The extract of the immature fruit of Poncirus trifoliata induces apoptosis in colorectal cancer cells via mitochondrial autophagy

Sun-Young Kim,Ho-Kun Yi,Bong-Sik Yun,D-Yol L,d,Pyung Hn Hwng,d,H-Ryong Prk,Min Sun Kim,d,*

a Research Institute of Clinical Medicine of Jeonbuk National University,Biomedical Research Institute of Jeonbuk National University Hospital,Jeonju,54907,South Korea

b Department of Oral Biochemistry,Institute of Oral Bioscience,School of Dentistry,Jeonbuk National University,Jeonju,54907,South Korea

c Division of Biotechnology and Advanced Institute of Environment and Bioscience,Jeonbuk National University,Iksan-si,54596,South Korea

d Department of Pediatrics,Jeonbuk National University Medical School,Jeonju,54907,South Korea

e Department of Food Science and Biotechnology,Kyungnam University,Changwon-si,51767,South Korea

ABSTRACT The immature fruits of Poncirus trifoliate are used as a medicine for the treatment of gastrointestinal disorders, inflammation, and allergies in East Asia.However, their effect on colon cancer cells remains unclear.We investigated the effect of the immature fruit of P.trifoliate extract on colorectal adenocarcinoma.The extract of the immature fruit of P.trifoliata inhibited the proliferation of CT-26 cells compared with untreated cells and it induced autophagy and apoptosis through the protein kinase B/mammalian target of rapamycin and 5′-AMP-activated protein kinase pathways.The number of autophagic vacuoles and autophage markers increased in response to the extract.At later time-points, apoptosis increased dose/time-dependently.In CT-26 cells pre-treated a pan-caspase inhibitor prior to P.trifoliata immature fruit extract treatment,we did not find any change in pro-caspase 3 and pro-PARP levels.Additionally,in cells pre-treated autphage inhibitor,SQSTM1/p62 and LC3AB,pro-caspase 3 and pro-PARP levels did not change.Our results indicate the molecular mechanisms that the extract of the immature fruit of P.trifoliata induces apoptosis in colorectal carcinoma cells by inducing mitochondrial autophagy.In this study,we provided a draft for further investigate the use of MEPT for colorectal cancer inhibition.

Keywords:Anticancer Autophagy Apoptosis Mitochondria Poncirus trifoliata

1.Introduction

Cancer is one of the leading causes of death worldwide and colorectal cancer is one of the three most common causes of cancer death[1].Colon adenocarcinoma,one of the common tumors,occurs in the lining of human colon [2].Conventional therapy for colon cancer includes surgery, chemotherapy and radiotherapy;however, the results of these treatments are not satisfactory due to their severe side effects,low survival rate,high relapse rate and limited clinical outcomes[2,3].Therefore,it is necessary to develop novel and effective therapeutic strategies.Recent studies have focused that several bioactive natural compounds found in food have anti-carcinogenic and anti-oxidant activities and, in in vitro studies, inhibit cancer cell proliferation [4,5].Notably, the antitumor drugs derived from natural products have many advantages such as their capability to target multiple cancers, low toxicity,limited side effects and lower chances to generate drug resistance[2].

Poncirus trifoliata (L.) Raf.(also called Citrus trifoliata) is a member of the Rutaceae family that has long been used as an antiinflammatory and anti-allergic medicine to treat gastrointestinal disorders (such as indigestion and constipation) and pulmonary diseases(such as chest fullness,chest pain,bronchitis and sputum)in East Asia[6].Recent studies have shown that the pharmacological effects of P.trifoliata vary depending on the maturity and active constituents (methanol [7], ethanol [8], 25-methylmelianodiol/21β-methylmelianodiol [9] and aqueous extract [10]) of its fruit.The effects of the mature form of P.trifoliata on inflammatory and cancer cells are various [11].However, to our knowledge, there is no research on the anti-colon cancer activity of the immature fruits of P.trifoliata.Therefore, we evaluated the antitumor effect of the methanol extract of immature P.trifoliata(MEPT)fruit on the colorectal carcinoma cell lines and explored the molecular mechanisms of MEPT.This study may support for the basic study and clinical trial of MEPT.

