chlorogenic acid is the ester of caffeic acid and quinic acid in shikimate pathway , which is commonly found in some plants , such as honeysuckle , cortex eucommiae , semen coffea arabica , and green tea .
chlorogenic acid has antibacterial , antiviral , clear free radicals , and antitumor effects . in recent years , the effective anticancer activity and low toxicity of chlorogenic acid
kurata et al .   showed that the inhibition of tumor cell proliferation effect of chlorogenic acid was enhanced with increasing dose ; they speculated that this inhibition of tumor cell proliferation may be obtained by enhancing the activity of the dna ladder and caspase-3 as well as increasing the expression of c - jun .
showed that the in vitro experiments show that the anticancer mechanism of cga contains inhibition of cell growth , regulation of cell cycle , and induction of apoptosis pathways , such as ( 1 ) to reduce ros expression to reduce cell growth / reproduction signal transduction pathway of nf-b , ap - l , and mapks to reduce cancer cell viability , ( 2 ) to improve the activity of the nad ( p)h and gst , ( 3 ) to inhibit the expression of tetradecanoyl method wave alcohol acetate ( tpa ) , in order to reduce the c - jun nh2-terminal kinase , p38 kinase , and mapk kinase-4 to prevent cancer transformation , and ( 4 ) to stimulate the expression of nf - e2-related factor and the activity of gst regulated by nrf2 downstream cascade links antioxidant response element ( are ) to inhibit the growth of cancer cells [ 6 , 7 ] .
chlorogenic acid is considered to be an effective cancer chemical repellant because of its significant inhibitory effect on colorectal cancer , liver cancer , and laryngeal . in this paper ,
the biological gene chip technology was used to detect the variation of a whole set of gene sequences in breast tumor - bearing mice cells in the dynamic treatment cycle with cga .
the differential genes were screened by using genomics profiling , which was also the potential target gene associated with tumor disease according to the gene go characteristic quality .
further verification testing must be designed , such as accurate quantitative pcr technology and western blot technology , to verify the potential target point in the treatment of chlorogenic acid for tumors .
  emt-6 mice breast tumor lines were preserved by key laboratory of transplant engineering and immunology , ministry of health , west china hospital , sichuan university .
female balb / c mice , weighing 17~18  g , and originally purchased from experimental animal center of sichuan university , have been bred in the ordinary small animal housing of the same center where the feeding conditions comply with gb 14925 - 2001 with the ambient temperature of 19  3c and humidity of 55  15% .  
co2 incubator ( mco-15ac , sanyo , jp ) , biological safety cabinets ( nu-425 , nuaire , usa ) , electronic balance ( jy12001 , sartorius , usa ) , high - speed centrifuge ( tgl-16 g , shanghai , china ) , pcr ( abi-9700 , abi , usa ) , hybridization oven ( g2545a , agilent , usa ) , scanner ( g2565ba , agilent , usa ) , spectrophotometer ( nd1000 , nanodrop , autoclave ) , quantitative pcr instrument ( bio - rad , usa ) , iq2-cryogenic high - speed centrifuge , standard 96-well plate , clean bench , sujing group aetna , chemidoc xrs + system medium ( gibco , china ) , calf serum ( minhai , lanzhou , china ) , 0.25% trypsin ( gibco , usa ) , double antibiotics ( penicillin and streptomycin sulfate ) ( north china ) , cy3 nhs ester ( lot no .
pa13105 ) , ge healthcare , aautp ambion ( am8436 ) , low rna input linear amplification kit ( lot no .
51885242 ) , 51885327 gene expression wash buffer kit ( including wash buffers 1 and 2 ) , stabilization and drying solution ( lot no .
g2534 - 60003a ) , 4 * 44k mouse microarray , hybridization cgamber ( lot no .
depc , oligo , 5x reaction buffer , riblock rnase inhibitor , dntp mix , revertaid m - mulv , 2*taq master mix novoprotein , iqtm sybr green supermix , bio rad .
revertaid first strand cdna synthesis kit , fermentas , fluorescent imager dna ladder , novoprotein , chlorogenic acid for injection ( cga , lot no .
061101 , 30  mg ) , white powder for injection ( content of 99.87% ) , offered by sichuan jiuzhang biological chemical technology development co. ltd .
docetaxel for injection ( dx , lot no . 10112611 , 20  mg ) , purchased from jiangsu hengrui medicine co. ltd .
ifn-2b ( 20110908 , 500  iu ) purchased from anhui anke biotechnology ( group ) co. ltd .
cga freeze - dried powder was dissolved with sterile saline to make concentrations of cga sample solution that were 1 , 0.5 , and 0.25  mgml , respectively . to use dx solution , 20  mg of dx injection powder was diluted with 1.5  ml of special solvent . to this , about 8.0  ml of saline was added to be stock solution of 2  mgml .
