Antagonistic Effect Of Soyasaponins On The Inhibition Of Gap Junctional Intercellular Communication By Cancer Promoter

BackgroundCancer is one of the leading causes threatening the health and life of human beings.Modern Nutrition Researches show that daily diet and cancer are closely related.It is proposed that one-third of cancer incidences are closely associated with diet.Unbalanced diet, excessive intake of three high (high fat, high protein, high sugar) and one low(low dietary fiber) food is one of the causes increasing the incidence of cancer of urban and rural residents.Studies have found that dietary pattern rich in soy protein other than meat protein significantly reduced the incidence of cancer. Soyasaponins(SS),a bioactive substance abstracted mainly from soybeans and other legume plants, have been proven to have significant tumor suppressor activities by experiments from cell culture,animal models and human studies.It is also found that different structural types of soyasaponins exhibit distinct anticarcinogenic activities.Soyasapogenol(SG) is the metabolites of soyasaponins in the gastro-intestinal gut. Three types of SG (A, B and E) were found currently. Recent studies have shown that SGs have higher bioactivites as compared to soyasaponins.Although many studies have shown that soyasaponins have antitumor activities, its mechanisms were scarely known. In general, the progress of carcinogenesis includes three stages:priming stage, promoting stage and advanced stage.The promoting stage is the only one that can be reversible as determined by environmental factors.This stage provides a good opportunity for cancer chemoprevention. During the promoting stage, the gap junctional intercellular communication (GJIC) between cancer initiator cells and normal cells is inhibited. The cancer initiator cells thus lose their own inhibition and multiply and express malignant genotype, and finally become cancer cells. A large amount of researches indicate that GJIC composed of connexins (Cx) play an important role in the process of carcinogenesis and cancerometastasis.The gene expression of Cx as well as Cx molecule amounts are decreased and disappeared in various types of tumor cells. The functions of GJIC are subsequently defected and disappeared, leading to formation of malignant tumor. Many cancer-promoting agents such as Tetradecanoylphorbol Acetate (TPA) downregulate Cx gene expression and inhibit the function of GJIC.A lot of substances with anticarcinogenic activities upregulate Cx gene expression and promote the function of GJIC, and thus prevent the occurrence of cancer. For example, ginsenosides can inhibit tumor growth through inducing Cx gene expression and enhancing GJIC.ObjectivesThe preventive effects on cancinogenesis and inhibitive effects on tumor growth of soyasaponins as well as their structural similarity to ginsenosides all indicate that soyasaponins may play roles in GJIC regulation and promotion.However, no report concerning the effects of soyasaponins on GJIC functions and its mechasnims is found till now. Therefore, in this study, the effects of different structural types of soyasaponins on the multiplication, apoptosis and GJIC of HepG2 cells were investigated by using the scrape-loading and dye transfer(SLDT), single cell gel electropherosis(SCGE),flow cytometry (FCM),and MTT assay as techniques. Furthermore,the antagonistic effects of different structural types of soyasaponins on the inhibition of GJIC of rat normal liver cells BRL by cancer promoter were also investigated. We aimed to explore the molecular mechanisms of anticarcinogenic activities of soyasaponins so as to provide scientific proof for developing anticancer drug product and advocating balanced diet, and to promote the development and utilization of soybean resources.MethodsPart I:Effects of soyasaponins with different chemical structures on cell proliferation,apoptosis and gap junctional intercellular communication of HepG2 cells1 Preparation of soyasapogeninsReferring to methods by Gurfinkel etc.