Anti-proliferation , pro-apoptosis and anti-migration 2 effects of ginkgolic acid C 13 : 0 isolated from ginkgo 3 biloba exocarp in MCF-7 and 4 T-1 breast cancer cells 4

Ginkgolic acids (GA) have been reported to exhibit anticancer properties, however, 15 the mechanisms remain unclear. This study aims to investigate the mechanisms of GA C13:0 16 that was isolated from Ginkgo biloba exocarp (GBE) for anti-proliferation, pro-apoptosis and 17 anti-migration effects in human MCF-7 and mouse 4T-1 breast cancer cells. The cytotoxic 18 effect, apoptosis induction and migration inhibition were measured using MTT, TUNEL and 19 Wound healing assays. The expression of mRNA and protein were determined using qPCR 20 and Western blot. Our results showed that no cytotoxicity was found at concentrations of 21 C13:0 below 100μM. The effects of GA C13:0 was further demonstrated by up-regulation of 22 the Bax/Bcl-2 apoptosis pathway and the expression of Apaf-1 protein in the mitochondria. In 23 addition, GA C13:0 also suppressed cell migration and epithelial to mesenchymal transition 24 (EMT) with the increase of E-cadherin expression accompanied by the decrease of Snail, 25 MMP-2, MMP-9 and Vimentin expression. Moreover, GA C13:0 induced cytochrome P450 26 (CYP) 1B1 expression in aryl hydrocarbon receptor (AhR) pathway. Notably, the 27 up-regulation of CYP1B1 also might play a pivotal regulatory role in mitochondrial and EMT 28 pathways in MCF-7 and 4T-1 cells. Our results may have implications for the development of 29 anticancer agents containing GA as functional additives. 30


Introduction
Breast cancer is the leading common neoplasm in women and cause significant rate of morbidity and mortality around the world [1].The initiation and progression of breast cancer are ascribed to approximately 80% of external environmental factors, involving radiation, carcinogens, diet and so on.However, changes in dietary consumption appear to exert a great effect [2].Some studies have shown that women consuming a high-fat diet are prone to enhance the risk of breast cancer compared with that in a low-fat diet in the Asian countries [3].Epidemiological studies indicate that more intakes of plant food reduce the risk of breast cancer [4,5].More and more studies also suggest that the prevention of cancer are strongly associated with the ingestion of natural bioactive compounds that exist principally in the organism of edible plants such as fruits, seeds and leaves [6,7].Therefore, it is a prior necessity seeking the natural agents from edible plant for anticancer strategies [8].
Ginkgo biloba (GB) is an ancient edible and medicinal plant and has been cultivated extensively in the Asian countries [9].The valuable organism of GB mainly includes leaves and fruits.EGB761 derived from Ginkgo leaves have reported to have efficient anticancer activity in the global drug development [10].The fruits of GB are known as ginkgo nuts for functional foods, which have been used for the treatment of malignant neoplasm in the traditional Chinese medicine [11].About 75% of the total fruit weight of the GBE, which are often treated as discarded waste have been reported to provide significant anticancer effects in vitro [12].In order to develop the resources of Ginkgo biloba more fully, GBE pharmacological effects have been exploited and attributed to the composition of flavonoids (kaempferol, quercetin and isorhamnetin) and terpene trilactones (bilobalide and ginkgolide) as well as phenolic acids (mainly GA).GA are mixtures of GA homologues (C13:0,C15:1,C17:2,C15:0,C17:1), which are abundant in GBE exhibit a wide range of interesting pharmacological effects, involving antioxidant [13], anti-inflammatory [14], anti-HIV [15] , anti-bacterium [16] and anticancer activities [17,18].Unfortunately, GA have been reported to contain toxic and allergenic potential [19].Thus, the maximal concentration of GA monomer compound has been restricted to 5ppm for application in the National Pharmacopoeia monographs.However, studies have shown that GA C13:0 at the concentrations of 25µM (8ppm) and 100µM (32ppm) are used for inhibiting invasion and migration in MCF-7 and MDA-MB-231 (human breast cancer cells) [20].GA C15:1 and C17:1 at 100µM (25ppm) and 40µM (11ppm) are used for pro-apoptosis and anti-migration in A549 and H1299 (human lung cancer cells) [17], and Tac8113 (human tongue squamous carcinoma cells) [21], suggesting that the maximal concentration of GA may be reassessed for pharmacological effects on anticancer activity.GA C13:0 belongs to the alkyl phenols and offers a higher suppressed migratory behavior compared with the other GA homologues in MCF-7 breast cancer cells [20].
Moreover, GA C13:0 also inhibits migration through suppression of NEMO sumoylation and NF-κB activity in MDA-MB-231 breast cancer cells [20].However, no studies have provided conclusive mechanisms of action regarding the effects of GA C13:0 in human MCF-7 and mouse 4T-1 breast cancer cells.In our previous study, the fruit of GB extract showed inhibited proliferation in MCF-7 and MDA-MB-231 breast cancer cells and that accompanied by the enhancement of CYP1B1 mRNA expression [22].In this study, we isolated GA C13:0 from the GBE and investigated the anti-proliferation, pro-apoptosis and anti-migration effects as well as the possible pivotal regulatory role of CYP1B1 in breast cancer cells.