2.Materials and methods

2.1.Cell line and cell culture

The murine colorectal carcinoma cell line CT-26 and the human colorectal carcinoma cell line HCT-116/ DLD-1 were purchased from the Korea Cell Line Bank(Seoul,Korea).The cells were maintained in DMEM(GIBCO BRL Life Technologies,Gaithersburg,MD,USA)supplemented with 10%heat-inactivated fetal bovine serum(FBS), 300 μg/mL L-glutamine, 100 U/mL penicillin G and 100 μg/mL streptomycin (all from GIBCO BRL Life Technologies).Cell cultures were incubated at 37°C under 5%CO2.Equal numbers of cells(5×104cells/well)were seeded in 24 well plates and allowed to attach,after which cells were treated with MEPT(0,1,5,10 or 20 μmol/L).Untreated control cells were treated with the corresponding volume of absolute methanol.Cells in mid-log phase cultures were used for the experiments, upon harvesting, washing (twice)and resuspension in culture medium.

2.2.Preparation of immature fruit extract of P.trifoliata

The content of immature fruits of P.trifoliata (10 kg) were extracted with 70%aqueous methanol at room temperature(20-25°C/ 68-77°F) for one day.The extract was concentrated under reduced pressure to obtain a residue, which was resuspended in distilled H2O (2 L) and extracted with hexane and ethyl acetate.The ethyl acetate-soluble portion was subjected to silica gel (230-400 mesh, Merck, Kenilworth, NJ, USA, ? 10 × 17 cm)column chromatography using a stepwise solvent system of chloroform/methanol(from 100:1 to 20:1;V/V).The active fraction was re-chromatographed on a silica gel column(? 5×11 cm)and eluted with chloroform/methanol(50:1;V/V).Active fractions were chromatographed on a Sephadex LH-20 column (? 2.5 × 80 cm, GE Healthcare,Bio-science AB,Sweden)and eluted with methanol and then on an ODS Sep-Pak C18cartridge(10 g,Alltech Associates Inc.,Deerfield,IL,USA)and eluted with 80%aqueous methanol to finally obtain a cluster of glabretal triterpenes(102 mg).MEPT is a complex of glabretal triterpenes partially purified by an isolation procedure shown in Fig.1E,not a pure compound.

2.3.Proliferation assays

CT-26 cells (1 × 103) were seeded in 96 well plates and incubated overnight.After treating the cells with various concentrations of P.trifoliata fruit extract at the appropriate time points, 10 μL of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)reagent was added to each well carefully along the sides of the well and the cells were incubated for 3-4 h.The plates were then spun down,and dimethyl sulfoxide(DMSO)was added.Absorbance at 540 nm was measured using a microplate reader (VersaMax ELISA microplate:Molecular Device,San Jose,CA,USA).

2.4.Nuclear(Hoechst)staining

CT-26 cells (2 × 104) were seeded in 24 well plates, incubated overnight and treated with P.trifoliata fruit extract for 24 h.The cells were then washed with PBS and fixed with 3.7%paraformaldehyde(PFA; Sigma Aldrich) for 10 min at room temperature.Fixed cells were washed with PBS and 200 μL of a Hoechst 33258 Staining solution(Sigma-Aldrich)was added.The cells were then incubated for 30 min at room temperature.After incubation, the cells were directly observed under a fluorescence microscope(IX71;Olympus,Center Valley,PA,USA)at 40×.

2.5.Monodansylcadaverine(MDC)staining

Autophagic vacuoles were detected with MDC by incubating the cells with MDC(50 μmol/L)in PBS at 37°C for 20 min.The cells were then washed with PBS, fixed in 4% PFA for 20 min, and observed using the IX71 microscope with an UV filter.