(2005),the soyasapogenin A(SG-A) and soyasapogenin B (SG-B) were prepared by firstly dissolving total soyasaponins with methanol,secondly hydrolyzing them with concentrated sulfuric acid, and finally separating them by using C-18 Sep-Pak solid-phase extraction column.The purity of SG-A and SG-B abstracts was 57.3% and 62.6%, respectively.2 The Experiment of Cell proliferation inhibiton (MTT assay)The HepG2 cells were cultured and passaged by using DMEM high glucose culture medium containing 10% fetal bovine serum in 25cm2 culture flasks.The cells in logarithmic phase were reseeded in 96 well culture plates with the density of 1×104 cells/mLand each well contained 200μL of culture medium.,The cells were treated with different concentrations(10,20,40,80,100,160,200,400μg/mL) of soyasaponins with various chemical structures (SG-A, SG-B and TS) after adherence in the cultural condition of 37℃and 5% CO2 humidity. The cells were also treated with culture medium containing no soyasaponins as negative controls. Wells containing culture medium but no cells were prepared as blank. Six wells were replicated for each point of concentration and time.After culturing for another 24,48,and 72 hours,20μL of MTT were added to each well,and re-cultured for another 4 hours. Culture supernatants were carefully removed. Each well was added with 200μL of DMSO and was detected for absorbance at 570 nm. The trials were replicated three times.The inhibition rate of cell proliferation was calculated as follows:inhibition rate(%)=(absorbance of control-absorbance of treatment)/ absorbance of controls×100.3 Detecting IC50In the MTT method, is the absorbance of the control group OD value of drug required to reduce by half the concentration of IC50.This study used the SPSS software Probit model to calculate the probability unit.4 Detecting apoptosisCollection negative control group and 400μg/mL Soyasaponins(SG-A, SG-B and TS) respectively role 24h,48h,72h cells, use Annexin V-FITC/PI kit specifying FCM for cell apoptosis analysis.5 Scrape-loading and dye transfer technique (SLDT)(1)HepG2 cells were seeded in 30mm Petri dish, at 37℃,5% CO2 incubator for 24h after treatment adherence,respectively, a normal control group (DMEM medium) and three drug group(100μg/mL TS,100μg/mL SG-A and 100μg/mL SG-B),each dealing with the parallel set of three cell culture dishes, incubated for 90 min.(2) Treated cells were washed three times PBS buffer.(3)Cells in 35mm2 Petri dish, cut with the scalpel pressure three mark.(4) Culture dishes were added to 1mL fluorescent yellow (LY, lmg/mL), incubated for three min, discard LY.(5) Washed with PBS buffer two times.(6) By adding 4% formaldehyde solution and fixed 4min, remove LY(7) In the fluorescence microscope, observing, photographing, measuring migration distance by Photoshop.6 Statistical AnalysisSPSS13.0 software with data on cell proliferation and apoptosis rate of the data analysis because of variance analysis and single factor analysis of variance,results in the form, P<0.05 indicated statistical significant difference.Part II:Effects of soyasaponins with different chemical structures on gap junctional intercellular communication of BRL cells1 Effects of soyasaponins on growth inhibition of BRL cellsBRL cells containing 10% fetal bovine serum and two anti-in DMEM high glucose on 25cm2 culture flask were cultured and passaged.In the logarithmic phase of cell density by 1×104/mL cells were seeded in 96 culture plates, each well 200μL, at 37℃,5% CO2 incubator, after 24h cell adherence,various concentrations (50-2000μg/mL) of soyasaponins (SG-A, SG-B and TS), also set the negative control without drugs and non-inoculated control cells.Continue to train at 24,48, 72h set up each time point for each concentration of 6 repeat holes.At the end of each time point in each well plate by adding 20μL MTT, after 4h incubation supernatant carefully absorb every hole by adding 200μL DMSO,in the microplate reader (U.S.BioTek Elx808) on the determination