Isolation and comparison of GA C13:0 in MCF-7 and 4T-1 cells
In this study, the MTT assay guided isolation of GA C13:0 from GBE in MCF-7 and 4T-1 cells was evaluated for the anticancer effects.As shown in Figure .1A,the ethanol extract of GBE showed the stronger anti-breast cancer activity with the IC50 value of 283.05 and 179.31µg/mL compared with the water extract with the IC50 value of 691.86 and 440.32µg/mL in MCF-7 and 4T-1 cells, respectively.Thus, ethanol was chosen as the optimal solvent for further extraction.Among the four fractions of the ethanol extract, Fr.1, the fraction of petroleum ether, exhibited the higher cytotoxicity with the IC50 values of 140.21 and 117.23µg/mL (Figure .1B),compared with the other fractions and selected for purification.Isolation of Fr.1 afforded nine subfactions.Subfr.4 exhibited the higher cytotoxicity with the IC50 values of 108.62µg/mL and 95.77µg/mL and selected for further purification (Figure .1C).Subfr.4 divided into five fractions.Subfr.4-2, the ingredient was GA C13:0 (Figure .1F,S1 and S2), which was identified using HPLC-MS presented the highest cytotoxicity with the IC50 values of 53.82µg/mL and 42.51µg/mL in MCF-7 and 4T-1 cells, respectively (Figure .1D).These results suggested that the GA C13:0 might be responsible for the cytotoxic effect of the GBE extract and the other fractions and subfractions with lower cytotoxicity were not further isolated.Some studies demonstrate that other phenolic acids with different side chain structures, such as gallic acid, chlorogenic acid, caffeic acid and salicylic acid have significantly cytotoxic effects in MCF-7 and MDA-MB-231 cells, but through different mechanisms of inhibition [23,24].Besides GA C13:0, there are many special active compounds of flavonoids and terpenoids in GBE.To further determine the predominant effect of GA C13:0, the comparison of GA C13:0 and the special standard compounds, involving ginkgolic acids (GAs), quercetin, kaempferol, isorhamnetin, bilobalide and ginkgolide B were examined in MCF-7 and 4T-1 cells.The lethal effect of these compounds at the concentration of 400µM was evaluated using trypan blue dye exclusion assay.As shown in Figure .2A and 2B, the lethal effect of GA C13:0 was higher than GAs group insignificantly, with cell death rate of 90.71%, 87.34% and 95.57%, 91.22% in MCF-7 and 4T-1 cells, respectively, and had more significant difference compared with the control and other active groups.
Therefore, our results demonstrated that the GA C13:0 had a powerful cytotoxic effect in MCF-7 and 4T-1 cells and represented a source of excellent natural anti-breast cancer compound in GBE.

