Inhibitory Effects And Mechanisms Of 30 Saponins On The Proliferation Of Erα-positive And Triple-negative Human Breast Cancer Cells

Objective:Firstly,the inhibitory effects of 30 saponins on the proliferation of breast cancer cells with different ERαphenotypes were tested and compared to determine whether they were selective or not,and to understand the structure-activity relationship so as to lay a foundation for the discovery of new anti-breast cancer drugs.In addition,the combination of MAPK signaling pathway activator EGF and PPI on MCF-7/ERαcells further clarified the mechanism of PPI in selectively inhibiting estrogen receptor positive breast cancer cells,and preliminarily clarified whether PPI targeted ERα.Methods:1.The inhibitory effect of saponins on the proliferation of ERα-positive MCF-7 cells and ERα-negative MDA-MB-231 cells was detected by modified MTT assay.The samples with activity selectivity for proliferation inhibition were further tested for proliferation inhibition on MCF-7/ERαoverexpressing ERαand its carrier cell MCF-7/ERα（V）.2.The modified MTT method was used to test the effects of PPI,Endoxifen and EGF on the proliferation of MCF-7/ERαand its carrier cells after 3,12 and 48 h.3.Western Blot was used to test the effect of PPI,Endoxifen and EGF on the expression of RTK/MAPK/p-ERαpathway protein in MCF-7/ERαcells after 3 and 24 h.4.The affinity of PPI for ERαwas detected by fluorescence polarization method based on coumarin（CS）as the tracer molecule.Results:1.Spirosanols:PFD36（NEW）is a newly discovered spirostanols,and its IC₅₀ of inhibiting MCF-7 and MDA-MB-231 cell proliferation is 18.67μmol/L and 14.09μmol/L,respectively.2.The inhibitory effect of isosorbide dioscin on the proliferation of MCF-7 and MDA-MB-231 cells:the IC₅₀ of Paris polyphylla saponin E（PFV11）was 3.14 and 0.63μmol/L,respectively;The IC₅₀ of Paris polyphylla saponin B（PDE24）was 5.18 and 0.86μmol/L,respectively.The IC₅₀ of dioscin gracilis（PFV14）was 25.00 and 2.82μmol/L,respectively.The IC₅₀ of Paris polyphylla saponinⅰ（PPI）was 3.83 and 9.95μmol/L,and that of Dioscorea zingiberensis saponin（PCT-33）was 3.21 and 4.85μmol/L,respectively.Pariposide A（PCT31）and PFV21（NEW）have no cytotoxic activity to MCF-7,but have strong cytotoxic activity to MDA-MB-231,with IC₅₀ of 11.53μmol/L and 5.01μmol/L;Parisyunnanoside G（PFV17）and Diosgenin have no cytotoxicity to MCF-7 and MDA-MB-231.The inhibitory effects of the above compounds on the proliferation of MCF-7/ERα（V）and MCF-7/ERαcells:the IC₅₀ of Paris polyphylla saponin E（PFV11）was 11.57 and 14.99μmol/L,respectively;The IC₅₀ of Paris polyphylla saponin B（PDE24）was 2.83 and 5.86μmol/L,respectively.The IC₅₀ of dioscin gracilis（PFV14）was 5.44 and 13.81μmol/L,respectively.The IC₅₀ of Paris polyphylla saponinⅰ（PPI）was 3.83 and 0.64μmol/L,and that of Dioscorea zingiberensis saponin（PCT-33）was 16.42 and 17.46μmol/L,respectively.3.The inhibitory effect of isoproterenol on the proliferation of MCF-7 and MDA-MB-231 cells:IC₅₀ of Paris polyphylla saponin VII（PDE23）was 4.49 and 2.24μmol/L,respectively;The IC₅₀ of Paris polyphylla saponin H（PCT-39）was 3.08μmol/L and4.85μmol/L,respectively.On the other hand,Ypsilandroside D（PFD37）with hydroxyl substitution at C27 has no cytotoxicity to MCF-7,but has a certain cytotoxicity to MDA-MB-231,and its IC₅₀ is 13.75μmol/L..Pennogenin has no cytotoxic activity to MCF-7 and MDA-MB-231.4.Seven kinds of furostanol saponins have no cytotoxicity to MCF-7 and MDA-MB-231.5.Tetracyclic triterpenoids-cucurbitacin compounds:cucurbitacin B（PFS-12）and cucurbitacin E（PFS-2a）have no cytotoxicity to MCF-7,but have strong cytotoxicity to MDA-MB-231,with IC₅₀ of 0.003 and 