of 570nm absorbance (A), The experiment was repeated 3 times.Cell proliferation was calculated as follows: inhibition rate(%)=(A value of the control group-experimental group A value)/control group A values×100.2 Scrape-loading and dye transfer technique (SLDT)(1)BRL cells were seeded in 30mm cell culture dish, at 37℃,5% CO2 incubator for 24h after the drug treatment adherence Drug treatment:A.Incubated 90min:take the 30mm cell culture dish adherent growth of BRLcells, the agent phorbol ester tumor promoters (TPA) with different forms of soyasaponins (TS,SG-A and SG-B)and BRL cells were incubated 90min.Divided into 11 treatment groups, including a normal control group (plus DMEM),1 two positive control group (plus 50ng/mL phorbol ester TPA),9 on Drug treatments that 50ng/mL TPA plus 100μg/mL TS,50ng/mL TPA plus 200μg/mL TS,50ng/mL TPA plus 400μg/mL TS;50ng/mL TPA plus 100μg/mL SG-A,50ng/mL TPA plus 200μg/mL SG-A,50ng/mL TPA Add 400μg/mL SG-A;50ng/mL TPA plus 100μg/mL SG-B, 50ng/mL TPA plus 200μg/mL SG-B,50ng/mL TPA plus 400μg/mL SG-B.Each treatment set three cell culture dishes, experiment was repeated 3 times B.Preincubation 24h:take the growth in 30mm cell culture dish adherent normal epithelial cells of rat liver BRL,respectively, with different forms of (TS,SG-A, SG-B) different concentrations(100,200,400μg/mL) of soyasaponins pretreatment 24h, treatment group settings Ibid, at the end of 90min, to join the cancer-promoting agent phorbol ester (TPA), incubated for 90min(2) In 35mm2 cell culture dish, we cut with a scalpel pressure three marks. (3)Each dish were added to 1mL Lucifer Yellow (1mg/mL),incubated for 3min, the LY absorb discarded. (4) Washed with PBS buffer two times.(5)By adding 4formaldehyde solution and fixed 4min, remove formaldehyde.(6) In the fluorescence microscope, observed and photographed and measured the migration distance.3 Single cell gel electrophoresis (SCGE)(1)The passage of BRL cells were seeded in 24 culture plates, at 37℃,5% CO2 incubator 24h adherent cells was conducted after 6 treatments:a negative control group (plus the same amount of DMEM culture medium), a solvent control group (plus an equal amount of 0.5% ethanol),a positive control group (plus 50ng/mL TPA) and 3 TPA dealt with soybean saponin group (50ng/mL TPA+100μg/mL TS, 50ng/mL TPA+200μg/mL TS,50ng/mL TPA+400μg/mL TS).Each treatment group set four cell culture hole repeat experiment was repeated 3 times.After experimental treatment, trypsin digestion, centrifuged to collect cells;(2) Completed the preparation of 0.5% LMP into 37℃water bath pot.(3)20μl PBS cells were resuspended with 80μl 0.5% low melting point agarose gel mixed shop.(4) Until the gel solidified, the gel placed in the cell lysate 1.5h (4℃dark).(5)Slides home before electrophoresis electrophoresis liquid aside for 20 min, liquid level above the slide 2 mm, so that DNA helicase.(6) Electrophoresis,electrophoresis adjust fluid levels, so that current is 300mA, voltage 25 V, time 20 min.(7) Finished the electrophoresis gel and the fluid placed in each 5min, a total of three times,dried.(8) Stained gel before dropping 1μg/mL EB staining 5 minutes(9)Observed under fluorescence microscope(515nm excitation wavelength) observations, each cell count of 100 cells were recorded comet cells.Comet cell rate =(comet cells/total cells observed) x 100%.4 Statistical AnalysisSPSS13.0 software data with ANOVA,the results in the form x±s,P<0.05 indicated statistical significant difference.SPSS13.0 software using single cell gel electrophoresis data for the whole x 2 test, that is, RxC table data x2 test, and then for a number of multiple comparisons between sample rate, P<0.05 indicated a significant statistical differenceResultsPart I:Effects of soyasaponins with different chemical structures on cell proliferation,apoptosis and gap junctional intercellular communication of HepG2 cells1 Soyasaponins on proliferation of HepG2 cellsAs Table 1-1 shows, the different structure of soybean saponin on HepG2 cell proliferation