the stock solution was diluted to be sample solution with the concentration of 0.25  mgml with saline before use .
the anabiotic emt-6 breast tumor lines were implanted subcutaneously into the right forelimb of mice .
the tumor - bearing mice were obtained until the tumor grew to 1  cm  1  cm  1  cm . then took out the tumor , cleaned it with ns , weighed it , cut it into pieces , and placed it in a homogenizer .
ns ( about 1  :  4 ) was added to the homogenizer , and cell suspension was obtained after fast homogenate .
0.2  ml of the cell suspension was inoculated subcutaneously into the right forelimb of mice and was randomized .
30 female babl / c mice were randomly divided into 6 groups ( n = 5 ) , including normal saline ( negative ) group , cga high - dose group ( 20  mgkg ) , cga middle - dose group ( 10  mgkg ) , cga low - dose group ( 5  mgkg ) , dx positive control group ( 5  mgkg ) , and ifn-2b positive control group ( 5 million ukg ) .   after being inoculated for 24 hours , the ns group ,
cga groups , and ifn-2b positive control group were continuously subcutaneously administered for 12 days .
the tumors were taken after 24  hrs of the last administration , then weighed , cut into pieces , frozen in liquid nitrogen , and stored at 70c after segmentation package . the lysis / binding buffer was added , 1  ml per 0.1  g tissue , for homogenate on ice to prevent rna degradation .
adding homogenate additive ( 1/10 ) into the homogenate ; the homogenate was vortexed , mixed , and kept on ice for 10  min
. then added phenol and chloroform mixture in the same volume as lysis , vortex them for 30  s , centrifuged them in 10,000  g for 5 minutes at room temperature . added the absolute ethanol to the supernatant , then vortex and mixed them , made them going through the purification column repeatedly , centrifuged them at 10000  g for 15 seconds , discarded the supernatant .
added 350  l wash i , centrifuged them for 5  s to purify the column , then centrifuged at 10,000  g for 15  s , discarded filtrate . added 10  l dnase i and 70  l buffer rdd to the film , placed them for 15  min , and then successively added 350  l wash 1 and 500  l wash 2 , centrifuged them for 5  s , cleaned the purification columns two times .
each cleaning must be centrifugal at 10,000  g for 15  s and discard the filtrate . added 100  l 95c preheated nuclease - free water to spin column and placed the spin column in a new collection tube after the second cleaning .
the total rna would be obtained after centrifuging at the maximum speed for 30  s and then stored at 70c .
100  l rnase - free water was added to dissolve the total rna , then mixed with 350  l buffer rlt and 250  l ethanol .
( the preparation of rlt : 14  ml original rlt can be added to 140  l of -mercaptoethanol . )
transferred the sample to the rneasy column , centrifuged at 10000  g for 30  s , and abandoned filtrate .
clean the rneasy minicolumn twice with 500  l buffer rpe , centrifuged at 10,000  g for 30  s and 2  min , respectively , and discard the filtrate . add 40  l rnase free water to the column , and centrifuge 10000  g for 1  min . repeat the operation once more
prepare the electrophoresis buffer 50x tae , which was processed with decp and autoclaves  a 1% agarose gel was prepared after adding on amount of agarose to lx tae electrophoresis buffer  run on a gel for 15  min , then observe and picture the gel over the gel imager  lab - on - chip .
the primers were designed through the 01190 software and synthesized by sangon biotech ( shanghai ) .
the primer information of these four genes was shown in table 1 . with the template of sample cdna
, the differentially expressed genes were verified to use sybr green by real - time fluorescent quantitative pcr .
the conditions of the q - pcr reactions were subjected to 94c for 10  min , followed by 40 cycles at 94c for 15  s and 54.5c for 30  s , and finally 72c for 45  s. the expression levels of gsk-3 , apc , and -catenin in test samples were detected with gapdh as the reference gene and calculated by 2 relative quantification method , which showed the differential expression of different groups compared to the ns group .
after isinfecting the reagent bottle , boxes , and gloves under uv light for 30  min , the tissues were homogenized in the trizol reagent .
the homogenate was incubated at 1530c for 5  min , to which chloroform was added , then vortex them for 15  s , and centrifuged separating the mixture for 15  min in low temperature . transferred the upper supernatant into another tube , added 0.5  ml isopropanol and mixed them at 1530c , incubated them for 10  min , 4c , then centrifuged them at 12000  g for 10  min , discarded supernatant , and added 1  ml 75% ethanol into the tube , centrifuged them at 7500  g for 5  min , discarded the supernatant , and dried them in the air for 35  min . added 20  l depc deionized water to dissolve the rna and stored them at 70c . 