2.6.Protein extraction and western blot analysis

CT-26 cells were treated with 0, 1, 5, 10 μmol/L of MEPT and adriamycin 2 μg/mL [2] as positive control and were harvested and lysed in a lysis buffer(50 mmol/L Tris-HCl, 150 mmol/L NaCl,1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and protease inhibitors).Membranes and debris were pelleted upon centrifugation at 16300 g at 4°C for 30 min and the supernatants were used as whole cell extracts.The protein concentration in cell lysates was measured using a protein quantification kit from Bio-Rad.Twenty micrograms of proteins per lane was loaded onto 12%SDSpolyacrylamide gels and transferred to a PVDF membrane.Proteins in Fig.5 were separated on 4%-15%SDS-PAGE Mini-PROTEAN TGX Precast Gels(Bio-Rad Laboratories,Hercules,CA).Non-specific antibody binding sites were blocked upon incubation with 5% skim milk in Tris-buffered saline with Tween 20(TBST).The membrane was then probed with a primary antibody.The immunocomplexes were detected using Immobilon Western chemiluminescent HRP substrate (Millipore, MA, USA), and immunosignals were captured and analyzed by a Las-3000 Luminescent Image Analyzer (Fuji Film, Tokyo, Japan).The following antibodies were used: anti-caspase 3 and cleaved caspase 3 (Santa Cruz Biotech,Santa Cruz, CA, USA); poly ADP-ribose polymerase (PARP, from Cell Signaling Technology, Beverly, MA, USA), anti-actin (from Sigma Aldrich)and anti-phosphorylated(p)-AMP-activated kinase(AMPK)-a, anti-mammalian target of rapamycin (mTOR), anti-Beclin,anti-SQSTM1/p62,anti-ATG-5,anti-microtubule-associated protein I light chain-3AB(LC3AB),anti-p-protein kinase B(Akt)and anti-Akt(all from Cell Signaling Technology,Beverly,MA,USA).

2.7.Preparation of cytosolic and mitochondrial fractions

The CT-26 cells (5×106) were seeded on a 100 mm dish and cultured for 24 h.The cells were treated with P.trifoliata at the indicated times.The mitochondrial fraction was isolated from the cytosolic fraction of CT-26 cells using a mitochondria/cytosol fractionation kit (Biovision, Mountain View, CA).The cells were harvested and washed with ice-cold PBS and then suspended with cytosol extraction buffer mixture and incubated on ice for 10 min.Then, the cells were homogenized in an ice tissue grinder and centrifuged again at 4°C.The supernatant and pellet,respectively the cytosolic fraction and mitochondrial fraction, were collected and stored at -80°C.Western blots were probed with anticytochrome c and anti-second mitochondrial-derived activator of caspase (Smac)/direct inhibitor of apoptosis protein-binding protein with low pI(DIABLO)(all from Santa Cruz Biotech,Santa Cruz,CA,USA).

Fig.1.MEPT inhibits survival and proliferation of CT-26 cells.CT-26 cells(A),HCT-116 cells(B)and DLD-1 cells(C)were treated with 0,1,5,10 or 20 μmol/L of MEPT for different time-frames(0,1,2,and 3 days);*P ＜0.05,**P ＜0.01,***P ＜0.001 vs.untreated cells in same day.The proportion of surviving/proliferating cells was analyzed with the MTT assay.(D)CT-26 cells were treated with(a)0;(b)1;(c)5;and(d)10 μmol/L MEPT:the cells showed shrinkage and growth inhibition in a concentration-dependent manner (magnification, ×400).Scale bar correspond to 100 μmol/L in representative images.(E) Diagram of the procedure to prepare MEPT.Results are expressed as the mean of three independent experiments.Columns and error bars indicate mean±SD.*P ＜0.05,**P ＜0.01,***P ＜0.001 vs.untreated cells.

2.8.Flow cytometric assays for Annexin V-FITC/PI

CT-26 cells were seeded(1×106cells/well)in 6-well plates and incubated overnight.After treating the cells with various concentrations of P.trifoliata fruit extract at the appropriate time points,the apoptosis was determined using the Annexin V-FITC/PI kit(BD Biosciences,San Jose,CA,USA)according to the manufacturer’s protocol.Following treatment with MEPT at 0,1,5,and 10 μmol/L for 48 h, apoptosis was analyzed using flow cytometry (FACSCalibur;BD Biosciences).

2.9.Statistical analyses

To determine the statistical differences in the inhibition rate of CT-26 cells after treatment with different concentrations of MEPT, we used one-way analysis of variance (ANOVA).Comparisons between control and treated groups were performed using Student’s t-test.P values of less than 0.05 were considered significant.

