The Effect And Mechanism Of Aqueous Extract Of Myrrh On Proliferation,Migration,Invasion Of HER2-Positive Breast Cancer Cells SK-BR-3

Objective:In the present study,we used SK-BR-3 cells and a xenograft model to investigate the anti-tumor effects and mechanisms of aqueous extract of myrrh on proliferation,migration,invasion of HER-2 positive breast cancer SK-BR-3 cells in vitro and vivo experiment.Methods:1 Human breast cancer SK-BR-3 cells were seeded in 24 culture plates.We set up a system of different concentrations of aqueous extract of myrrh and negative control group.Wound healing assay and transwell assay were used to determine the effect of aqueous extract of myrrh on migration of SK-BR-3 cells.Invasive analysis was done by transwell assay which were covered 30ug matrigel upside for the above breast cancer cells.Secondly,SK-BR-3 cells were exposed to different concentrations of aqueous extract of myrrh for 24h,we used immunofluorescence analysis and western blot analysis to detect the expression of E-Cadherin,N-Cadherin,Vimentin and the change of Cytoskeleton and filopodia on the cell membrane in each group.2 Human breast cancer SK-BR-3 cells were seeded in 24 culture plates.SK-BR-3 cells were exposed to different concentrations(0ug/ml,50ug/ml,100 ug/ml,200ug/ml)of aqueous extract of myrrh after 24h,48h,72h,96h,120h,144h,we observed proliferation of SK-BR-3 cells.We used trypan blue staining to detect the number of living cells of all groups and then drew growth curve.SK-BR-3 cells were exposed to Oug/ml,50ug/ml,100ug/ml,200ug/ml,400ug/ml,800ug/ml of aqueous extract of myrrh for 24h and 48h.We used trypan blue staining to detect the numbers of living cells of all groups.We used Flow Cytometry(FCM)to detect the cell cycle and apoptosis of each group.Further,we used western blot analysis to detect the influence of expression of CyclinA,BCL-2,BAX and Cleaved-Caspase8.3 SK-BR-3 cells were exposed to different concentrations(Oug/ml,50ug/ml,100ug/ml,200ug/ml)of aqueous extract of myrrh and we used western blot analysis to detect the protein expression of HER2,P-HER2,ERK,P-ERK,MEK,P-MEK,AKT,P-AKT in molecular pathways.4 In vivo experiments,we first implanted SK-BR-3 into the forth mammary gland of BALB/c mice to establish breast cancer models.After tumors grew up,we stripped tumors and transplanted to subcutaneous of 30 BALB/c mice.Then,BALB/c mice were randomly assigned to three groups:control group were gave normal saline by gavage;low-dose group(100mg/kg/d aqueous extract of myrrh by gavage)and high-dose group(200mg/kg/d aqueous extract of myrrh by gavage)n=10 per group.Every two days we measured the size of all tumors and recorded,all mice were killed after 23 days.Then we stripped tumors and measured tumors weight.PCNA and vimentin protein expression levels were detected by Immunohistochemical assay.Results:1 After treatment with aqueous extract of myrrh,the migration of the SK-BR-3 cells were inhibited:In the wound healing assay,SK-BR-3 cells were exposed to concentrations of 50ug/ml,100ug/ml and 200ug/ml of aqueous extract of myrrh for 24h,compared with the control group,the inhibition rate of the migration distance were 21.6±0.401%?36.9±2.81%?38.7±2.08%respectively(as show in Fig.1A),the difference compared with the control group is significant(P<0.05).In transwell chamber migration assay,SK-BR-3 cells were exposed to concentrations of 50ug/ml,100ug/ml and 200ug/ml of aqueous extract of myrrh for 18h,SK-BR-3 cells numbers that had migrated to the lower chambers were 1839.33±142.73/well in the control group,in 50ug/ml?100ug/ml?200ug/ml groups,SK-BR-3 cells numbers that had migrated to the lower chambers were 1773.67±184.27,1024.67±146.55,779.00±43.28/well respectively.100ug/ml?200ug/ml groups compared with the control group,the difference is significant(As show in Fig.IB).But 50ug/ml group compared with the control group,the difference is not significant(P>0.05).2 Aqueous extract of myrrh inhibited invasion of HER-2 positive breast cancer SK-BR-3 cells:After treatment with aqueous extract of myrrh for 24h,the number of cells invaded through the Matrigel-coated filter was 505.33±77.98/well in control group.In the concentration of 50ug/ml,100ug/ml and 200ug/ml of aqueous extract of myrrh groups,the number of cells invaded through the matrigel-coated filter were 482.33±33.50,239.67±27.47,209.33±46.31/well respectively.100ug/ml?200ug/ml groups compared with the control group,the difference is significant(P<0.05).But 50ug/ml group compared with the control group,the difference is not significant(P>0.05).3 Aqueous extract