| Multiple myeloma is a malignancy of plasma cells that accumulate in the bone marrow,secret antibody,and interfere with the production of normal blood cells.Now it is the second most common hematological malignancy in the U.S.with median survival of 3-4 years.In this decade,proteasome inhibition revolutionized myeloma therapies,in especial,bortezomib can induce high and quality response rates by effectively targets the constitutive proteasome subunit ?5 of 26S proteasome.However,myeloma cells acquire resistance to bortezomib remains a major concern to bortezomib administration.The second generation proteasome inhibitors and other small ubiquitin-like molecule(NEDD8)activating enzyme inhibitor,MLN4924,are development and bring to ongoing clinical trails.Ckslb(Cyclin-dependent kinases regulatory subunit 1),is an essential protein for normal cell division and growth have been demonstrated through various genetic and biochemistry experiments in different species.It is expressed at physiologically high levels have been found in different cancers,such as hepatocellular carcinoma,colorectal cancer,lung cancer,oral squamous cell carcinoma and breast cancer et al..In multiple myeloma(MM),amplification of region 1q21,including Cks1b gene,was identify further subgroups of poor prognosis,aggressive clinical course and few therapeutic options(Shaughnessy J,2005;Mikala G,2005).Chang et al.found Ckslb amplification is associated with transformation from MGUS to MM and progression to plasma cell leukaemia(PCL)(Chang H,2007),Our previous studies on 70 high-risk genes model also showed that Ckslb was inversely associated with survival in newly diagnosed MM,and Cks1b over-expression promotes MM cell drug-resistance,providing direct evidence of crucial role of Ckslb in MM progression(Shi L,2010).Furthermore,immunohistochemistry in bone marrow of 60 cases of relapsed/refractory MM patients showed that nuclear expression of Ckslb is an adverse prognostic factor for patients with MM treated with bortezomib therapy(Chen MH,2012).Clinic data and in vitro OE cell lines both performed that Ckslb is a drug resistance gene for bortezomib,but if it is a multiple drug resistance gene,if it is cross resistance with other ubiquitin-proteasome system inhibitors is not clear so far.In this study,we used lentivirus vector-mediated Cks1b-cDNA transfection MM cells to detect Ckslb induced the mechanism of drug resistance to different anticancer drugs.We found that Ckslb is a selective drug resistance gene to ubiquitin like protein system inhibitors,but not a multiple drug resistance gene to variety anticancer drug through P-glycoprotein family.Furthermore,we identified down-regulated neddylation of Cull-N8 is the mechanism of MLN4924 resistance.Objective:To explore if Ckslb can play as a drug marker in multiple myeloma therapy.Results:1,Ckslb is a ubiquitin-like protein system drug resistance gene,no cross-resistance to other anticancer drugsElevated expression of Ckslb has been linked to a poor prognosis in MM.so,it shows overexpression of Ckslb play a key role in MM which is closely related to diagnose and cancer development.To investigate drug resistance for Ckslb and responsible for MM relapse,more anti-cancer drug,such as NAE inhibitor:MLN4924,fluoropyrimidine antimetabolite:5-FU,topoisomerase and DNA growth inhibitor:tetracycline,and cyclophosphamide(CYC),were tested in overexpression of Ckslb(OE)OCI-MY5,KSM28-PE,and XG1 cell line.Untreated and empty vector(EV)-transfected cells with or without drugs served as controls.After drugs treatment for 48 hours,respectively,a dose-dependent decrease of viability of cell growth was observed in all cells.The results obtained from Fig.1.1 represent that cells growth inhibition with drugs treatment in Ckslb OE cells is less,compared with EV-transfected controls.But,it is obviously different between MLN4924 treated two cells only,not 5-FU,tetracycline,and CYC.5-Fu,tetracycline,and CYC have effective anticancer,however,drug-resistance has obsession to application in clinics[1].It is reported that the mechanism of drug resistance is mainly involve in multidrug resistance associated proteins(MRPs),which is as