Anticancer Activity Of Buxus Alkaloids On Targeting The Degradation Of Mutant P53 And Traditional Chinese Medicine For Osteoarthritis Bone Remodeling

Malignant neoplasia is a broad group of diseases involving unregulated cell growth.Cancer is a serious disease,which threatens to human health and life.p53 gene has showed a high mutation rate in all kinds of cancers.Over 50%of cancers are p53 mutations.They are more common in particular lung cancer,colorectal cancer and breast cancer.The molecular chaperones of Hsp90,Hsp70 protein are important factor in the stability of mutant p53 in cancer cells.It was accompanied by the continued high expression of p53 mutation and Hsp90/Hsp70 in the occurrence and development process of cancers.Therefore,it has become a hot anticancer drugs research area that targets mutant p53,Hsp90 and Hsp70.The Buxus alkaloids(KBA01/KBA02)extracted from B.Microphylla and B.austroyunnanensis.Firstly,we screened the Buxus alkaloids for anti-tumor activity in HCT116 cell(wild-type p53)and HT-29 cell(mutant p53 R273H).It was found that the Buxus alkaloids were more toxic in HT-29 cells with IC50 value of 22.48 ±1.30,compared with HCT116 cells with IC50 value of 6.00±2.03.Additionally,the western blot analysis suggested Buxus alkaloid reduced protein levels of mutant p53,Hsp90 and Hsp70.However,the real-time PCR results showed that the mRNA levels of mutant p53,Hsp90 and Hsp70 had been upregulated marginally.KBA01 in combination with Hsp90 inhibitor BIIB021 in HT-29 cell,KBA01 enhanced the effect of BIIB021 killing HT-29 cell.Cell cycle analysis,suggested that HT-29 cells was blocked in S-G2/M phase after 10μM KBA01/KBA02 treatment for 48h and 72h.It indicates that the cell cycle arrest caused the inhibition of tumor cell growth by Buxusal kaloids.In order to verify whether the Buxus alkaloids activated HSF1 expression,eGFP-HSE-transfected 3T3-Y9 cells were used.As we all know heat sock response(HSR)can be activated by Hsp90 inhibitor and protease inhibitor,thereby activating transcription of HSF1.There was no expression of eGFP fluorescence,when we treated 3T3-Y9 cell by the boxwood alkaloid.In addition,the western blot analysis suggested that Buxus alkaloids upregulated the expression of HSF1 protein.Therefore we concluded Buxus alkaloids may not activate HSR.In summary,this part of study found that Buxus alkaloids were simultaneously targeting degradationof mutant p53,Hsp90,Hsp70 protein.And it does not activate HSF1 protein expression.This indicating Buxus alkaloids has broad prospects in Cancer therapyas potential anti-tumor drugs.The main pathological features of osteoarthritis are damage of articular cartilage degeneration,reactive hyperplasia of bone edge and articular cartilage,imbalance of subchondral bone synthesis and catabolism.The process of subchondral bone remodeling dominated in bone resorption in early stages,lately developing of increased bone formation,resulting in subchondral bone sclerosis.For a long time,the main method of treatment for OA was to reduce drastically the joint activities and excessived weight to delay progression of disease.But it does not improve bone tissue lesions radically.Therefore,the research on molecular mechanisms of bone remodeling and pathway of bone resorption stimulating factor had become a hot topic in the study of OA.Firstly,we constructed a rat model of osteoarthritis in vivo,then treated a month by using gavage method.Taken the subchondral bone of rats for HE stained,pathological analysis suggested that OA had healed.Extract proteins from subchondral bone to do western blotting,found that the drugs treatment group’s OPG protein expression had been upregulated,indicating that traditional Chinese medicine increased the expression of OPG protein.The real-time PCR analysis obtained RANKL/OPG ratio decreased,indicating that the traditional Chinese medicine had significantly decreased the number of OC and the activity of OC.While PKA mRNA high expression and gp130 mRNA did not change significantly,indicating that the process of traditional Chinese medicine for treatment,the rate of bone formation in vivo is far greater than bone resorption.PKA mRNA is highly expressed,while gp130 mRNA did not change significantly.It indicating that after traditional Chinese medicine treatment,the rate of bone formation is far greater than bone resorption in vivo.To further study the specific mechanisms of bone remodeling,we simulated the organism microenvironment in vitro.Maturation MC3T3-E1 and RAW264.7 be induced and co-culture.Due to the complexity of Chinese medicines,drug-containing serum was been made.Using the principles of drug-containing serum,we try to find the direct intrinsically linked between traditional chinese medicines and subchondral bone remodeling.After co-cultured cells in treatment of containing serum,we observed the increased of OPG protein expression and the decreased of protein RANKL/OPG ratio.It suggested that the traditional Chinese medicine indirectly inhibit the differentiation and maturation of OC by regulating the ratio of RANKL/OPG.Then it induced OC apoptosis.The real-time PCR analysis indicating that gp130 gene has been significantly increased,while PKA gene did not change after the containing serum treatment.It indicating that after containing serum treatment,gp130 gene expression is significantly suppression,thereby inhibiting the IL-6 signal pathway in the cell,and also inhibits the proliferation and differentiation of OC and bone resorption.Therefore,we can drawn a conclusion that the traditional Chinese medicine can be beneficial to the healing of osteoarthritis.