The Anti-breast Cancer Effect Of Arenobufagin And Its Underlying Mechanism

Breast cancer has been the most commonly diagnosed cancer in the world,and is the leading cause of female cancer death.Up to now,effective treatment strategies are still lacking.Arenobufagin is one of the main anti-cancer active ingredients of the traditional Chinese medicine‘toad venom’.However,the anti-cancer effect of arenobufagin and its underlying molecular mechanisms have not been clearly elucidated.In this thesis,we systematically tested the anti-cancer effect of arenobufagin and its underlying molecular mechanisms in breast cancer.The main research contents and results are as follows:（1）Arenobufagin inhibits breast cancer cell proliferation and induces apoptosis.MTT and colony formation experiments showed that arenobufagin could significantly inhibit the proliferation of MDA-MB-231 and MCF7 breast cancer cells,and the IC₅₀values at 48 h were 27.72±1.55 n M and 33.11±1.55 n M,respectively.Flow cytometry assay showed that arenobufagin could induce apoptosis of breast cancer cells in a dose-dependent manner.Western blot experiments further proved that arenobufagin activated the exogenous apoptosis pathway associated caspase-8 protein and the endogenous apoptosis pathway associated caspase-9 protein,and induced the cleavge of their downstream PARP protein,which indicated that arenobufagin could activate both endogenous and exogenous apoptotic pathways.（2）Arenobufagin regulates the CIP2A/PP2A/c-Myc/signaling pathway.In the research of anti-cancer mechanism of arenobufagin,western blot results showed that arenobufagin could quickly clear the c-Myc protein,which is considered to be an oncoprotein that plays a key role in the occurrence and development of a variety of cancers（including breast cancer）,and is currently lack of target drugs.It is of great significance to study the molecular mechanism of arenobufagin-triggered down-regulation of c-Myc.The results of semi-quantitative PCR and cycloheximide（CHX）chase experiments showed that arenobufagin could down-regulate the expression of c-Myc protein at the post-translational level.Further studies demonstrated that proteasome inhibitor MG132 significantly inhibited arenobufagin-triggered c-Myc degradation,suggesting that arenobufagin induced the degradation of c-Myc via the proteasome pathway.E3 ubiquitin ligase plays a key role in the degradation of ubiquitinated substrates.Using western blotting experiments,we found that arenobufagin significantly increased the expression of E3 ubiquitin ligase Fbw7 that mediated the c-Myc degradtion.It was reported that protein phosphatase 2A（PP2A）pathway activation induced Fbw7-mediated c-Myc degradation and the threonine 58（T58）of c-Myc protein played a key role in this degradation pathway.Western blotting results further showed that arenobufagin significantly decreased the endogenous PP2A inhibitory protein CIP2A,and another target protein of PP2A,AKT was also inhibited by arenobufagin,indicating that arenobufatin might activate PP2A.In addition,PP2A activator DT061 also down-regulated the expression of c-Myc protein.Then,the c-Myc T58 mutation vector was constructed and transfected into breast cancer cells.The result showed that T58 mutation could inhibit the degradation of c-Myc protein induced by arenobufagin.The above results indicate that arenobufagin induces c-Myc degradation through the E3 ubiquitin ligase Fbw7-mediated proteasome pathway by activating PP2A.（3）Arenobufagin sensitizes the anti-breast cancer activity of docetaxel（Doc）.Doc is a commonly used chemotherapeutic drug for the treatment of breast cancer.The next step in this thesis is to study the effect of combined use of arenobufagin and Doc in the treatment of breast cancer and the molecular mechanisms involed.The results of MTT and immunoblotting assays showed that arenobufagin could synergistically sensitize the anti-breast cancer activity of Doc in both MDA-MB-231and MCF7 breast cancer cells via inhibiting the target protein c-Myc and AKT of PP2A.In summary,our thesis showed that arenobufagin could inhibit proliferation and induce apoptosis in breast cancer cell.It was reported for the first time that the anti-breast cancer activity of arenobufagin was related to its effect on CIP2A/PP2A/c-Myc signaling pathway followed by Fbw7-mediated proteasomal degradation of c-Myc.In addition,our study also found that arenobufagin could sensitize the anti-breast cancer effect of Doc.In conclusion,as a natural small molecule compound that significantly inhibits the expression of oncoprotein c-Myc,arenobufagin has an important application and development value against breast cancer or other c-Myc-related cancers.