members of this family's C-branch,might be of particular interest because they are able to efflux nucleoside analogs used in the treatment of cancers.To further explore the function of Ckslb that elevated expression of Ckslb is not cross-resistance to 5-FU,Etoposide,and Dox.Expression of MDR1,BCRP,and MRP1,which were mitotic checkpoint protein MAD2 and ABC transporter family members,were tested by western in OCI-MY5 and KSM28PE(OE and EV)cells.The results show it is almost no difference between Ckslb OE and EV cells.In other words,cks1b OE don't up-regulated expression of MDR1,BCRP,and MRP1,compared with EV cell(Fig.1.2).Consistently,Ckslb OE don't promoted a higher efflux of the hydrophilic eFluxx-IDTM gold fluorescent dye from MM cells,compared with control cells(Fig.1.3).Then,it demonstrated that drug resistance Ckslb induced don't show by classic drug efflux transporters in MM cells.2,Overexpression Ckslb blocked MLN4924,Bortezomib-inhibited cell growth and blocks MLN4924 and Bortezomib induced the apoptosis,respectively,but not CTCMLN4924,Bortezomib-drug resistance is appeared in Ckslb OE MM cell through viability assay.In order to further verity the functional role of MLN4924 and Bortezomib in the ckslb,colony assay is applied in the experiment.The number of colony formation are significant decrease(22.92,16.76,7.23,and 1.46,respectively)with increasing dose of MLN4924 treatment(DMSO,10nm,50nm,100nm)in OCI-MY5 EV cell for 21 days.Furthermore,cancer cells ckslb OE showed obviously no decrease in their capacity to form colonies for MLN4924 tested at low dose(24.90,21.28,19.61,and 17.34)(Figure 2.1),compared with non-treated controls.So,the same results were presented in KSM28PE cell(23.50 vs 24.23,15.21 vs 21.12,8.80 vs 19.34,and 2.34 vs 17.68).In the meanwhile,ckslb OE also block Bortezomib inhibit increase of colony formation(OCI-MY5:EV and OE,23.17,13.94,7.21,2.30 vs 24.51,20.67,15.96,14.90;KSM28PE:EV and OE,22.30,15.96,6.82,2.11 vs 24.04,18.55,17.11,14.32).cancer cells cks1b OE showed a significant increase in colony formation indicating that Ckslb OE promote cancer cell proliferation,compared with EV-transfected cells.To answer whether Ckslb conferred drug resistance is related with decreased apoptosis,the same two cell lines were treated by different dose of MLN4924,Bortezomib,and Dox treatment for 48 h,respectively.Apoptotic cells were stained by the APC conjugated Annexin-V and determined by flow cytometry.As shown in Figure 2.2a-b,Ckslb OE decreases cell apoptosis after addition of anti-cancer drugs,compared with controls,but not Dox(2.2c).The function of Cks1b will also be demonstrated in vivo.The compound was administered to nude mice bearing subcutaneous OCI-MY5 xenografts and monitored tumor growth rate.MLN4924 was administered by subcutaneous injection once daily to mice bearing OCI-MY5 xenografts,and inhibition of tumor growth was calculated on the 20th day of treatment(Fig.2.2).It was found a time-dependent increase of tumor volume in OCI-MY5 EV,OCI-MY5 OE,OCI-MY5 EV plus 60mg/kg MLN4924(1 time/day),OCI-MY5 OE plus 60mg/kg MLN4924 groups(1 time/day),OCI-MY5 OE plus lmg/kg Bortezomib groups(2 times/week),OCI-MY5 OE plus lmg/kg Bortezomib groups(2 times/week).Ckslb OE obviously blocked MLN4924 administered inhibited tumor growth in OCI-MY5 OE group,compared with that of OCI-MY5 EV group(Fig.2.3).3,Overexpression of Ckslb blocks the decrease of cullinl/N8 with MLN4924 and Bortezomib treatment,but not CTXCullin-RING E3 ubiquitin-Ligases(CRLs)are multi-subunit complexes composed of a catalytic site and a substrate recognition module nucleated around cullin scaffold protein.These cullins recruits a specific substrate-recognition module such that CRL complexes are modular[1].In particular,cullin/NEDD8(neural-precursor-cell-expressed developmentally downregulated 8,N8)complexes was formed by conjugation of the ubiquitin-like protein N8 on the cullin subunit stimulates substrate polyubiquitination.It is known MLN4924 can decrease expression of cullin/N8 and ubc12/N8 as NAE inhibitor[2],but,it is unknown for cullin/N8 in the ckslb OE and EV MM cells with MLN4924 treatment.In the current experiments,first,it