Effect Of Astragalus Mongholicus And Salvia Miltiorrhiza On Proliferation And Apoptosis In Hormone Sensitive (MCF-7) And Insensitive (MDA-MB-231) Breast Cancer Cell Lines

Background and objectiveBreast cancer is one of the malignant tumour which hazards women's health seriously.Breast cancer incidence and mortality vary considerably by world regions.In general,the incidence is high(greater than 80 per 100,000)in developed regions of the world and low(less than 30 per 100,000),though increasing,in developing regions;it increasing sharply in some big cities of China.Chinese herb was managed as one kind of adjunctive therapy of breast cancer.As rising of replacement medicine and hormone replacement therapy(HRT)increasing risk of breast cancer in post-menopause women,chinses herb was managed to treat tumor and climacteric syndrome frequently in other countries.The effects of some decoction and herb were similar to phytoestrogens,herb to active blood and tonify are the most common.Astragalus mongholicus is rich in flavone and isoflavone,which are the effective component of estrogenic effect.Astragalus mongholicus was generally applied to lung cancer,gastric carcinoma,breast cancer to relieve adverse reaction and in coordination with chemotherapy,treat myelosuppression,boost immunological function and improve quality of life.Related studies suggested that Astragalus mongholicus or its extracts could inhibit proliferation and induce apoptosis of some tumor cell lines.But others discovered Astragalus mongholicus may promote tumor cell growth.Salvia miltiorrhiza is the representative medicine to active blood, the pharmacologic action of tanshinone,the liposoluble constituent of salvia miltiorrhiza,is resemble to phytoestrogen.Various kinds praeparatum were managed as adjunctive therapy of mammary adenocarcinoma widespread.Some study and clinical application indicated that Salvia miltiorrhiza,its extract,its compound preparation all had effect to inhibit tumor,simultaneously some found Salvia miltiorrhiza could promote tumor metastasis of experimental animals.Phytoestrogens(PE)are a group of plant-derived substances that are structurally or functionally similar to estradiol,concluding isoflavones,Lignans and coumestans.The estrogenic effects of phytoestrogen is 10 000 to 14 000 fold less potent than 17beta-estradiol (E2)after binding with estrogen receptor(ER).As occupying ER competive, low concentration PE antagonize estrogen,middle dose have some estrogenic activity,high dose display phytoestrogenic activity.Interest in phytoestrogens has been fueled by epidemiologic data that suggest a decreased risk of breast cancer in women from countries with high phytoestrogen consumption.High genistein circulation levels are associated with reduced breast cancer risk.However,some foreigen scholar suggested PE could potentially increase the risk of breast cancer or interact with tamoxifen or aromatase inhibitors.Phytoestrogens may serve as chemopreventive agents while at the same time being capable of promoting growth in estrogen receptor positive cancer cell lines. Phytoestrogen intake might be ill advised for patients at an increased risk for hormone-dependent cancers,cancer patients,or cancer survivors. There is a paucity of clinical trial data of Some Chinese herb similar to PE demonstrating either safety or efficacy in patients with breast cancer or those with tamoxifen coadministration.The purpose of this study was to investigate the efficacy and safety of Astragalus mongholicus and Salvia miltiorrhiza in hormone sensitive (MCF-7)and insensitive(MDA-MB-231)breast cancer cell lines with physiological dose E2 or pharmacologic dose TAM. PartⅠPreliminary experiment:Effect of Astragalus mongholicus and Salvia miltiorrhiza on proliferation of human breast cancer cells MCF-7 in vitroObjectiveThe aim of the study was to find the optimal experimental concentration of Astragalus mongholicus and Salvia miltiorrhiza on human breast cancer cells MCF-7 with MTT assay.Materials and methodsHormone sensitive breast