1.24μmol/L,respectively.Cucurbitacin B（PFS-12）has cytotoxic activity to MCF-7/ERα（V）,and its IC₅₀ is 24.72μmol/L;However,it has no cytotoxic activity to MCF-7/ERα.The IC₅₀ of cucurbitacin E（PFS-2a）on MCF-7/ERα（V）and MCF-7/ERαproliferation inhibition were 16.60 and 36.16μmol/L,respectively.Cucurbitacin L（PFS-8）,mogroside I E2（PFD-24）,PFB-33（NEW）,PFD-25（NEW）and PFD-26（NEW）have no cytotoxic activity on MCF-7 and MDA-MB-231.Tetracyclic triterpenoid-dammarane compound:PDE-22（ginsenoside Rg1）has no cytotoxic activity to MCF-7 and MDA-MB-231.6.In MCF-7/ERα（V）cells:EGF at the concentration of 1,10 and 100μg/m L can promote the proliferation of MCF-7/ERα（V）,and the proliferation rate is 20%after 48hours.Compared with PPI alone group,PPI combined with EGF had no effect on cell viability after 3h,12h and 48h;Compared with Endoxifen alone,the cell viability increased from 74.26%to 92.70%after 10μM Endoxifen combined with 1ng/m L EGF for 48 hours（P<0.05）.7.In MCF-7/ERαcells:EGF at 1,10 and 100μg/m L can promote the proliferation of MCF-7/ERαcells,and the proliferation promotion rate is 20%after 48 hours.Compared with PPI alone group,the cell viability increased from 92.75%to 96%after3 hours of treatment with 0.3μM PPI combined with 100ng/m L EGF（P<0.05）,and increased from 64.66%to 83.52%after 3 hours of treatment with 1μM PPI combined with 100ng/m L EGF（P<0.01）.After 3μM PPI combined with EGF1,10 and 100ng/m L for 48 hours,the cell viability increased from 64.79%to 81.76%,89.04%and 81.61%respectively（P<0.01）,and the cell viability did not change significantly after PPI combined with EGF at other concentrations.Compared with Endoxifen alone,the cell viability of Endoxifen combined with EGF did not change significantly after 3,12 and48 hours.8.After overexpression of ERαin MCF-7 cells,the expression level of HER2 protein was down-regulated（P<0.001）,and the phosphorylation levels of ERK and p38 in MAPK pathway were up-regulated（P<0.01）,but the phosphorylation levels of JNK and ERαdid not change significantly.After 24h,the expression levels of ERαand C-myc proteins were up-regulated(P9.In MCF-7/ERα（V）cells:Compared with the control group,EGF decreased the expression level of HER2 protein（P<0.001）and increased the expression levels of P-ERK（P<0.01）and C-myc（P<0.05）after 3 hours of treatment;After 24 hours of EGF treatment,the expression level of HER2 protein was down-regulated（P<0.01）.Compared with the control group,1μM PPI increased the expression level of p-ERK protein after 3 hours（P<0.01）.After 24h,the expression level of p-ERK protein was down-regulated（P<0.01）.The phosphorylation level of MAPK-related protein and ERαdid not change obviously after 3 hours of treatment with 3μM PPI,but the expression level of C-myc protein was significantly increased after 24 hours of treatment（P<0.001）.Compared with PPI alone group,1μM PPI combined with100ng/m LEGF decreased the expression levels of HER2 and p-ERK protein after 3h and 24h（P<0.01）.3μM PPI combined with 100ng/m LEGF decreased the expression level of HER2 protein after 3h and 24h（P<0.01）,and decreased the expression level of p-ERK protein after 3h（P<0.05）.After 24 hours,the expression level of C-myc protein was up-regulated（P<0.01）.30μM Endoxifen increased p-ERK and P-P38（P<0.05,P<0.01）after 3h and 24h.Compared with Endoxifen alone,the relative expressions of P38,ERK,JNK,AKT,ERαand their phosphorylated proteins,HER2and C-myc did not change significantly after 30μM Endoxifen combined with 100ng/ml EGF for 3 hours.After 24 hours,the expression levels of HER2 and p-ERK protein were down-regulated（P<0.01,P<0.001）.10.in MCF-7/ERαcells:compared with the control group,EGF decreased the expression level of HER2 