Effects Of Celastrol On Leukemia Cell Proliferation And Its Molecular And Structural Mechanisms Via Inhibition Of HSP90

BACKGROUNDS AND AIMSLeukemia,a hematopoietic system of malignant tumors,is one of the top ten high incidences of cancer in China,and the incidence trends to increasing year by year,which of the prognosis is poor.With the development of modern medical technology,leukemia treatment has made great progress,but there are still shortcomings.Therefore,it is urging to explore new treatment strategies.At present,the treatments of leukemia are divided into the following categories:radiotherapy,chemotherapy,targeted therapy,immunotherapy and hematopoietic stem cell transplantation.Each method has its own advantages and disadvantages.Radiotherapy and chemotherapy have a certain effect only for some specific type of leukemia,and the patients are difficult to tolerate side effects resulting from the functions of some normal cells in their bodies are destroyed while tumor cells are killed at the same time.Immunotherapy has little side effects,so patient compliance is good.But it exists a certain risk for involving in cell manipulation in vitro,the treatment of cumbersome and the difficulty of quality control.Hematopoietic stem cell transplantation is currently the only way to cure leukemia.Autologous transplantation is not limited by donor sources,and has fewer complications,while the transplant may be the remaining tumor cells,resulting in high recurrence rate.Allograft may seriously affect the quality of life due to long-term use of immunosuppressive drugs for patients.Targeted therapy has relatively fewer side effects because of its identified target,but the tumor is often caused by multiple factors.So,the therapeutic effect is often not as good as expected.Based on the above analysis,it is an important development direction and breakthrough point for leukemia treatment strategy in future to find a(or some)targeted molecule(s)with a wider range.The role of heat shock protein 90(HSP90)in the development of tumor has been paid more and more attention in recent years.In tumor cells,HSP90 proteins can be more than ten times higher than normal cells,which help tumor cells survive in the harsher environment.Many of HSP90 client proteins are involved in tumor formation and progression.In all types of leukemia,HSP90 expression was significantly increased related to the poor prognosis.HSP90 inhibitors and other treatment of leukemia drugs in combination,can improve the treatment of leukemia.Therefore,inhibition of HSP90 may become a new strategy for leukemia targeted therapy.However,there is no HSP90 inhibitor in clinical application so far,indicating that the new discovery of HSP90 inhibitors and their mechanism still need to be further studied.Celastrol is a triterpenoid small molecule compound extracted from Tripterygium wilfordii,with a variety of biological activities.Our group members in 1990 s found that celastrol can induce apoptosis in lymphocytic leukemia cells,opening the research on celastrol in leukemia treatment.In recent years,researchers using gene expression signature(GES)technology have confirmed that celastrol is a novel HSP90 inhibitor.However,the mechanism by which celastrol inhibits HSP90 in the treatment of leukemia is still unclear.In this study,the mechanisms of celastrol in the treatment of leukemia werer discussed in detail from the aspects of the molecular and structure mechanisms of HSP90 inhibition.METHODSIn this study,the effects of celastrol on the proliferation in various kinds of leukemic cells and the HSP90 inhibition were observed systematically.The bioinformatics technique was used to analyze and predict the combination of celastrol and HSP90,as well as possible key groups.The uses of chemical,molecular biology,cell biology,bio-thermodynamics and other research methods and experimental techniques,were applied to explore the molecule and structure mechanisms of celastrol’s HSP90 inhibition.The study involves the main methods as follows:1,Cell cultureLeukemia cell lines were routinely cultured in RPMI1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin,and were maintained at 37℃,5% CO2 and proper humidity.Depending on the cell growth,replace the fresh medium.Cells in logarithmic growth phase were used for the experiment.2,Cell count by flow cytometry with CFSE labeled cell microspheres as internal referenceThis technology is a single-platform cell counting technique based on the flow cytometry with CFSE labeled cell microspheres as the internal reference.Cells were labeled with green fluorescent substance CFSE to prepare fluorescent microspheres.A certain amount of labeled cells was added into the cells to be counted.At the same time,the cells dead were labeled with red-fluorescent dye 7-AAD.The samples were separated from the cell microspheres by different fluorescence detection channels.The number of live cells,dead cells and total cells in samples were counted and obtained by calculation.3,Western blotThe total protein was extracted from different leukemia cells and the protein concentration was determined.A certain amount of protein was taken and subjected to SDS-PAGE.PVDF incubated with the corresponding primary antibody,and the corresponding secondary antibody.The band was collected by chemiluminescence imaging system,and the gray value of the band was analyzed.4,Molecular dockingTaking the Auto Dock software as an example,the main steps are as follows: the PDB files of celastrol and HSP90 protein are transformed into the format which can be recognized by software.The file is imported into the analysis software to pretreat the protein,such as removing the ligand molecule,repairing the amino acid residue,hydrogenation,charge,etc.,so that it can be recognized by the software Auto Grid;then,run Auto Grid to generate Grid file;and set the software operating parameters and run Auto Dock software and analyze the results.5,Human full-length HSP90 prokaryotic and eukaryotic expression vector constructionTarget genes were amplified by PCR and subjected to agarose gel electrophoresis.The PCR products collected were cutting by two different restriction sites and were ligated with plasmids.The plasmids were extracted and identified by DNA sequencing.6,Expression and Purification of wild-type and mutant HSP90 ProteinsUsing