of myrrh inhibited epithelial-mesenchymal transition of HER-2 positive breast cancer SK-BR-3 cells:Immunofluorescence analysis and western blot analysis displayed that SK-BR-3 cells were exposed on a concentration of 100ug/ml,200ug/ml aqueous extract of myrrh after 24h,the expression of E-cadherin significantly increased and the expression of the N-cadherin and Vimentin was inhibited significantly(As show in Fig.2A,B).4 Aqueous extract of myrrh destroyed the Cytoskeleton of SK-BR-3 cells and decreased filopodia on the cell membrane:Immunofluorescence analysis also found the Cytoskeleton of SK-BR-3 cells was destroyed and filopodia on the cell membrane was decreased significantly which were exposed on a concentration of 100ug/ml,200ug/ml aqueous extract of myrrh after 24h compared with the control group(As show in Fig.2C).5 Aqueous extract of myrrh could inhibite proliferation of SK-BR-3 cells:SK-BR-3 cells were dealt with different concentrations(50ug/ml,100 ug/ml,200ug/ml)of aqueous extract of myrrh for different time(24h,48h,72h,96h,120h,144h)respectively,we tested the cell viability change with trypan Blue exclusion assay and drew the growth curve.SK-BR-3 cells were dealt with different concentrations(50ug/ml,100ug/ml,200ug/ml,400ug/ml,800ug/ml)of aqueous extract of myrrh for 24h,48h respectively.We found the proliferation of SK-BR-3 cells was inhibited by aqueous extract of myrrh in a dose-and time-dependent manner.Further,Immunofluorescence analysis also found the fluorescence intensity of PCNA got weaken in the groups of aqueous extract of myrrh compared with the control group(As show in Fig.3B).6 Aqueous extract of myrrh could induce S?G2/M cells cycle arrest of HER-2 positive breast cancer SK-BR-3 cells:SK-BR-3 cells were dealt with different concentrations(50ug/ml,100 ug/ml,200ug/ml)of aqueous extract of myrrh for 96h,the proportion of SK-BR-3 cells at the S phase was increased 35.24±1.33%in control group,55.55±0.79%in 50ug/ml aqueous extract of myrrh group 68.62±0.63%in 100ug/ml aqueous extract of myrrh group.Western blot assay examined aqueous extract of myrrh induced the accumulation of cyclinA(As show in Fig.4C).7 We applied flow cytometry to analyze the apoptotic after treatment with aqueous extract of myrrh:The data demonstrated that the ratios of apoptotic were 0.93±0.83%in control group,2.00±0.36%in the 50ug/ml aqueous extract of myrrh group,2.00±0.53%in the 100ug/ml aqueous extract of myrrh group,9.63±2.20%in the 200ug/ml aqueous extract of myrrh group for 72 h,respectively.200ug/ml aqueous extract of myrrh group compared with the control group,the difference is significant(P<0.05).Western blot assay examined aqueous extract of myrrh induced accumulation of Bax and Cleaved-caspase-8,deseased expression of Bcl-2(As show in Fig.4F).8 Aqueous extract of myrrh could regulate multiple moleculars:We used western blot analysis to investigate the changes of between the control and aqueous extract of myrrh treated.Aqueous extract of myrrh dose-dependently inhibited the total and phosphorylation of HER-2,Akt,MEK and ERK in SK-BR-3 Cells(As show in Fig.5).9.Aqueous extract of myrrh inhibits tumor growth in vivo:We detected tumor after mice were treated with myrrh for 23 days,compared with the control group,the mice treated with aqueous extract of myrrh had a smaller mean xenograft tumor volume(control group 5933.18±780.70mm3,100ug/ml aqueous extract of myrrh group 3419.52±384.84 mm3,200ug/ml aqueous extract of myrrh group 2447.53 ±400.81mm3)(P<0.05).compared with the control group,the mice treated with aqueous extract of myrrh had a lighter mean xenograft tumor weight(P<0.05).(control group 5.07 ±0.72g,100ug/ml aqueous extract of myrrh group 3.04±0.36g,200ug/ml aqueous extract of myrrh group 2.02±0.53g).10.In vivo,aqueous extract of myrrh suppresses PCNA and vimentin expression.The Proliferating Cell Nuclear Antigen(PCNA)and vimentin of the aqueous extract of myrrh group was significantly lower than that of the control group(As show in Fig6D).Conclusions:1 The results showed that aqueous extract of myrrh suppressed the metastasis of SK-BR-3 cells in vitro through inhibited EMT?destroyed Cytoskeleton and decreased filopodia on the cell membrane.2 Aqueous extract of myrrh inhibited the tumor proliferation in vitro through induction of S?G2/M arrest of the cell cycle and apoptosis.3 Aqueous extract of myrrh suppressed the proliferation and metastasis of SK-BR-3 cells through inactivating expression of total and phosphorylated HER-2,Akt,ERK,and MEK.4 Aqueous extract of myrrh suppressed the growth of SK-BR-3 cells in vivo.