cancer MCF-7 cells were cultured in RPMI1640(10%calf serum,penicillin 100U/mL,phytomycin 100ug/mL),MCF-7 cells in logarithmic growth phase inoculated in 96 well mask at 1×10e4/ml concentration,MTT assay was used to evaluate the proliferation of MCF-7 cells of Astragalus mongholicus or Salvia miltiorrhiza at kinds of concentrations.Optical density data were expressed as mean±standard deviation(SD),and a one-way ANOVE was performed for group comparison.All statistical tests were two-sided and P＜0.05 was considered to indicate statistical significance.ResultsCompared with blank group,Astragalus mongholicus could inhibit the proliferation of MCF-7 cells at any concentration(P＜0.05).Dose exceeded 2×10^-5g/ml,the effect was dose dependent;dose smaller than 2×10^-5g/ml, the effective curve was irregular.Salvia miltiorrhiza at concentration of 1×10^-2g/ml and 1×10^-6g/ml could inhibit the proliferation of MCF-7 cells(P＜0.05),Others dose was uneffective.There is linear relationship when dose from 1×10^-5g/ml to 1×10^-2g/ml.ConclusionIn this study Astragalus mongholicus was selected at concentration from 2×10^-1g/ml to 2×10^-5g/ml;Salvia miltiorrbiza was selected at concentration from 1×10^-1g/ml to 1×10^-5g/ml.PartⅡEffect of Astragalus mongholicus on proliferation and apoptosis in hormone sensitive(MCF-7)and insensitive(MDA-MB-231)breast cancer cell linesObjective The aim of the study was to investigate the effect of Astragalus mongholicus on proliferation and apoptosis in hormone sensitive(MCF-7) and insensitive(MDA-MB-231)breast cancer cell lines with physiological dose E2 or pharmacologic dose TAM.Materials and methodsHormone sensitive(MCF-7)and insensitive(MDA-MB-231)breast cancer cells were cultured respectively in RPMI1640(10%calf serum,penicillin 100U/mL,phytomycin 100ug/mL),the effects of different dose of Astragalus mongholicus on proliferation and apoptosis was observed in hormone sensitive(MCF-7)and insensitive(MDA-MB-231)breast cancer cell lines with physiological dose E2 or pharmacologic dose TAM,the proliferation of MCF-7 and MDA-MB-231 cells were evaluated with MTT assay,cell apoptotic rate were measured with flow cytometry and DNA ladder,cell cycle were observed by flow cytometry.Data were expressed as mean±standard deviation(SD),and analysis of variance was performed for group comparison of measurement data,chi square test was used for enumeration data.All statistical tests were two-sided and P＜0.05 was considered to indicate statistical significance.ResultsWith physiological dose E2,Astragalus mongholicus did not promot MCF-7 cells proliferation at any concentration.As time lasting, Astragalus mongholicus showed better inhibitory effect than TAM(P＜0.05).At 2×10^-1g/ml concentration Astragalus mongholicus increased the rate of apoptosis to 16.7%.Combined with TAM,Astragalus mongholicus showed dose dependent inhibitory effect on MCF-7 cells(P＜0.05).The rate of apoptosis is 22.1%at 2×10^-1g/ml concentration.After cocultured 24 hours,Astragalus mongholicus significantly increased the proliferative phase(percent S-phase)and decreased G0/G1 andG2/M phase(P＜0.05)at high concentration(2×10^-1g/ml)with physiological dose E2 or combined with TAM,at 2×10^-2g/ml concentration,it accumulating cells in G0/G1 and G2/M phase,decreasing the proliferative phase(percent S-phase)(P＜0.05).The influence was different in insensitive(MDA-MB-231)breast cancer cell lines at distinctive concentration.Astragalus mongholicus inhibited cell proliferation and increasing the rate of apoptosis at high concentration(2×10^-1g/ml);inhibited cell proliferation and increased the proliferative phase(percent S-phase)at 2×10^-2g/ml,2×10^-3g/ml concentration;but promoted MDA-MB-231 cell proliferation at 2×10^-4g/ml,2×10^-5g/ml concentration.ConclusionThe findings suggested it was safety for patients with breast cancer or administered TAM to use Astragalus mongholicus clinically.Astragalus mongholicus inhibit MCF-7 cell proliferation in coordination with TAM in the initial stage,it may be beneficial for patients with