protein after 3h and 24h（p<0.01）,and increased the expression level of p-p38 protein after 3h（p<0.01）.The expression level of p-ERK protein was down-regulated after 3 hours of treatment with 1μM PPI（P<0.05）,and up-regulated after 24 hours of treatment（P<0.01）.After 3 hours of treatment with 3μM PPI,the relative expression level of ERαprotein has no obvious change,and the expression level of p-ERαprotein is down-regulated（no statistical significance）,while after 24 hours of treatment,the expression levels of ERαand C-myc protein are down-regulated（P<0.01,P<0.001）,and the level of p-ERαprotein has no obvious change.Compared with PPI alone group,1μM PPI combined with 100 ng/ml EGF increased the expression levels of p-p38 and p-ERK proteins for 3 hours（P<0.01,P<0.05）.After 24 hours,the expression levels of HER2 and p-ERK protein were down-regulated（P<0.001,P<0.05）.3μM PPI combined with 100 ng/ml EGF for 3 hours decreased the expression levels of HER2 and p-ERK protein（P<0.01,P<0.05）and increased the expression level of p-ERαprotein（P<0.05）.After 24 hours,the expression level of HER2 protein was down-regulated（P<0.001）and the expression level of C-myc protein was up-regulated（P<0.05）.Compared with the control group,30μM Endoxifen decreased the expression level of p-ERK protein（P<0.05）and increased the expression level of p-p38 protein（no statistical difference）after 3h,but decreased the expression levels of HER2 and C-myc protein（P<0.01,P<0.05）and increased the expression level of P-after 24h.Compared with 30μM Endoxifen alone,30μM Endoxifen combined with 100 ng/ml EGF decreased the expression level of HER2protein for 3 hours（P<0.05）.However,the relative expression of HER2,P38,ERK,ERαand its phosphorylated protein and C-myc did not change significantly after 24hours of joint action.11.The curves of fluorescence polarization values of PPI,FLv,Endoxifen,E2 and CS competitive binding ERαshowed that the fluorescence polarization values decreased with the increase of sample concentration,and their half effective competitive binding concentrations(IC₅₀)were 3.24×10-7,2.76×10-6,1.41×10-6 and 1.55×10-7M respectively.The relative affinity of E2 and ERαis 100%,and the relative binding forces of PPI,FLv,Endoxifen and ERαare 47.84%,17.03%and 33.33%respectively.Conclusions:In this study,we found that the structure-activity relationship of steroidal saponins against the proliferation and inhibition of human breast cancer cells was as follows:The aglycones had no cytotoxic activity,and the ring opening of the aglycone F ring of dioscin caused the steroidal saponins to lose their cytotoxic activity.The oxygen bridge modification on the diosgenin of iso-spirostanol and the existence of hydroxyl（-OH）substitution at C27 would also weaken the cytotoxic activity.Multiple carbon（C）sites on its parent nucleus connected with sugar groups and the existence of hydroxyl substitution might make the compound lose cytotoxic activity.2.Among the isospirostanol dioscin compounds,Paris saponin E（PFV11）,Pariposide A（PCT31）and PFV21（NEW）had strong inhibition on the proliferation of triple negative breast cancer cell line（MDA-MB-231）and had good selectivity.The C5and C8 positions of Pariposide A（PCT31）and PFV21（NEW）were both connected by an oxygen bridge,and there was a certain number of glycosyl substitutions at C3,but no modification at other positions.Paris saponin B（PDE24）and dioscin gracile（PFV14）showed strong inhibition on the proliferation of MDA-MB-231 and MCF-7/ERα（V）cells,indicating that the cytotoxic activities of these two compounds were inversely proportional to the expression of ERα.The above compounds can be used as natural candidate compounds against