Huaier Sensitizes Radiotherapy And Endocrine Therapy Of Breast Cancer Through Regulating DNA Repair Pathway

ObjectiveBreast cancer is a kind of common malignancy and ranks in the first position in females,which is a serious threat to women's health.At present,breast cancer is defined as a systemic and chronic disease,thus,a comprehensive treatment strategy should be performed including surgery,chemotherapy,radiotherapy,endocrine therapy and target therapy,et al.Among these therapeutic methods,radiotherapy and endocrine therapy play critical roles and are extremely important in the comprehensive treatment of breast cancer.Radiotherapy acts as one of the effective comprehensive methods since breast cancer is sensitive to radiation.Radiotherapy is mainly applied before,in or after the surgery,and also in the palliative treatment or in the metastasis of breast cancer.Radiotherapy can significantly reduce the local recurrence and the metastasis of breast cancer.Besides,radiotherapy is a key strategy in symptomatic treatment of breast cancer bone metastasis.Along with the development and application of CRT(Conformal Radiation Therapy),IMRT(Intensity Modulated Radiation Therapy),IGRT(Imaging Guided Radiation Therapy)and respiratory gating technique,radiotherapy becomes a more accurate therapy,and plays even widely therapeutic effects,with reduced toxic side effect and enhanced gain ratio.As we know,breast cancer is closely related to estrogen level.Endocrine therapy is of importance for estrogen receptor positive breast cancer.At present,it is believed that a better curative effect and better prognosis would be achieved if endocrine therapy is used earlier and primary after surgery.Endocrine therapy of breast cancer mainly includes drugs of ER antagonist such as tamoxifen,and drugs degrading ER,such as fulvestrant.In conclusion,radiotherapy and endocrine therapy is critical strategies in breast cancer treatment,and great benefit would be gained if the two strategies be used properly.In fact,however,resistance and side effect come closely along with the long-term use of the two strategies,which seriously limited its wide application in breast cancer.So,how to sensitize radiotherapy and endocrine therapy becomes a focus of breast cancer comprehensive treatment.More recently,Chinese medicine gains more and more attention as its multi-antitumor effect and low toxicity.Huaicr,an extract of Trametes robiniophila murr mainly containing glycoprotein,is a kind of TCM which has been used in China for many years.Previous investigations suggest that Huaier displays multiple biological effects,such as inhibiting tumor proliferation,invasion,metastasis,and regulation of immune function.Huaier is widely used in hepatocarcinoma and breast cancer clinically,but there is no research in whether Huaier had a sensitization effect in breast cancer radiotherapy and endocrine therapy.The objective of this study is to uncover the effect of Huaier on radiotherapy and endocrine therapy,which may provide an experimental basis for clinical usage.MethodsSince Huaier plays multiple biological effects,to investigate whether there is a radiosensitization effect of Huaier,MCF-7 cells were treated with 8 mg/mL Huaier for 72 h,after which RNA was extracted.Then HTA 2.0 gene expression array was used and the differential genes were analyzed in Gene Ontology(GO)method to speculate Huaier's effect on radiotherapy.Next,according to the gene expression array,clone formation assay was used for a further verification.Since there is 4"R" rules related to radiotherapy reaction,flow cytometry was used to detected alteration of cell cycle after radiotherapy with or without Huaier.A further western blot experiment was performed to make sure the specific mechanism of cell cycle changes.Immunofluorescence staining was used to detected y-H2AX foci to evaluate the conditions of double stands breaks(DSBs)of DNA.And a further western blot experiment was used to uncover the specific mechanism.To research whether there was a sensitization effect of Huaier on endocrine therapy,we collected several microarrays related to tamoxifen and fulvestran resistance on PUBMED,and analyzed using GCBI,a network analysis tool.The microarrays samples included MCF7 cells