3.Results

3.1.MEPT inhibits the growth and proliferation of colorectal carcinoma cells

We first examined the effect of MEPT on the growth of CT-26,HCT-116 and DLD-1 cells.We used different concentrations of MEPT (0, 1, 5, 10, and 20 μmol/L) for different time-lengths (0, 1,2,and 3 days).As shown in Fig.1A-1C,the growth of CT-26,HCT-116 and DLD-1 cells significantly decreased upon treatment with MEPT in a dose-dependent manner over the three-day period of the experiment (P ＜0.05).In CT-26 cells, after treatment for 24 h, the highest growth inhibition(58.40%±4.74%)occurred at 10 μmol/L,but the inhibitory effects at 10 and 20 μmol/L did not differ significantly(Table 1).After treatment with 20 μmol/L MEPT for 48 h,CT-26 cell growth was approximately 74.14%±1.89%of the control as the most marked inhibition rate.After 48 h of treatment,CT-26 cells shrank in a dose-dependent manner and showed a significant decreased in the number of live cells(Fig.1D).The preparation method for this compound is presented in Section 2(Fig.1E).

Fig.2.MEPT inhibits CT-26 cell growth and proliferation via the suppression of the AkT/mTor pathway.(A)Cells were treated with MEPT at different concentrations for 6 h.Images show Western blots relative to the expression of P-Akt,P-AMPKα and P-mTOR in CT-26 cells.(B)Western blot showing the expression of P-AMPKα and P-mTOR in cells treated with different concentrations of MEPT for 8 and 12 h.Actin was used as loading control.The graph is representative of three independent experiments.Columns and error bars indicate mean±SD.*P ＜0.05,**P ＜0.01 vs.untreated cells.

Table 1 Inhibition rate of CT-26 cells after treatment with different concentrations of MEPT(n=6).

3.2.MEPT inhibits the growth and proliferation of CT-26 cells via the Akt/mTOR and AMPKα pathway

To understand the molecular mechanism of MEPT inhibitory effect on CT-26 cell growth and proliferation,we performed western blotting assays.We found that the phosphorylation of Akt and mTOR significantly decreased,while that of AMPKα increased in CT-26 cells treated with increasing concentrations of MEPT(Fig.2A).Additionally, the levels of the phosphorylated AMPKα increased in a time-dependent manner(Fig.2B).

3.3.MEPT induces apoptosis in CT-26 cells

Increased doses of MEPT led to nuclear condensation and fragmentation compared to the control group by Hoechst 33342 stain(Fig.3A).Additionally,we found that the expression of pro-caspase 3 and pro-PARP was significantly downregulated by MEPT in a dosedependent manner, and that cleaved caspase 3 was upregulated similar to CT-26 cells with adriamycin treatment (Fig.3B).Fig.3C shows that MEPT treatment was accompanied by the upregulation of cytochrome c and Smac/DIABLO in the cytosol and their downregulation in mitochondrial pellets.Presence of cytochrome c and Smac/DIABLO in the cytosol were strongly observed,beginning 12 h after treatment with MEPT.Furthermore,the expression of cleaved caspase 9 was promoted in CT-26 cells.MEPT treatment of cells increased apoptotic cell populations compared with untreated control as determined with flow cytometric analysis (Fig.3D).These results indicate that MEPT induces apoptosis in CT-26 cells in a dose-dependent manner.

3.4.MEPT induces autophagy in CT-26 cells

MDC staining showed the increase in the number of MDClabeled vesicles(autophagic vacuoles)upon MEPT treatment for 8 h in a dose-dependent manner (Fig.4A).Notably, the expression of Beclin-1, SQSTM1/p62, ATG-5 and LC3AB, autophagy-related key proteins,was significantly upregulated after MEPT treatment,similar to adriamycin treatment, and was stronger after 8 h of treatment than after 12 h (Fig.4B), suggesting that MEPT induces autophagy.

3.5.MET promotes CT-26 cells apoptosis via the mitochondrial pathway

We found that MEPT treatment decreased the levels of procaspase 3 and pro-PARP, and increased those of SQSTM1/p62 and LC3AB in a time-dependent manner (Fig.5).To further investigate MEPT-induced apoptosis, we pre-treated CT-26 cells with either carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk; a pan-caspase inhibitor) or 3-methyladenine(3-MA;a type III phosphatidylinositol 3-kinases[PI3K] inhibitor) for 1 h prior to MEPT treatment.We found no changes in pro-caspase 3 and pro-PARP levels upon Z-VAD-fmk treatment and no changes in SQSTM1/p62 and LC3AB,pro-caspase 3 and pro-PARP levels upon 3-MA treatment.