a260/a280 value was determined by uv with 1  l extracted from each of rna which was diluted to 100  l with te buffer .  

the voltage of conditions electrophoresis was 80  v , and running time was 40  min .
the rna samples prepared in section 3.3.2 were diluted with depc water suitably and mixed with an equal volume of sample buffer , then heated for 4  min in boiling water , cooled on ice for 2  min , and then centrifuged in 1200  rpm for 5  s. the sample was spotted on plate to be carried on the electrophoresis .
the total reaction volumes of rt - pcr reactions were 20  l . the system consists of 2  l rna , 1  l oligo(dt)18 primer , 9  l rnase - free water , 4  l 5x reaction buffer , 1  l riblock rnase inhibitor , 2  l 10  mm dntp mix , and 1  l revertaid m - mulv .   the reverse transcription system liquids of the above were subjected to 42c , 60  min , and 70c , 5  min , in a pcr instrument .
briefly , the presence or absence of the only bands was observed at 496  bp as the standard showed the quality of cdna .
if there is one and only one band , the pcr product cdna was qualified , and q - pcr can be the next .
the rt - pcr amplification system had 25  l liquid of total reaction volumes , in which consists of 10  l 2x taq master mix , 2  l cdna , 0.75  l forward gapdh primer pair and 0.75  l reverse gapdh primer , and 11.5  l rnase - free water .
pcr reactions were subjected to 94c for 10  min , followed by 40 cycles at 94c for 15 s and 54.5c for 30 s and finally 72c for 45  s. 1  l cdna was diluted to 100  l with sterile water on standby .
each sample should go through the three parallel tests , taking the mean value to calculation .  

, we can screen the optimal annealing temperature by real - time quantitative pcr reactions .
the melting curve can be used to assess the specificity of the reaction in the quantitative pcr instrument . if there are multiple peaks on the melting curve , which indicates nonspecific products such as primer dimer , they are amplified along with specific products simultaneously , which also indicates the primers of the reaction need to be redesigned . 

cdna was selected as the template and set eight 10-fold dilution series of points each dilution was repeated three times . the equation of the linear regression line has obtained the logarithmic value of template initial concentration as the abscissa and the ct value as the vertical coordinate .
standard curve correlation coefficient ( r ) or the coefficient of determination ( r ) can be used to evaluate the degree of linearization with the specific requirements of r > 0.990 and r > 0.980 .
as is shown in figure 1 , the luminance ratios of 28  s and 18  s are greater than or equal to 2 in electrophoresis graph , which preliminary determines that total rna was qualified .
the result has shown that the quality of rna extracted from each group of tumor tissue samples meets the requirement of further detection of gene chip
. then the differential genes closely related to the tumor cell suppression in the course of treatment were screened by the time series analysis , go analysis , and pathway analysis and validated with q - pcr . 

( 1 ) the results of od values . as is shown in table 4 , the a260/a280 value of rna was from 1.63 to 2.18 , which meets the quality requirements of rna .  

as is shown in figure 4 , the formaldehyde denaturing electrophoresis showing the 28s and 18s were clear without dna impurity band and degradation rna , which meets the requirements of the experiment . 