inhibition rate of time and concentration dependent, with time, inhibition of cell proliferation was more significant (F=2085.956,P<0.001);with Soyasaponins concentration increased, cell growth inhibition was significantly higher (F=2117.180, P<0.001) (Figure1-1,Figure 1-2).SG-B and SG-A's inhibitory effects on cell proliferation was significantly higher than TS (F=687.155, P<0.001).2 Cell proliferation inhibitory concentrationsThe role of different structures of soyasaponins HepG2 cells after 72h there are significant differences in median inhibitory concentration (Figure 1),SG-A and SG-B of the IC50 values of less than TS (P<0.05).3 Soyasaponins (400μg/mL) on apoptosis of HepG2 cellsEffect of soyasaponins could be detected after HepG2 cells apoptosis significantly (Table 1-3).After 24 hour's treatment, SG-A, SG-B and TS-induced apoptosis were significantly higher than early (P<0.05), three different structures of soyasaponins induced apoptosis show an early difference(SG-B> SG-A> TS).4 Soyasaponins on HepG2 cells affected GJICAs can be seen from Figure 1-8,HepG2 cells gap junctional intercellular communication is very weak, consistent with those reported in different Soyasaponins structure can not increase human hepatoma HepG2 cells gap junctional intercellular communication.Part II:Effects of soyasaponins with different chemical structures on gap junctional intercellular communication of BRL cells1 Effects of soyasaponins on growth inhibition of BRL cellsFigure 2-1 shows, the different structure of soyasaponins(SG-A, SG-B,TS) at different concentrations of BRL cell proliferation result, when the concentration was 1000-2000μg/mL, SG-A and SG-B group of BRL cells was significantly decreased, TS Group BRL cell proliferation rate has dropped, so this part of the experiment the concentration of soyasaponins set 100～400μg/mL.2 Detect the structures of soyasaponins on TPA inhibition of BRL gap junctional intercellular communication function by SLDT1 Using two different methods of drug treatment, namely:the BRL cells with TPA and different forms of soyasaponins 90min incubation and incubated with BRL cells for 24h, the results have shown that normal rat liver cells BRL has a very strong function of GJIC, cancer-promoting agent PMA(10ng/mL) completely inhibited the GJIC function, and different forms of soyasaponins BRL cells can restore gap junctional intercellular communication.Further study showed that the treatment of pre-incubated 24h, BRL cells may restore the role of gap junctional communication is more apparent.3 Effects of soyasaponins on gap junction intercellular communication of BRL cells by SCGEBy the x2 test, compared with the control group, except TPA50+400 was no significant difference, the, the rest trailing in all groups were significantly higher (P<0.05).Compared with the solvent, in addition to TPA+400 was no significant difference, the rest trailing in all groups were significantly higher than the solvent group (P<0.05).The test can be observed with different concentrations of TS cells on the TPA inhibition of GJIC function BRL enhancement.Conclusions1.Three forms of Soyasaponins can inhibit the proliferation of human hepatocellular carcinoma HepG2 cells in a concentration and time dependent. SG-A and SG-B inhibited the activity of HepG2 cells was stronger than soy saponins TS.2.Three forms of soyasaponins could induce apoptosis in HepG2 cells, after 24h, the main early apoptosis, early apoptosis inducing activity of size: SG-B>SG-A>TS.3.Human hepatoma HepG2 cell gap junction communication is weak, this experiment with a concentration of 100μg/mL Soyasaponins not observed in HepG2 cells in their function of gap junctional intercellular communication enhancement.4.Normal rat liver BRL cells have a very strong GJIC function, lOng/mL phorbol ester tumor promotion agents completely inhibited gap junctional intercellular communication.Three forms of soyasaponins can antagonize phorbol ester BRL cells inhibition of gap junctional intercellular communication.