyzed by immunoblotting to determine the levels of Akt, phospho-Akt, Erk, phospho-Erk, CREB and phospho-CREB. As an internal control, GAPDH were used. (b) The ratio of pAkt/Akt, pErk/Erk, and pCREB/CREB was calculated. N.T, non-treated control. The data represent the means of five independent samples. Significantly different compared with N.T (Quercitrin * p < 0.05, ** p < 0.01, *** p < 0.001; Minoxidil # p < 0.05, ## p < 0.01, ### p < 0.001).

Quercitrin Activated Receptor Tyrosine Kinases and Non-Receptor Tyrosine Kinases
The receptor tyrosine kinase and its receptor activation pathways were well proved for the activation of Akt and Erk/MAPK pathways [14]. Therefore, we investigated the phosphorylation of tyrosine kinases using RTK dot blotting in cultured DPCs treated with quercitrin for 30 s and 1 min. Total of 71 phosphorylated proteins were examined and 18 proteins were detected with significant increase by quercitrin treatment (p < 0.05), 8 receptor tyrosine kinases and 10 non-receptor tyrosine kinases including Src family proteins (Figure 9). Molecules 2020, 25, x 8 of 14

Quercitrin Activated Receptor Tyrosine Kinases and Non-Receptor Tyrosine Kinases
The receptor tyrosine kinase and its receptor activation pathways were well proved for the activation of Akt and Erk/MAPK pathways [14]. Therefore, we investigated the phosphorylation of tyrosine kinases using RTK dot blotting in cultured DPCs treated with quercitrin for 30 s and 1 min. Total of 71 phosphorylated proteins were examined and 18 proteins were detected with significant increase by quercitrin treatment (p < 0.05), 8 receptor tyrosine kinases and 10 non-receptor tyrosine kinases including Src family proteins (Figure 9).

Quercitrin Activated Receptor Tyrosine Kinases and Non-Receptor Tyrosine Kinases
The receptor tyrosine kinase and its receptor activation pathways were well proved for the activation of Akt and Erk/MAPK pathways [14]. Therefore, we investigated the phosphorylation of tyrosine kinases using RTK dot blotting in cultured DPCs treated with quercitrin for 30 s and 1 min. Total of 71 phosphorylated proteins were examined and 18 proteins were detected with significant increase by quercitrin treatment (p < 0.05), 8 receptor tyrosine kinases and 10 non-receptor tyrosine kinases including Src family proteins (Figure 9). Figure 9. Quercitrin stimulated the receptor tyrosine kinases and non-receptor tyrosine kinases in cultured hDPCs. 100 nM of quercitrin was treated for appropriate times (0, 0.5, 1 min). Whole cell lysates were analyzed by immunoblotting to determine the level of phospho-tyrosine following manufacturer's instruction. Total of 71 types of tyrosine kinase were analyzed. The 8 receptor tyrosine Figure 9. Quercitrin stimulated the receptor tyrosine kinases and non-receptor tyrosine kinases in cultured hDPCs. 100 nM of quercitrin was treated for appropriate times (0, 0.5, 1 min). Whole cell lysates were analyzed by immunoblotting to determine the level of phospho-tyrosine following manufacturer's instruction. Total of 71 types of tyrosine kinase were analyzed. The 8 receptor tyrosine kinases and 10 non-receptors tyrosine kinases were displayed (Significantly different compared with 0 min, p < 0.05). Positive, biotin-conjugated IgG.

Quercitrin Stimulated Hair Growth Ex Vivo
The effect of quercitrin on human hair growth was investigated in human hair follicle organ culture model. Quercitrin significantly increased the hair growth compared with non-treated control ( Figure 10). On day 4 of culture, quercitrin increased the length of hair shaft by 62.4% (5 µM) and 70.0% (10 µM) compared with non-treated control. A positive control minoxidil showed a comparable result. The effect of quercitrin on human hair growth was investigated in human hair follicle organ culture model. Quercitrin significantly increased the hair growth compared with non-treated control ( Figure 10). On day 4 of culture, quercitrin increased the length of hair shaft by 62.4% (5 µM) and 70.0% (10 µM) compared with non-treated control. A positive control minoxidil showed a comparable result.
(a) (b) Figure 10. Effect of quercitrin on hair growth in human hair follicle organ culture. In order to evaluate the effect of quercitrin, the anagen human hair follicle were prepared and cultured for 6 days. Quercitrin was treated at concentrations of 5, 10 µ M. (a) At day 4 and 6, the cultured hair follicles were photo-documented. (b) The hair shaft growth was analyzed. Minoxidil was used as a positive control. The data represent the means of sixteen follicles. Significantly different compared with N.T (* p < 0.05, ** p < 0.01, *** p < 0.001).