Assessment of cytotoxicity and anti-proliferation of GA C13:0 in MCF-7 and 4T-1 cells
To analyze the cytotoxic and anti-proliferative effects in the MCF-7 and 4T-1 cells, the cells were treated with GA C13:0 at various concentrations for 24h and the cell viability was determined using MTT assay, trypan blue dye exclusion assay and anti-proliferative assay.As shown in Figure .1E,the cytotoxic effect of GA C13:0 was more significant at concentrations over 200µM and the cell viability rate were less than 40% compared with the control group in MCF-7 and 4T-1 cells.However, no significant cytotoxicity was noted at concentrations of GA C13:0 below 100µM and the cell viability rate were more than 85% compared with the control group.Additionally, we also observed that the death rates were 91.45% and 95.02% at concentration of 400µM, 59.75% Virtually, in the National Pharmacopoeia monographs, the concentration of GA C13:0 has been restricted to 5ppm (16µM) for pharmaceutical agents.Studies have reported that the concentrations of GA C13:0 over 25µM with no cytotoxicity are used for MCF-7 and MDA-MB-231(human breast cancer cells) [20], A549 (human lung cancer cells) and SMMC7721 (human liver cancer cells) [25].However, our results suggested that no cytotoxic effect of GA C13:0 was observed at concentrations below 100µM and the anti-proliferation of GA C13:0 appeared increase in a dose dependent manner compared with the control group in MCF-7 and 4T-1 cells.

Apoptosis induction of GA C13:0 in MCF-7 and 4T-1 cells
After incubation with GA C13:0 at concentrations ranging from 200µM to 25µM for 24 h, TUNEL and DAPI staining assay was performed to determine whether GA C13:0 could induce apoptosis in MCF-7 and 4T-1 cells.The expression of related proteins in Western blot analysis showed that the concentration of GA C13:0 at 100µM significantly induced Bcl-2 protein expression level decrease, the Bax and Apaf-1 protein expression levels increase in MCF-7 and 4T-1 cells (Figure .4C).Apoptosis, a mode of programmed cell death, has a strict and complex signal transduction pathway and is induced through the activation of either extrinsic receptor mediated apoptosis pathways or intrinsic mitochondria-dependent pathways [27,28].Bax, a pro-apoptotic protein, has been regulated by an anti-apoptotic protein of Bcl-2, which is located in the outer mitochondrial membrane.
Mitochondria-associated Bcl-2 suppresses Bax/Bak oligomerization and the formation of pores through which cytochrome C can be released into the cytosol, and initiate with Apaf-1 formation of the apoptosome [29].The Bax/Bcl-2 ratio and Apaf-1 expression increase have been considered as an indicator of apoptosis in the intrinsic mitochondria death pathways [30,31].Overall, our results illustrated that the concentration of GA C13:0 at 100µM significantly induced apoptosis in MCF-7 and 4T-1 cells through increased the Bax/Bcl-2 ratio and Apaf-1 protein expression levels in mitochondrial death pathway.

Anti-migration activity of GA C13:0 in MCF-7 and 4T-1 cells
To assess the effect of GA C13:0 on the migration activity of MCF-7 and 4T-1 cells with no anti-proliferative effect, wound healing assay was performed and the expression of related proteins, involving Snail, MMP-2, MMP-9, Vimentin and E-cadherin were examined.As shown in Figure .5Aand 5B, GA C13:0 inhibited migration significantly at concentrations ranging from 50 to 25µM for 48h compared with the control group.
Western blot analysis showed that GA C13:0 (50µM) decreased the expression of Snail, MMP-2, MMP-9, Vimentin, and increased E-cadherin expression significantly in MCF-7  In the process of cancer metastasis, EMT is the initial process in cancer progression and is triggered by the expression changes of genes of epithelial and mesenchymal specific in cancer cells.The occurrence of EMT allows epithelial cells lose their adhesion and acquire the capacity of invasion and migration [32].More evidence for the EMT is regulated mainly by several epithelial proteins (E-cadherin and keratins) and mesenchymal proteins (vimentin and MMPs).MMP-2 and MMP-9 are closely associated with the processes of invasion and migration in various cancer cells [33].Thus, the decrease of E-cadherin expression and the increase of Vimentin, MMP-2 and MMP-9 expression are representative characteristic of EMT [34].Studies also have reported that GA C13:0 exerts anti-EMT effects by inhibited the NF-κB activity and down-regulation of uPA, PAI-1 and MMP-9 expression on MDA-MB-231 cells [20].GA C15:1 also suppress migration and EMT transition by decreased protein expression of Snail, Vimentin, MMP-2 and MMP-9 on A549 and H1299 cells [17].Therefore, our data suggested that the concentration of GA C13:0 at 50µM suppressed migration and EMT effects through up-regulated E-cadherin and down-regulated Snail, MMP-2, MMP-9 and Vimentin protein expression in MCF-7 and 4T-1 cells.