Fig.3.The effect of MEPT on apoptosis of CT-26 cells.(A) CT-26 cells were stained with Hoechst 33342 to assess cellular apoptosis and examined under a fluorescent microscope(magnification,×40);(a)0 μmol/L;(b)1 μmol/L;(c)5 μmol/L;and(d)10 μmol/L MEPT.Scale bar corresponds to 2 μm in representative images.(B)Pro-caspase 3,cleaved caspase 3,and pro-PARP expression in CT-26 cells,treated with increasing concentrations of MEPT.(C)Smac/DIABLO,cytochrome c,and cleaved caspase 9 presented at 3,6,12,and 24 h after MEPT treatment(10 μmol/L).(D)CT-26 cells were stained with ANNEXIN V and analyzed by flow cytometry.The right bottom quadrant represents Annexin V-stained cells(early-phase apoptotic cells).The top right quadrant represents PI-and Annexin V-stained cells(late-phase apoptotic cells).Actin was used as loading control.The graph is representative of three independent experiments.Columns and error bars indicate mean±SD.*P ＜0.05,**P ＜0.01 vs.untreated cells.

Fig.4.Effect of MEPT on autophagy in CT-26 cells.(A) Representative images of MDC staining of CT-26 cells following treatment for 8 h with MEPT at the following concentrations:(a)0 μmol/L;(b)1 μmol/L;(c)5 μmol/L;and(d)10 μmol/L.Punctate fluorescence in the cytoplasm indicates formation of autophagic vacuoles(magnification,×20).Scale bar corresponds to 25 μm in representative images.(B)Western blot showing the expression of Beclin-1,SQSTM1/p62,ATG-5,and LC3AB in CT-26 cells treated with different concentrations of MEPT for 8/12 h and adriamycin 2 μg/mL of 8 h as a control.Actin was used as loading control.The graph is representative of three independent experiments.Columns and error bars indicate mean±SD.*P ＜0.05,**P ＜0.01 vs.untreated cells.

Fig.5.Western blot analysis of apoptosis and autophagy markers in MEPT-treated CT-26 cells.CT-26 cells were incubated with 10 μmol/L Z-VAD-fmk(pan-caspase inhibitor)or 5 mmol/L of 3-MA(autophagy inhibitor)for 1 h,and then with MEPT(10 μmol/L)for 12 and 24 h.SQSTM1/p62,LC3AB,pro-caspase-3,pro-PARP,and β-actin were then assessed.Control cells were not treated with Z-VAD-fmk or 3-MA.Actin was used as loading control.The graph is representative of three independent experiments.Columns and error bars indicate mean±SD.*P ＜0.05,**P ＜0.01 vs.untreated cells.

4.Discussion

The fruits of P.trifoliata have various phytotherapeutic activities and have therefore been used for long time in Eastern medicine.However, the underlying anticancer mechanisms of the immature fruit of P.trifoliata in colorectal carcinoma are not yet clear.Recently, several studies have shown that the pharmacological effects of these fruit depend on their maturity.Specifically, the extracts from the mature fruit have been reported to be effective on cancer cells or tissues (human breast adenocarcinoma [7,12],human hepatocellular carcinoma cells [13,14], mouse lymphoma[9] and human promyelocytic leukemia [10]).On the other hand,the effects of the immature fruit of P.trifoliata have been reported in rat benign prostatic hyperplasia[8],human oral cancer cell[15]and rat hepatoma[16].

In this study,to check the response for MEPT in colorectal carcinoma cells,we treated three different types(the murine colorectal carcinoma cell line CT-26 and the human colorectal carcinoma cell line HCT-116/DLD-1)with MEPT.CT-26 is one of the most widely used models of syngeneic mouse tumors that share molecular properties with aggressive,undifferentiated and refractory human colorectal cancer cells [17].In vitro models with HCT-116/ DLD-1 human colon cancer cell lines were commonly used to study drug response and cancer biology[18].In this study,the most sensitive inhibitory effect was achieved against CT-26 cells after MEPT treatment.In CT-26 cells treated above 5 μmol/L for 48 h, the number of live cells was significantly reduced(under 50%),and 20 μmol/L MEPT or above resulted in significant cell death.Therefore,we performed experiments with MEPT concentrations up to 10 μmol/L in CT-26 cells.The authors have reported the inhibition of cell growth and proliferation, and the promotion of cell death by the extract from the immature fruit of this plant on the murine colorectal carcinoma cell line CT-26 and the human colorectal carcinoma cell line HCT-116/DLD-1.Additionally,we found that MEPT treatment induces an early mitochondrial autophagic response followed by apoptosis in CT-26 cells.

