Network Pharmacology-based Prediction Of Chinese Herbs In Treating Metastatic Breast Cancer Patients

Objective:Breast cancer(BC)is one of the most common cancers threatening the female health,whose mortality is second only to lung cancer among female malignances.Chinese Herbal Medicine(CHM)plays a critical role in treating cancers.However the mechanisms of CHM in treating BC are extremely complicated and need to be explained in depth for its complex ingredients and targets.In this study,we retrospectively selected metastatic breast cancer(MBC)patients in stage Ⅳand divided them into two groups based on whether they had received CHM treatment or not.We compared the difference of survival benefits between the two groups after following ups and related statistics.In further research,we analyzed the CHM ingredients in treating MBC patients with the method of Network Pharmacology and predicted the treating targets based on their molecular structures.Furthermore,we concluded the main mechanisms of each Chinese herb in order to provide evidence for doctors to choose.In the end,we carried experimental studies to verify the anti-breast cancer effects and mechanisms predicted of Trichosanthes Kirilowii-one of the effective herbs above which are highly related to survival benefits in CHM group patients.Methods:1.Cases collection: Patients with metastatic breast cancer between June 2004 and December 2011 in Tianjin Medical University Cancer Hospital were retrospectively studied.Information about the patients was collected from inpatient and outpatient medical records in Tianjin Medical University Cancer Hospital or from direct patient follow-up visits.We divided the patients into CHM group and non-CHM group.2.Survival analyses: We adopted Kaplan-Meier curves and Cox regression analysis to compare the survival benefits of two groups.We conducted statistics of the median survival time and 1-,3-and 5-year survival rates for the CHM and non-CHM groups and judged whether CHM was an independent proactive factor.3.Network Pharmacology analysis: The CHM formulas were put into statistical analysis.The herb using frequencies were calculated.Spearman bivariate correlation analysis was used to determine the correlation between herbs and survival time.Online searching based on TCMSP and TCMID was conducted to find active ingredients and targets.BC related targets were found on TTD.We matched the targets of BC and herb ingredients.Then the ingredients-targets networks were constructed for these herbs using Cytoscape software.The meaning targets were concluded as several classic signaling pathways.4.According to the results predicted above,we chose Trichosanthes Kirilowii Maxim as study object.Add different concentrations of TKE into cultured SKBR-3 cells.Trypan Blue experiment was performed to observe the anti-proliferation effect of TKE on BC cells after 48 h.5.Select cucurbitacin B(CuB)and set up different concentration groups on SKBR-3 cells.Observe the number and morphology change after 24 h.Trypan Blue experiment and CCK-8 assay were conducted to the anti-proliferation effect in different time.6.The experimental groups were set as above.Wound-healing assay and Transwell assay were conducted to explore effect of CuB on migration of SKBR-3 cells.7.The experimental groups were set as above.We use Flow Cytometry to detect the apoptosis-inducing effect of CuB on SKBR-3 cells.8.The experimental groups were set as above.We use Flow Cytometry to detect cell-cycle distribution changes of SKBR-3 treated by CuB.9.Western-blot assay was carried out to detect protein expression changes of SKBR-3 treated by CuB.Results: 1.Survival analysis:(1)Overall 182 MBC patients were brought into the study.Among that 78 were CHM using patients and 104 under non-CHM treatment.(2)The median survival time of CHM group(55 months)was longer than that of the non-CHM group(23 months).The 1-,3-and 5-year survival rates for the CHM and non-CHM groups were 96.0%,69.0%,44.0%,and 72.1%,36.5%,24.7%,respectively(p< 0.001).(3)Cox regression analysis results revealed that,among the protective factors,CHM was an independent one.The hazard ratio(HR = Exp[β])of CHM treatment was 0.591 and the associated 95% confidence intervals ranged from 0.395 to 0.885(p < 0.05).2.Network Pharmacology analysis:(1)Spearman bivariate correlate analysis showed that 10 herbs with high correlation coefficients with improved survival benefits were Cervus Nippon Temminck(NT),Ginger Charcoal(GC),Citri Reticulatae Pericarpium Viride(RP),Phytolaccae Radix(PR),Licorice(Lic),Trichosanthes Kirilowii Maxim(KM),Citri Reticulatae Folium(CR),Panax Notoginseng(PN),Epimedium Herb(EH),Fritillariae Thunbergii Bulbus(FTB).(2)After searching on TCMSP and TCMID,we got 1192 ingredients and 2736 targets in the 10 herbs.A total of 120 candidate targets about BC were found through TTD.Finally there were 254 ingredients and 81 targets related to BC treatment in 10 herbs demonstrated by Ingredient-target network(c I-c T).(3)We summarized the major targets and found that ER,HSP90,TOP and TOP-II related proteins were the main ones.There were also targets in apoptosis pathway on which ingredients in herbs such as Trichosanthes Kirilowii Maxim could act.3.Experimental confirm:(1)The anti-proliferation effect of TKE on SKBR-3 cells: SKBR-3 cells were collected 48 h after treated with groups of TKE(2.5、5、10、20μg /m L).CCK-8 assay revealed that the growth of BC cells was depressed most obviously under the concentration of 20μg /m L.(2)The anti-proliferation effect of CuB on SKBR-3 cells: Set up different concentrations of CuB groups(30、60、120 n M).CCK-8 assay showed the OD values in the 3 groups above were decreased in a dose-dependent and time-dependent manner(p<0.0001).What’s more Trypan Blue experiment showed the similar result(p<0.0001).(3)The effect of CuB on apoptosis and cell cycle of SKBR-3 cells: Set up different concentrations of CuB groups(30、60、120、240 n M).The Flow Cytometry detected that the apoptosis cells increased.The apoptosis ratio was most significant in the 240 n M group-59.63(±0.85)%.The cells were blocked in G2/M phase in cell cycle.(4)The effect of CuB on SKBR-3 migration: Wound-healing assay: From the picture we can observe the anti-migration effect of CuB on SKBR-3 cells.The migration distance were reduced compared with blank control group(p<0.0001).Transwell assay: The numbers of migrated SKBR-3 cells were decreased significantly after treated with concentrated of CuB for 12 hours compared with negative group(p<0.0001).(5)Verify of CuB on anti-BC mechanism: Western-blot analysis showed that the expression on proteins related to the process of proliferation and apoptosis has changed a lot after treated with CuB.In the high CuB group(120n M),the expression of AKT,Ras and Bcl-2 were decreased.Meanwhile,Caspase-9,a protein promoting apoptosis,had increased.Conclusions: 1.The clinical part of the study showed CHM could improve the survival benefits of MBC patients significantly.2.Network Pharmacology analysis demonstrated that herbs related to survival benefits mainly worked through acting on molecules such as ER,HSP90 and TOP-II.3.CuB could indeed play an anti-BC effect through suppressing the process of proliferation and migration and inducing apoptosis.The mechanisms of that were partly consistent with the predicted results by Network Pharmacology.So the Network Pharmacology could provide us novel methods and ideas to study CHM anti-tumor mechanisms.