Effects of CYP1A1, CYP1B1 and AhR expression of GA C13:0 in MCF-7 and 4T-1 cells
To further ascertain the mechanism of inhibition effect of GA C13:0 in MCF-7 and 4T-1 cells, after treatment with GA C13:0 at 50µM for 0, 6, 12 and 24h, the mRNA and the related AhR is a ligand-activated transcriptional factor, which binds to specific aromatic hydrocarbons regulates the expression of downstream genes including CYP1 family members: CYP1A1 and CYP1B1 [35].Distinct from CYP1A1, which is detected in only a few tumors, CYP1B1 has been proposed as a potential target on the basis of investigation that this enzyme has catalyzed activation of various compounds to metabolites, which induce DNA damage have anticancer activity [21].CYP1B1 has catalyzed 17b-estradiol to 4-hydroxyestradiol metabolite, which induce cellular damage has been implicated in the initiation stages of mammary tumors [36].CYP1B1 and CYP1A1 also are shown to metabolise eupatorin to the flavone cirsiliol and catalyze conversion of diosmetin to flavone luteolin, which exhibits inhibitory action in MDA-MB-468 breast cancer cells [37,38].In our study, the results indicated that GA C13:0, which belongs to the aromatic hydrocarbons could up-regulate the expression of AhR and induce CYP1B1 increase in AhR signaling pathway in MCF-7 and 4T-1 cells.However, in contrast to CYP1B1, the expression of CYP1A1 had no significant changes compared with the control group.The reason for these may be attributed to the alkyl phenolic structure of GA C13:0.Moreover, the anticancer activities of metabolites, which were generated from GA C13:0 due to CYP1B1 catalytic role are required further studies to be confirmed.
Additionally, CYP1B1 has supposed to play a key role by targeting multiple components of the cell apoptosis and metastatic pathways that are involved in many tumor cells, including prostate cancer [39], endometrial cancer [40] as well as breast cancer [41].CYP1B1 plays a vital regulatory role to affect EMT and activate Wnt/β-catenin signaling via regulation of Snail, Zeb2,Twist1, MMPs, E-cadherin and Vimentin in MCF-7 breast cancer cells [41].CYP1B1 also has induced apoptosis through regulate the expression of caspase-1, tumor necrosis factor receptor superfamily, member 9 (Tnfrsf-9), CD27 molecule as well as Harakiri, which is Bcl-2 interacting protein on PC-3 prostate cancer cells [39].In this study, our results indicated that the

Chemicals, standards and reagents
HPLC-grade methanol was purchased from TEDIA (Fairfield,OH, USA).All other solvents were of analytical grade.Column chromatography was performed over silica gel Anti-β-actin and other secondary antibodies were obtained from En Jing (Beijing, China).

Cell lines and culture conditions
The MCF-7 and 4T-1 breast cancer cells were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China).The cells were carrying green fluorescent protein (GFP) in the cytoplasm.The cells were grown and maintained in RPMI-1640 cell culture medium supplemented with 10% fetal bovine serum and 1% antibiotics in a tissue culture apparatus with atmosphere at 37˚C containing 5% CO2.All experiments were repeated three times independently and the representative images were shown.

Cytotoxic assay (MTT)
The cytotoxic effect of extracts, fractions, subfractions, subfraction-4 and GA C13:0 were measured using MTT.The MCF-7 and 4T-1 cells (1×10 5 /well) were cultured in sterile 96 well plates and treatment with samples containing different concentrations.After the plate was incubated for 24h, 10µL/well MTT reagents were added to each well and incubated for 4h.The medium was carefully removed and formazan crystals were dissolved in 110µL of DMSO.
The optical density was determined at 490 nm with SpectraMax190 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).The 50% inhibitory concentration (IC50) was defined as the most cytotoxicity.