Discussion
The hair follicle is one of the most energy-consuming organs in humans so energy-genesis processes are highly activated in human hair follicle cells. In DPCs, mitochondria strongly contribute to energy generation [15], and the enhancement of mitochondrial function affects hair growth in vitro and in vivo [16]. Therefore, mitochondria and their energy production are regarded as important for promoting hair growth. Figure 10. Effect of quercitrin on hair growth in human hair follicle organ culture. In order to evaluate the effect of quercitrin, the anagen human hair follicle were prepared and cultured for 6 days. Quercitrin was treated at concentrations of 5, 10 µM. (a) At day 4 and 6, the cultured hair follicles were photo-documented. (b) The hair shaft growth was analyzed. Minoxidil was used as a positive control. The data represent the means of sixteen follicles. Significantly different compared with N.T (* p < 0.05, ** p < 0.01, *** p < 0.001).

Discussion
The hair follicle is one of the most energy-consuming organs in humans so energy-genesis processes are highly activated in human hair follicle cells. In DPCs, mitochondria strongly contribute to energy generation [15], and the enhancement of mitochondrial function affects hair growth in vitro and in vivo [16]. Therefore, mitochondria and their energy production are regarded as important for promoting hair growth.
The mitochondrial energy generation is determined by mitochondrial membrane potential (∆Ψ), which in turn is tightly conjugated with positive steady state of NAD/NADH coupling. Quercitrin treatment was found to increase not only NADH production, but also mitochondrial membrane potential (Figures 1b and 2). In this context, our data strongly suggest that quercitrin could stimulate mitochondrial activities in human DPCs, which might be related to hair follicle morphogenesis [17].
The Bcl-2 family proteins were shown to manage cell senescence and administer cellular apoptosis [18]. Among the family members, Bcl2 in particular protects cells from stress challenges [19] and higher Bcl2 level prevents apoptosis [20]. In hair follicles, interestingly, the Bcl2 level is significantly higher in DPCs than other cell types [21]. In addition, high level of Bcl2 is essential for maintaining anagen and keeping DPCs function properly [22]. As mentioned above, Bcl2 level was increased and Bad level was decreased by quercitrin treatment (Figure 3). Our data demonstrate the possibility that quercitrin could maintain the hair follicle in anagen state.
In hair follicle, growth factors-such as bFGF, KGF, PDGF-AA, and VEGF-were reported to activate hair cycle and growth [8] and to be secreted by DPCs. Especially, KGF is one of the DPCs' signal that participates in instructing hair germ to proliferate and induce a new hair cycle [23]. In the present study, quercitrin was found to stimulate the production of these growth factors in cultured hDPCs (Figures 4-6). It could be predicted that the growth factors contributing to prolong the anagen and support hair growth could be supplied to hair bulb in higher level by quercitrin treatment.
As previously described, quercitrin activated Akt and Erk/CREB signaling in DPCs. The activation of CREB is strongly conjugated with mitochondrial activation, facilitating mitochondrial gene expression and cell survival through phosphor-CREB (Ser133) binding to CRE site in the mitochondrial D-loop DNA [24]. The cellular improvements are accompanied by corresponding expression of CREB-targeted genes, such as Bcl2 (cell survival) and PGC-1α (mitochondrial biogenesis) [25]. These findings are consistent with our data that quercitrin stimulated Bcl-2 expression and mitochondrial function via activation of CREB.
Furthermore, many growth factors were reported to be regulated by MAPK/CREB signaling pathway. VEGF expression is regulated by PI3K/Akt-NF-κB cascade [26] or MAPK/ERK pathway [27]. The promoter region of the KGF gene has transcriptional regulatory element for activating transcription factor (ATF) family of CREB proteins [28], and CREB activation stimulate KGF gene expression in DPCs [29]. bFGF and PDGF-AA genes are also the target of early growth response gene-1 (EGR1) which is activated by Erk phosphorylation [30].
The receptor tyrosine kinase or related non-receptor tyrosine kinases, for example Src family proteins, are possibly involved in the activation of Akt and Erk pathways and the regulation of PI3K and MAPK pathways by Src family proteins are well-established [31]. As shown in Figure 9, several receptor tyrosine kinases and non-receptor tyrosine kinases were activated by quercitrin treatment. Among those, Src family proteins-such as Csk, FRK, Hck, and SRMS-are of special interest because they, as non-receptor tyrosine kinase, not only interact with the protein-tyrosine kinase receptors at cell membrane but also participate in numerous signaling pathways. Further studies for unveiling the role of Src family proteins in the activation of DPCs by quercitrin would be challenging.
In this report, we have found that quercitrin activated the energy metabolism and enhanced the production of growth factors including bFGF and KGF. We also found that Bcl2 and Ki67 levels were upregulated and Bad level was downregulated. It seems plausible that all these stimulations are related to the activation of Akt, Erk, and CREB signal transduction pathways (Figure 7). The Erk inhibitor U0126 but not Akt inhibitor API-2 was found to inhibit the quercitrin-induced expression of growth factors and Bcl2, strongly implying the involvement of Erk activation in its mechanism of action. The cell proliferation marker Ki67 was decreased by both Erk inhibitor U0126 and Akt inhibitor API-2. In addition, several receptor tyrosine kinases and their conjugated tyrosine kinases were also activated by quercitrin as an upstream of Akt and Erk/CREB signaling ( Figure 9). Finally, quercitrin stimulated hair growth in a human hair follicle culture model, confirming our hypothesis.
We could not exclude, however, the possibility that the biological activities including hair growth promoting activity came from other constituents or unidentified ones of the HCE even though they took a little since other major components like chlorogenic acid and quercetin showed stimulatory activities in CCK-8 assay. Although quercitrin was our first choice for further investigation because of its abundance, investigating the roles of other major components of the HCE on hair growth might be interesting.
In conclusion, our data strongly suggest that quercitrin is the major active component of HCE and stimulates hair growth by enhancing the cellular energy metabolism and increasing the secretion of growth factors through the activation of MAPK/CREB signaling pathway, demonstrating the possible therapeutic potential of quercitrin for treating and/or preventing hair loss.