which acted as control,MCF7 cells which is resistant to tamoxifen,MCF7 cells which is resistant to fulvestrant,MCF7 cells which was 48h after treated with fulvestrant,and MCF7 cells which was silenced in ERa.The differential genes were get and an Gene Ontology(GO)analysis was performed to evaluate the importance of DNA repair function to endocrine therapy.MTT assay was used to evaluate the proliferation difference of tamoxifen and fulvestrant with or without combination of Huaier.Next,clone formation was used to further evaluated Huaier's effect on endocrine therapy.Flow cytometry was used to detect apoptosis and cell cycle change of endocrine therapy in combination with or without Huaier.Western blot was also used to uncover the mechanism.In vivo tumorigenesis array was also established under tamoxifen treatment with or without Huaier.The tumor size was measured and immunohistochemistry assay was conducted.According to result of western blot,cells overexpressing Rad51 or low-expressing Rad51 was established and a remedial experiment was carried out to further verify the role of Rad5 l in the sensitization effect of Huaier.Results1.Huaier sensitize breast cancer cells to radiotherapy.Gene expression assay of MCF-7 cells after treatment of Huaier at 8 mg/mL for 72 h indicated that,Huaier altered biological activities related to cell cycle,cell division and DNA repair,which ranks the top position.This suggested that Huaier might sensitize breast cancer to radiotherapy,so next we used clone formation assay to a further confirmation,which finally indicated that Huaier sensitize breast cancer to radiotherapy.SF2 value of Huaier group(radiotherapy combined with Huaier)was significantly reduced about 47.65%compared to control group(single radiotherapy)(p=0.01 8).Next,linear quadratic formula was used to analyze the ? and ? components The a and ? value suggested two significantly different curves using linear regression analysis(p<0.01).The result was the same in MDA-MB-468 cells.2.Huaier sensitized breast cancer to radiotherapy through altering cell cycle and interfering DNA repair pathway.Flow cytometry was used to analyze cell cycle progression and G0/G1 arrest was found when cells were treated with Huaier at 4 mg/mL for 24 h.Within the 24 hours after irradiation,MCF-7 radiated cells(6 Gy)were found to have a block at the G2/M phase,whereas in cells pre-treated with Huaier this radiation-induced G2/M phase blocking was not obvious.To determine the mechanism of G0/G1 arrest,we detected the protein level of cell cycle regulating proteins by western blot and were able to demonstrate that two important cycle-regulating proteins,Cyclin D1 and CDK4,were obviously decreased in both cell lines treated with Huaier,which may explain the G0/G1 cell cycle arrest for both cell lines.Huaier pre-treatment prolonged the persistence of DNA double-strand breaks(DSBs).?-H2AX foci were detected using immunofluorescence staining to evaluate conditions of DNA double-strand breaks(DSBs)at 0 h,0.5 h,4 h,8 h,and 12 h after 1 Gy irradiation in both MCF-7 and MDA-MB-468 cells.In cells without Huaier treatment,?-H2AX foci appeared most prominently at 0.5 h,decreased at 4 h,and almost cleared at 8 h,while in cells pretreated with Huaier,y-H2AX foci were not completely cleared even at 12 h.Thus,pre-treatment with Huaier prior to radiation seems to be associated with a longer interval of DSBs,which may contribute to its radiosensitization effect.Huaier interfered DNA repair through homologous recombination(HR)pathway Ku70 and Ku86 are markers of non-homologous end joining(NHEJ),while Rad51 is related to homologous recombination(HR).Results of western blot experiment showed the protein levels of Ku70,Ku86 and Rad51.In MCF-7 cells,pre-treatment of 4 mg/mL Huaier leading to a decrease in Rad51 but not in Ku70 and Ku86.In cells pre-treated with Huaier compared to cells of control,treatment with 6 Gy irradiation resulted in decreased Rad51 levels in a time-dependent fashion(0 h,2 h,6 h,24 h).However,protein levels of Ku70 and Ku86 were not changed in either group.These results were similarly found in MDA-MB-468 cells.3.Analysis of gene expression microarray sharing on the web indicates that endocrine therapy and resistance may closely