Trypan blue dye exclusion assay
This method was done as demonstrated previously [42].Briefly, cells were plated into 96 well plates (1×10 5 /well).After treatment with the samples for 24h, cells were harvested washing with phosphate-buffer-solution (PBS) to remove debris and treated with 1X trypsin/EDTA solution.The suspended cells were centrifuged for 5 min at 1200 rpm and then resuspended the cells in 100µL PBS to obtain single-cell suspension.The cell suspension was loaded into the hemocytometer with the trypan blue (0.4%, 100µL), which was to stain dead cells.The cell death rate was determined from the ratio of the number of nonviable (stained) cells to the total number of cells.

Anti-proliferative assay
The GA C13:0 was diluted in the medium with 0.05% DMSO and filtered with pore of 0.22µm and then stored in the dark at -20℃ until use.Cells were treated with the samples at various concentrations ranging from 400 to 50µM for 24h, and the control containing the medium with 0.05% DMSO.Cells percent survival (%) were counted and images were captured on inverted fluorescence microscope (Eclipse 90i, Nikon, Japan).

TUNEL assay
The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay for detection of apoptosis was performed by using Apoptosis Detection Kit (Millipore, Chemicon®, USA).According to the manufacturer's instructions, Cells were plated in 96 well plates (1×10 5 /well) and treated with GA C13:0 for 24h.Cells washed with PBS and fixed in 2% paraformaldehyde at 4°C for 30 min.Fixed cells were then permeabilized in 0.1% Triton X-100 and labeled with fluorescein 12-d UTP using terminal deoxynucleotidyl transferase.Apoptotic cells were observed using inverted fluorescence microscope.

Wound healing assay
Cells were plated into 24 well plates (5×10 5 /well) with serum-free medium overnight and 1ml pipette tip was used to vertically scratch the surface of cells at the center of each well.The wells were washed with PBS and images were taken under inverted bright microscope at 0h time point, then GA C13:0 was added to various groups and images were taken at 48h time point post-scratch at the same positions.The image software was used to calculate the percentage of gap closures.

Quantification of reverse transcription polymerase chain reaction (qPCR)
Total RNA was extracted using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and reverse transcription into the first-strand complementary DNA (cDNA) using the reverse transcriptase kit (Ta Ka Ra, China) according to the instructions.Primer pairs for CYP1A1, CYP1B1 and AhR are listed in

Western blot assay
Western blotting was done as described previously [43].Briefly, proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).The SDS-PAGE gel was transferred to polyvinylidene fluoride (PVDF) membranes.The membranes were incubated with different primary antibodies, followed with secondary antibody (horseradish peroxidase conjugated anti-rabbit IgG).Antibody binding was detected by chemoluminescence regent.Bands were scanned and quantified by automatic chemiluminescence image analysis system (Tanon Science and Technology Co., Ltd., Shanghai, China).All experiments were conducted in triplicate.

Statistical analysis
Data were expressed as the means ± standard deviation (SD) of three replications and were evaluated by one-way analysis of variance (ANOVA).Statistical analysis and bar graphs were performed by the GraphPad Prism version 5.0 (Graph PadTM Software, SanDiego, CA, USA).*P <0.05 and **P <0.01 were regarded as significant.

Conclusion
In summary, the present study demonstrated that the natural compound of GA C13:0, which was isolated from the GBE by cytotoxicity guided isolation exhibited anti-proliferation, pro-apoptosis and anti-migration effects in MCF-7 and 4T-1 cells.Meanwhile, the assessment of cytotoxicity of GA C13:0 indicated no cytotoxicity was found at concentrations below 100µM.Furthermore, the inhibition mechanisms of GA C13:0 that were involved with up-regulating Bax/Bcl-2 ratio and the Apaf-1 protein expression in the mitochondrial apoptosis pathway, down-regulating Snail, MMP-2, MMP-9, Vimentin and up-regulating E-cadherin protein expression to reveal anti-EMT effects and up-regulating CYP1B1 and AhR protein expression in the AhR pathway.Notably, the possible associations among the mitochondrial apoptosis pathway, EMT pathway as well as the vital regulatory role of CYP1B1 in MCF-7 and 4T-1 cells are needed further studies to be confirmed.Our results indicate that the more concentrations of GA maybe beneficial to apply in the natural anticancer agent research field.
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/link.