estrogen receptor positive by inhibiting MCF-7 cell proliferation and inducing apoptosis at some dose limit.Astragalus mongholicus is safe and potent to hormone insensitive breast cancer patients at high concentration by increasing the rate of apoptosis and inhibiting MDA-MB-231 cell growth.However,Astragalus mongholicus promoted MDA-MB-231 cell growth at low and less middle concentration,had adverse effect on the cell cycle by significantly decreased the resting phase G0/G1 phase)and increased the proliferative phase(percent S-phase)at less high concentration.Additional research on safety and quantitation of Astragalus mongholicus in serum,their interactions with plasma and cellular proteins,and their uptake in target tissues is necessary.PartⅢEffect of Salvia miltiorrhiza on proliferation and apoptosis in hormone sensitive(MCF-7)and insensitive(MDA-MB-231)breast cancer cell linesObjectiveThe aim of the study was to investigate the effect of Salvia miltiorrhiza on proliferation and apoptosis in hormone sensitive(MCF-7) and insensitive(MDA-MB-231)breast cancer cell lines with physiological dose E2 or pharmacologic dose TAM.Materials and methodsHormone sensitive(MCF-7)and insensitive(MDA-MB-231)breast cancer cells were cultured respectively in RPMI1640(10%calf serum,penicillin 100U/mL,phytomycin 100ug/mL),the effects of different dose of Salvia miltiorrhiza on proliferation and apoptosis was observed in hormone sensitive(MCF-7)and insensitive(MDA-MB-231)breast cancer cell lines with physiological dose E2 or pharmacologic dose TAM,the proliferation of MCF-7 and MDA-MB-231 cells were evaluated with MTT assay,cell apoptotic rate were measured with flow cytometry and DNA ladder,cell cycle were observed by flow cytometry.Data were expressed as mean±standard deviation(SD),and analysis of variance was performed for group comparison of measurement data,chi square test was used for enumeration data.All statistical tests were two-sided and P＜0.05 was considered to indicate statistical significance.ResultsWith physiological dose E2,Salvia miltiorrhiza at high concentration(1×10^-1g/ml)promoted MCF-7 cells proliferation at 24 and 72h(P＜0.05),had no influence at 48h.Salvia miltiorrhiza had inverse association on dose when inhibiting MCF-7 cell growth at other less concentration(P＜0.05).Combined with TAM,the promoting effectof high dose Salvia miltiorrhiza was negated at different degree,the promoting growth rate decreased at 24h,there was no promoting effect at 72h.Salvia miltiorrhiza inhibited MCF-7 cells proliferation at other lower dose (P＜0.05).The inhibitory effect was stronger than TAM at low concentration(1×10^-5g/ml)combined with TAM or not(P＜0.05).Salvia miltiorrhiza had no influence on MCF-7 cell cycle and induced cell apoptosis at high concentration(1×10^-1g/ml).The effect of Salvia miltiorrhiza inhibited MDA-MB-231 cell proliferation at 48h and promoted MDA-MB-231 cell proliferation at 72h was dose dependent.At 24h it inhibited growth at low dose(1×10^-5g/ml)but promoted proliferation at high dose(1×10^-1g/ml)(P＜0.05).Astragalus mongholicus increased MDA-MB-231 apoptosis rate at 1×10^-1g/ml,1×10^-2g/ml,but increased the proliferative phase(percent S and G2/M phase)and decreased the resting phase(G0/G1 phase)at 1×10^-4g/ml(P＜0.05)ConclusionThe data suggested effects of Salvia miltiorrhiza on MCF-7 cells was similar to phytoestrogen,it promoting hormone sensitive(MCF-7)breast cancer cell growth at high concentration and inhibiting cell proliferation at lower concentration.Salvia miltiorrhiza is a class of potent phytoestrogen.The safety and quantitation of Salvia miltiorrhiza used in clinic need further research.Salvia miltiorrhiza inhibited MDA-MB-231 cell proliferation depending on cell line and point of time and concentration especially at 48h at high concentration.Salvia miltiorrhiza may enhance MDA-MB-231 cell caryocinesis via activing ERβpartly.It should be cautious to use Salvia miltiorrhiza to hormone insensitive breast cancer patients,the exact evidence need additional study in vivo.