( 3 ) results of cdna quality test . as is shown in figure 5 ,
there is only one band at 496  bp , which indicates that the quality of the pcr product meets the requirements .
the pcr expression of gsk-3 , apc , and -catenin was shown in figure 6 in the tumor tissue of balc - b / emt-6 tumor - bearing mice after the treatment of dx ( 5  mg / kg ) , ifn ( 5  10  ukg-1 ) , cga ( 20  mg / kg ) , cga ( 10  mg / kg ) , and cga ( 5  mg / kg ) for 12 days . compared with the blank group , the expression of gsk-3 and apc was upregulated in group of 20  mg / kg dosage ( p < 0.05 ) and the expression of -catenin was downregulated in the same dosage group ( p < 0.05 ) .
( 1 ) as shown in table 3 , compared to the positive control group of cytotoxic anticancer drugs dx , biological response modifier ( brm ) ifn groups , and the negative group of ns , the three dosag of the cga groups showed better antitumor effect ( p < 0.05 ) ; especially the inhibition rate of the 20  mgkgdose group was more than 50% , which was equivalent to 59.92% of the dx group and 40.80% of the ifn group .
it is suggested that chlorogenic acid should be able to be a new good anticancer agent in future clinical application .
( 2 ) according to the data of the different time points in the inhibitory process acquired with the total rna that was qualified in figures 1 , 2 , and 3 the differential genes closely related to the wnt pathway in the course of treatment were screened by the time series , go , and pathway .
the different genes which were connected with cga treatment were extracted and were confirmed with q - pcr .
the downward trends of the tumor genomes 18 and 21 were screened by the logarithm of standardization and fitting the change course of genes with similar trends ; the genetic trend was closer to the fitted values of p smaller , such as figures 7(a ) and 7(b ) .
( 3 ) as shown in figure 7(a ) , the trend of genome 18 was confirmed to the anticancer process of cga . according to the analysis of kegg signaling pathway and the characteristics of
go in genome 18 , some downregulated genes were found which are related to the tumor suppression pathway , such as bdnf mediated mapk , cflar , cln3 ddit3 notch2 rps6 , sox9 , spn , and ppp1r13l .
the gene 21 time sequence diagram relates to the wnt pathway , mtor pathway , the notch pathway , and some immune - related pathways , such as b cell receptor pathway , t - cell receptor pathway , and metabolic pathway .
( 4 ) because our animal model was a breast cancer ( emt-6/balb - c ) , we focused on the influence of wnt pathway ( shown in figure 8) which was a special pathway that the breast cancer had . in particular ,
the gene glycogen synthase kinase 3(gsk-3 ) and downstream gene ubiquitin ligase e3(apc ) were upregulated . and
the two kinds of genes had closed relationship , in which the gene apc was upregulated by upstream gene gsk-3. and then , both genes could cause the down - stream gene -catenin to downregulate directly in the wnt pathway . as you know
, the cancer will be developing when the gene -catenin expression was upregulated . what is the gene -catenin ? from its go searching revealed , -catenin is a multifunctional protein , which can assist the cells react to extracellular signal and changes by interaction with the cytoskeleton .
this protein acts as a transcription ( transcription ) factor in the nucleus to promote cell division genes .
the accumulation of -catenin would lead to abnormal activation of the downstream transcription factors after transferring to the nucleus , which mainly cause tumor .
so , it is suggested that the -catenin inhibited was one of the ways to inhibit tumor growth . on the other hand ,
the upregulation of gsk-3 is a multifunctional serine / threonine class of protein kinase , which plays an important role in the wnt / wingless , pi3-kinase , and hedgehog signaling pathway with the physiological functions including transcriptional activation , cell proliferation , and cell differentiation , cell movement .
it can phosphorylate shaft protein and -catenin , cause the degradation of -catenin protein , and thereby inhibit the activation of the pathway .
the activated apc plays an important role in promoting complexes degradation in a fast , efficient , and highly selective way in the anaphase cell cycle , which also can phosphorylate shaft protein and -catenin , cause the degradation of -catenin protein , and thereby inhibit the activation of the pathway .
so the activation of gsk-3 and apc and downregulation of -catenin in the group of cga suggested that cga could inhibit tumor by activating gsk-3 and apc .
( 5 ) as shown in figure 6(a ) , the regulation confirmed by q - pcr in treatment process of cga for emt-6 breast cancer indicates groups of cga in each dose and dx can activate expression of gsk-3 , especially the cga 20  mg / kg group and the dx group can significantly upregulate gsk-3 ( p < 0.05 ) . as shown in figure 6(b ) , groups of cga at 20  mg / kg and 10  mg / kg and dx can activate expression of apc , but groups of cga at 5  mg / kg dose little to upregulation . as shown in figure 6(c ) , dx group , ifn group and cga 20  mg / kg group was inhibited gene -catenin .
  it is suggested that affecting the gene expression of gsk-3 , apc , and -catenin by chlorogenic acid in wnt pathway was one of the targets in the multipathway to antitumor of cga .
 
 objective . inhibiting gene -catenin and inducting genes gsk-3 and apc , promoting the tumor cell apoptosis in wnt pathway , by chlorogenic acid were discussed ( cga ) . method . 
 the different genes were scanned by the 444k mouse microarray chips . 
 the effect of the three genes was confirmed by rt - pcr technique with cga dosage of 5 , 10 , and 20  mg / kg . 
 result . 
 the expression of gsk-3 and apc was upregulated in group of 20  mg / kg dosage ( p < 0.05 ) and the expression of -catenin was downregulated in the same dosage ( p < 0.05 ) . 
 conclusion . 
 the results infer that the multimeric protein complex of -catenin could be increased by cga upregulated genes gsk-3 and apc , which could inhibit the free -catenin into the nucleus to connect with tcf . 
 so the transcriptional expression of the target genes will be cut to abnormal cell proliferation . 
 it is probably one of the ways that can stop the tumor increase by cga . 
1. Introduction
2. Instrument and Material
3. Methods
4. Results
5. Discussion
6. Conclusion