Figure 2 .
Figure 2. The comparison of ginkgolic acid (GA) C13:0 and the active compounds in MCF-7 and 4T-1 cells.The effect of GA C13:0 and the standards of Ginkgolic acids (GAs), Kaempferol, Quercetin, Isorhamnetin, Ginkgolide B and Bilobalide were examined in MCF-7 and 4T-1 cells at concentration of 400µM for 24h.Cell death (blue) was observed by trypan blue dye exclusion assay (A).The cell death rate was determined from the ratio of the number of nonviable (stained) cells to the total number of cells (B).The data were represented as the mean ± SD from three independent experiments.(*P < 0.05 and **P < 0.01 compared with 0.05%DMSO-treated control cells).

Figure 3 .
Figure 3. Assessment of cytotoxicity and anti-proliferation of GA C13:0 in MCF-7 and 4T-1 cells.Treatment with GA C13:0 in MCF-7 and 4T-1 cells at concentrations ranging from 400 to 50µM for 24h.Cell death (blue) was observed by trypan blue dye exclusion assay (A).Cell death rate was determined from the ratio of the number of nonviable (stained) cells to the total number of cells (B).The surviving cells with green fluorescent protein (GFP) were observed by inverted fluorescence microscope (C).The percent survival of cells were performed from the ratio of the number of GFP cells to the total number of cells (D).The data were represented as the mean ± SD of three independent experiments.(*P < 0.05 and **P < 0.01 compared with 0.05%DMSO-treated control cells).

PreprintsFigure 4 .
Figure 4. Apoptosis induction of GA C13:0 in MCF-7 and 4T-1 cells.Effect of GA C13:0 on apoptosis in MCF-7 and 4T-1 cells by DAPI, TUNEL and Western blot analysis.Treatment with GA C13:0 at concentrations ranging from 200 to 25µM for 24h, apoptotic cells were observed by DAPI and TUNEL assay using inverted fluorescence microscope.Red arrows showed the apoptotic cells and white arrows showed the unstained nuclear of cells following toxic insult of the high concentration of GA C13:0 at 200µM (A).The apoptotic cells were determined from the ratio of the number of apoptotic cells to the total number of cells.(B).Western blot analysis was detected for the expression of Bax, Bcl-2 and Apaf-1 after treatment with GA C13:0 at 100µM for 24h (C).The results were represented as the mean ± SD from three independent experiments.(*P < 0.05 and **P < 0.01 compared with 0.05%DMSO-treated control cells).

Figure 5 .
Figure 5. Anti-migration activity of GA C13:0 in MCF-7 and 4T1 cells.Wound healing assay was performed for evaluating the anti-migration effect of GA C13:0.The confluent monolayers of MCF-7

Figure 6 .
Figure 6.Effects of GA C13:0 on the modulation of CYP1A1, CYP1B1 and AhR expression in MCF-7 and 4T1 cells.MCF-7 and 4T1 cells were treated with GA C13:0 at 50µM for 0, 6, 12 and 24h, the mRNA was extracted and the CYP1A1, CYP1B1 and AhR were examined by qPCR (A).Western blot analysis was detected for the expression of CYP1A1, CYP1B1 and AhR, after treatment with GA C13:0 at 50µM for 24h (B).The results were represented as the mean ± SD from three independent experiments.(*P < 0.05 and **P < 0.01 compared with 0.05%DMSO-treated control cells).

Figure 7 .
Figure 7. Scheme for the possible mechanisms of CYP1B1 action.Diagram showed the mechanism of GA C13:0 on the multiple pathways and the possibility of associations among the mitochondrial

Table 1 .
The PCR reactions were performed using a Preprints (www.