Cell Viability Assay
The effect of quercitrin on the viability of DPCs was examined using CCK-8 assay (Dojindo, MA, USA) and JC-1 mitochondrial membrane potential assay (Abcam, Cambridge, UK) kits following the manufacturer's protocols. For examining the cellular energy metabolism, NAD(P)H generation was measured by CCK-8 assay. The absorbance at 450 nm was read using micro-plate reader (BioTek, Winooski, VT, USA). The mitochondrial membrane potential was measured by JC-1 staining. Briefly, after quercitrin treated DPCs were stained with 1 µM JC-1 solution, fluorescence intensities from JC-1 aggregate and monomer form were measured at 590 nm (535 nm excitation) and 530 nm (475 nm excitation), respectively, with Wallac Victor3 1420 (PerkinElmer, Waltham, MA, USA). Mitochondrial membrane potential (∆Ψ) was visualized by taking fluorescence images with EVOS™ FL Auto2 Imaging System (Thermofisher scientific, Waltham, MA, USA).

Quantitative Real-Time PCR
Quercitrin was treated at concentrations of 1, 10, 100 nM and 1 µM for 24 h, with non-treated cells served as control. Total RNA was extracted using Rneasy RNA extraction kit (Qiagen Inc., CA, Germantown, USA). cDNA synthesis was performed using cDNA synthesis kit (Phillkorea, Seoul, Korea) with ThermoCycler (R&D systems, Minneapolis, MN, USA), according to the manufacturer's protocol. cDNA samples obtained from control and treated cells were subjected to real-time(RT) PCR analysis.
TaqMan One-Step RT-PCR Master Mix Reagent (Life Technologies, Carlsbad, CA, USA) was used. The PCR reactions were performed on ABI 7500 Real Time PCR system following the manufacturer's instruction. The resulting data were analyzed with ABI software (version).

Western Blot Analysis
DPCs (1 × 10 6 cells/dish) were seeded in 100 mm dishes and cultured for 24 h. Quercitrin was treated at concentrations of 1, 10, and 100 nM for appropriate time. The cells were then lysed and total cellular proteins were prepared. 50 µg protein samples were analyzed by Western blotting with corresponding antibodies; Bcl2 (1:1000