relate to DNA repair.Considering the analysis result of the MCF7 microarray of Huaier,a conclusion might be prompted that Huaier may sensitize endocrine therapy.Microarrays sharing on PUBMED were analyzed using GCBI web tool.The differential genes were further analyzed using GO(gene ontology)method.Results indicated that DNA repair function changed significantly in cells which were resistant to tamoxifen or fulvestrant,in cells which were treated with fulvestrant for 48h,and in cells which were silenced in ERa.GO functions were arranged according to its significance of alteration,which indicate that DNA repair ranks in the top 50 position.Eventually,in the MCF7 cells treated with fulvestrant for 48h,the DNA repair function ranks the third position.Combined with the previous conclusion which indicated Huaier's role in regulation of homologous repair of DNA,we made a speculation that Huaier could sensitize endocrine therapy through interfering DNA repair pathway.4.Huaier sensitized endocrine therapy of breast cancerResults of MTT indicated that,inhibition rate was significantly decreased when cells were treated in combination of endocrine drugs and Huaier,compared to endocrine drugs alone.CDI value(Coefficient of drug interaction)<1,some value of CDI<0.7,suggested Huaier had sensitization effect on tamoxifen and fulvestrant.Clone formation assay indicate a much less clone number treated with tamoxifen or fulvestrant combined with Huaier.A further experiment was conducted to detect the alteration in apoptosis and cell cycle using flow cytometry.A significant apoptosis rate was observed in cell treated in endocrine drugs combined with Huaier.G0/G1 cycle arrest was more obvious in the combination group,with a significant down-regulation of cells in S phase.The above results indicated that Huaier could sensitize endocrine therapy of breast cancer.5.Huaier sensitized endocrine therapy of breast cancer through regulating DNA repair pathway.Western blot experiment indicated that tamoxifen,fulvetrant and Huaier could down regulate Rad51,interfering DNA homologous repair.However,compared to cells treated with Huaier or endocrine drugs singly,the combination of the two drugs led to a more significant down-regulation of Rad51,and also CyclinDl which plays important role in combination of Rad5 1 to BRCA2.?-H2AX level was significantly up-regulated in cells treated in combination of endocrine drugs and Huaier,which indicated a persistent exist of DNA break.In vivo experiments were established which showed that tumors treated with tamoxifen in combination with Huaier group was obviously smaller than that treated with single tamoxifen or Huiaer.Furtherly,a reverse experiment was performed.Rad51 was overexpressed or kncoched down in MCF7 and T47d,and MTT assay was used to indicate that antitumor effect of endocrine drugs was decreased in cells overexpressing Rad51 and increased in cells low-expressing Rad51.These all indicated that Rad51 could regulate the sensitivity of endocrine drugs in breast cancer.Conclusions(1)Huaier sensitizes radiotherapy through interfering DNA repair pathway by targeting Rad51 and downregulation of CyclinD1,which is an important cofactor in homologous repair.This lays a vital theoretical foundation for combination use of radiotherapy and Huiaer.(2)endocrine therapy and resistance of breast cancer is closely related to alteration of DNA repair function,which is an effective target to sensitize endocrine therapy.(3)Huaier downregulates Rad51 and reduce the combination of Rad51 to the DNA damage position.Thus,Huaier has sensitization effect to endocrine therapy of breast cancer.Significances1.Action mechanism of Huaier was furtherly clarified since Rad51 was confirm as a target of Huaier in this study.2.Alteration of DNA repair function was closely related to endocrine therapy,and this may provide a new idea for research and clinical management of endocrine resistance.3.Huaier could radiosensitize breast cancer cells,which would provide an experimental basis for clinically combination use of radiotherapy and Huaier.4.Huaier could sensitize breast cancer to endocrine therapy,which would provide an experimental basis for clinical use of combination use of endocrine therapy and Huaier.