Studies on autophagy and apoptosis in cancer cells have reported various results, and these two pathways are thought to cooperate, antagonize, or aid each other.In some reports,autophagy and apoptosis have been correlated with mTOR [19]and Beclin-1 [20].Autophagy plays a role as a promoter or suppressor of cancer formation, depending on the type and stage of the cancer,as well as the underlying genetic factors.For example,in studies on breast carcinoma, inhibition of autophagy has been shown to activate apoptosis [21,22], whereas contrasting results have been reached in colon carcinoma [23].In the present study,we demonstrated that MEPT has an anticancer effect similar to adriamycin on colon cancer.Specifically, we showed that MEPTinduced autophagy up to 12 h of treatment and enhanced apoptosis after 12 h.

Mitochondria-mediated apoptosis led to the activation of several proteins (Smac/DIABLO, cytochrome c,Bcl-2, cleaved caspase 9,etc.)associated with programmed cell death[24]and promoted the release of Smac and cytochrome c from the mitochondria to the cytoplasm[25].Cytochrome c forms the apoptosome complex,induces the activation of initiator caspase-9, and proceeds to the cleavage of pro-caspase-3[25].We detected increased contents of cytochrome c and Smac/DIABLO in the cytosol after 12 h of treatment.Similar effects were observed in the activation of caspase-9 in a time-dependent manner.In the MEPT-treated colorectal cancer cells, we observed the activation of both caspase-9 and caspase-3 with a successive PARP cleavage as a result of the release of apoptosis activators.

The PI3K/AKT/mTOR pathway has an important role in the activation of autophagy [26,27]; specifically, its inhibition promotes autophagy [28].mTOR inhibits autophagy under basal conditions and serves as a negative regulator of the autophagy machinery[29].mTOR levels are higher in colorectal cancer than in adjacent tissues[30],giving poor prognosis for stage II colorectal cancer[31].The mTOR signaling pathway is regulated by several upstream signals, including the AMPK and PI3K/Akt [32] pathways.Recently,AMPK signal activation[21]and PI3K/Akt signal inhibition[11,33]have been shown to enhance colorectal cancer cell apoptosis.In this study, we found that the expression of PI3K, p-AKT, and p-mTOR in CT-26 colorectal carcinoma cells decreased dose-dependently upon MEPT treatment, and the expression of p-AMPK increased dose-and time-dependently after MEPT treatment.

In addition, we investigated the expression of factors involved in autophagy initiation and autophagosome formation (Beclin-1,SQSTM1/p62, ATG-5, and LC3AB).We found that the expression of these factors increased early after MEPT treatment.However,the expression of apoptosis-related factors(pro-caspase 3 and pro-PARP) decreased at later time points.Additionally, we confirmed these findings upon treatment of the cells with 3-MA(an autophagy inhibitor), which negatively interfered with both autophagy and apoptosis.Therefore, we conclude that, MEPT induces autophagy,which,in turn,enhances cell death.

5.Conclusion

To our knowledge, our study describes for the first time that the extract of immature P.trifoliata triggers autophagy and apoptosis in CT-26 colorectal carcinoma cells.MEPT contributes to tumor suppression by inducing autophagy and apoptosis and inhibiting the proliferation of colorectal carcinoma cells via the activation of AMPK and the inhibition of the PI3K/Akt/mTOR pathways.Further,these effects continue through autophagy,mitochondrial signaling,and the release of cytochrome c and Smac/DIABLO protein with subsequent activation of the apoptotic process.Autophagy,in turn,induces cell death.Based on these results, we believe that MEPT could be tested as a useful natural compound for the treatment of colorectal carcinoma.

Declaration of Competing Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was supported by Fund of Biomedical Research Institute,Jeonbuk National University Hospital.