the wild-type HSP90 prokaryotic expression vector as template,the mutated prokaryotic expression vector plasmid was obtained by PCR method.The wild-type and mutant HSP90 prokaryotic expression plasmids were transformed into Arctic Express TM(DE3)prokaryotic host cells,and the target protein was induced by IPTG.The wild-type and mutant HSP90 fusion proteins with Tag were obtained by Ni-column affinity chromatography.The Tag was digested by protease to separated from HSP90 and purified by Ni column.The residual tagged fusion protein,Tags and proteases were removed,resulting in high purity wild type and mutant HSP90 proteins.7,Isothermal titration calorimetryHuman purified wild type and mutant HSP90 proteins were dialyzed through dialysis membrane,and protein concentrations were determined by BCA method to determine.MicroCal i TC200 instrument was applied to detect the combination of celastrol and wild type or mutant HSP90.The experiments were carried out as follows: a certain concentration of wild-type or mutant HSP90 proteins were added into the cell and a certain concentration of celastrol or 17-AAG was added into the syringe.Make sure the same concentration of DMSO with the diluent in the sample cell and syringe.The cell temperature was set at 25 ° C.A total of 13 titrations were carried out,with a titration rate of 3 μl each time and a titration interval of 180 s.The experimental data were fitted and analyzed with Origin 8.0 software.8,Cell transfectionCell transfection was performed according to Invitrogen Lipofectamine 2000 transfection reagent instructions.Cells were plated in 24-well plates so they will be 70%confluent at the time of transfection.Lipofectamine 2000 liposomes and transfected plasmid DNA were diluted with Opti-MEM medium to be the required concentration and incubated to form a DNA-liposome complex,which was added to the cells and transfected for 1-2 days.9,Co-immunoprecipitationThe transfected cells were collected and lysed with IP lysate to extract the total protein and the protein concentration was determined.The total protein was mixed with the anti-Flag antibody overnight,and the agar beads were added.After incubation,the beads were washed three times with cold PBS and then added to SDS loading buffer for Western blot analysis.10,Statistical analysisIn this study,all the data were expressed as mean ± standard deviation.The mean comparison between two groups was using the student t test,and the multiple-group was compared using the variance analysis.Each independent experiment was repeated at least 3times.RESULTSIn the first part,we found that celastrol has inhibitory effects on the proliferation of six leukemic cell lines commonly used in the study in a dose-dependent way.But the degree of proliferation inhibition is different in these leukemic cell lines.Three kinds of leukemia cells with obvious inhibitory effect on the proliferation of celastrol were selected,and it was found that the three leukemia cells were observed by the induction of the heat shock response(HSP90 inhibition).After the treatment with celastrol,the heat shock response was observed,and the expression of HSP70 was significantly increased,indicating that HSP90 was inhibited in the cells,but the expression levels of HSP90 and HSP40 did not change significantly.These results indicate that celastrol inhibits leukemia cell proliferation,possibly by inhibiting HSP90 function but not protein expression.In the second part,it was pleasantly surprised to find that there may exist a celastrol-binding pocket in HSP90 dimer with nucleotide-free a Apo state,which consists of two monomers of HSP90 dimer,that is,Tyr493,Val530,Gln531,Thr540,Val542,Lys546,Gly548 on a HSP90 monomer,and Lys582,Thr683 on another HSP90 monomer.The carboxyl group of celastrol forms two very strong salt-bridges with Lys546 from one monomer and Lys582 from another monomer.In order to confirm the results of bioinformatics prediction,we examined the key binding groups on celastrol and HSP90 in the third and fourth parts,respectively.In the third part of the study,we used three glycine-containing short peptides to replace the hydroxyl group of celastrol,and found that the effects of celastrol on leukocyte proliferation and induction of heat shock were lost.In the fourth part,we performed two mutations of the two key binding amino acid groups on HSP90,Tyr493 and Lys546,to study the effect of these two key groups on the direct binding between celastrol and HSP90 and on the HSP90 complex.Through the ITC experiment,we observed the combination of celastrol and HSP90 protein.It was found that celastrol could bind to HSP90 protein directly,while not to mutant HSP90 protein.The ability of celastrol combined with HSP90 protein was stronger than classical HSP90 inhibitor 17-AAG.By co-immunoprecipitation method,it was found that the expressions of Cdc37,Cdk4,HSP70 proteins in wild-type HSP90 complex were significantly decreased by celastrol treatment,while these changes were disappeared in mutant HSP90 complex.We also found that the ability of binding HSP90 mutation was significantly reduced in Cdc37,but retained in Cdk4 and HSP70.It suggested that the two amino acid sites on HSP90 may also be important for binding to Cdc37.CONCLUSIONTargeted therapy for leukemia requires a new strategy,and HSP90 may be involved in the development of various types of leukemia.Herbal extract celastrol has a pharmacological effect in the treatment of leukemia and has been shown to be a novel HSP90 inhibitor.Our previous studies and other studies have found that celastrol can inhibit leukemia cell proliferation,may be related to its inhibition of HSP90.In this study,the molecular and structure mechanisms of celastrol were studied on the proliferation of leukemia cells by inhibiting HSP90.Using the methods of cell biology,molecular biology and bio-thermodynamics,we first confirmed that celastrol could bind directly to HSP90 and that two amino acid groups on HSP90 and celastrol’s carboxyl group were play key roles in celastrol’s HSP90 Inhibition,and thus inhibit the proliferation of leukemia cells.This study provides a more solid theoretical basis for the treatment of leukemia with celastrol,and the pharmacological mechanism of celastrol and drug modification,which also lays the foundation for the clinical application of celastrol.