triple negative breast cancer.Paris polyphylla saponin I（PPI）had strong inhibitory effect on the proliferation of estrogen receptor positive cells and had good selectivity.In addition,the overexpression of ERαcould increase its sensitivity to PPI,and there was arabinose in the C3 glycosyl substitution,suggesting that Paris polyphylla saponin I could be used as a natural candidate compound for anti-estrogen receptor positive breast cancer.The specific structure-activity relationship required further study.3.Among the isospirostanol monoterpenoid saponins,Paris saponin VII（PDE23）has a strong inhibitory effect on the proliferation of MDA-MB-231 and has good selectivity;The C3 position of that aglycone is connecte with a certain number of glycosyl.The C27 position of pennogenin aglycone is introduced with OH to obtain Ypsilandroside D（PFD37）,which has strong proliferation inhibition effect on triple negative breast cancer cells and no proliferation inhibition effect on estrogen receptor positive cells.The two compounds can also be used as natural candidate compounds for anti-triple negative breast cancer.4.Among the tetracyclic triterpenoid saponins,cucurbitacin compounds cucurbitacin B and E have good selectivity and cytotoxic activity on triple negative breast cancer cells,and the cytotoxic activity is inversely proportional to the expression of ERα.It has a double bond between C1-C2 and C23-C24,and an acetate radical（-o Ac）at the C25position.They can also be used as natural candidate compounds against triple negative breast cancer.Dammarane-type compound ginsenoside Rg1（PDE-22）has no cytotoxic activity on breast cancer cells with different phenotypes.5.EGF,MAPK activator,could antagonize the proliferation inhibition of MCF-7/ERαby PPI,but did not affect the proliferation inhibition of MCF-7/ERα（V）by PPI,indicating that PPI was more inclined to interfere with MAPK signaling pathway to exert proliferation inhibition after ERαwas overexpressed.The inhibitory effects of Endoxifen on the proliferation of MCF-7/ERαand its carrier cells were not affected by EGF,so we speculated that its mechanism might not be related to the inhibition of MAPK signaling pathway.6.EGF rapidly antagonized PPI in down-regulating the phosphorylation levels of ERK,p38 and ERαof MAPK signaling pathway in MCF-7/ERα,and the detected phosphorylation site of p-ERαwas ser118,which was mainly activated by MAPK pathway.However,after 24h of action,EGF could antagonize PPI in down-regulating the expression level of C-myc protein,a target gene of MAPK/p-ERαpathway.None of the above-mentioned antagonists of EGF were present in MCF-7/ERα（V）.It was further proved that when breast cancer cells expressed sufficient ERα,Paris polyphylla saponin I exerted the anti-breast cancer cell proliferation effect by interfering with ERαnon-classical pathway RTK/MAPK/p-ERα.EGF did not affect the downregulation of ERαprotein expression by PPI,suggesting that PPI might directly degrade ERαprotein.7.Endoxin was able to up-regulate the phosphorylation of P38 and ERα（ser118）in MCF-7/ERαand its carrier cells,and the effect was not affected by EGF,suggesting that Endoxin could activate p38 pathway in MAPK pathway,and thus phosphorylate ERα.This might be the reason for the partial activation of ERα-positive breast cancer cells by Endoxifen.8.PPI has a certain affinity with the AF1 region of ERα,and the affinity intensity is greater than that of FLv and Endoxifen,which is about 1/2 of E2.PPI binding could down-regulate the expression of ERαprotein,suggesting that PPI might target and degrade ERα,and could be used as a candidate compound for targeted treatment of ERαpositive breast cancer.