was observed Cks1b OE up-graded expression of cullin/N8 and ubcl2/N8 complexes in OCI-MY5 and KSM28PE,compared with EV(Fig.3.1),then,a dose-dependent decrease of expression of above two complexes after verity dose of MLN4924(Onm,4nm,lOnm,and 30nm)treatment for 36 hours was found in all cells(Fig.3.2).Compared with those in EV cells,it is not obvious for decreased expression of complexes with adjacent dose of MLN4924 treatment in ckslb OE cells,respectively,indicated Ckslb OE blocks the decrease of cullin/N8 with MLN4924 treatment.Roland HJERPE et al.found an atypical NEDDylation is independent of classical NEDD8 enzymes,conserved from yeast to mammals,and triggered by an increase in the NEDD8 to ubiquitin ratio.In cells,NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8,whereas proteasome inhibition has the same effect by depleting free ubiquitin,such as,bortezomib and MG132[3].In order to investigate neddylation with Bortezomib in OCI-MY5 and KSM28PE,Bortezomib was added into above cells for 36 hours,respectively.It is observed that expression of Cullin/N8 in OCI-MY5 and KSM28PE were degraded with verity dose of Bortezomib(Onm,0.5nm,2nm,and 5nm)treatment step by step.Similar with MLN4924,it is not obvious for decreased expression of complexes with adjacent dose of Bortezomib treatment in OCI-MY5 and KSM28PE OE cells,compared with those in EV cells respectively,indicated Ckslb OE aslo blocks the decrease of cullin/N8 with Bortezomib treatment(Fig.5.3 a).But Dox can't do it(Fig.5.3 b).4,Overexpression of Ckslb increases MLN4924-resistance through inhibition of p21Cksl was found to be an essential cofactor for efficient Skp2-dependent ubiquitination of p27 Kipl,and regulated the substrates of SCFskp2[4,5].This results were demonstrated in the current experiment,it is firstly observed the expression of SCF skp2 substrates,such as,p21,p27,p57,p130,and CDT1 in OCI-MY5 OE cell were obviously lower than those in OCI-MY5 EV cell(Fig.4.1).Similar results for these KSM28PE cell lines were obtained.To further examine the function of ckslb in promoting cancer cell survival,specific shRNAs against Ckslb gene were designed.A tetracycline-inducible lentiviral expression system containing shRNA to ckslb was used to knockdown ckslb in tumor cells;Western-blots confirmed a remarkable down-regulation of ckslb proteins in OCI-MY5 and KSM28PE MM cells after transfection of these ckslb-shRNAs.Importantly,these substrates of SCF skp2 were significantly increased in knock down of cks1b cells,compared with Scr-transfected cells(Fig.4.2).Poor cancer patients often have low expression of SCFskp2 substrates,and MLN4924 can promote the cancer cell death through up-gradation of these substrates[6,7].In order to investigate the MLN4924 and Bortezomib-resistance in ckslb OE,four dose of MLN4924(0,4,15 and 30nm),Bortezomib(0,0.5,2 and 5nm),and CTC(0,5,15and 30nm)were used to treat the cancer cells for 36 hours.It is found MLN4924,Bortezomib,and CTC concentration-dependent increase of substrates of SCF skp2were showed in all cells,respectively(Fig.4.3).Compared with EV-transfecf cells,it is no obvious for MLN4924 and Bortezomib increase for protein expression ckslb OE in OCI-MY5 and KSM28PE,but not CTC,indicating that it showed ckslb OE induced MLN4924,Bortezomib-resistance by SCF skp2 pathway.Importantly,in contrast,p21,a known substrate of CRL/SCF[8],was little accumulated in a dose-dependent manner in all three lines of cancer cells treated with MLN4924 and Bortezomib(Figure 3,A and B),suggesting that p21 may play an essential role in MLN4924-induced senescence.To test this hypothesis,we used two pairs of cell lines:HCT116-p21+/+ versus HCT116-p21-/-and mouse embryonic fibroblasts(MEFs)with p21+/+ versus p21-/-background.5,Overexpression of Ckslb blocks MLN4924 induced senescence through inhibition of p21Some studies reported low dose of MLN4924 can induce clone HCT116,H1299,and U87,in addition to apoptosis,senescence,which was associated with DNA damage response[8].We therefore determined whether Cks1b can block pharmacological inactivation of CRL/SCF E3 ligase by MLN4924 induce senescence.Indeed,four concentration MLN4924(DMSO,10 nm,50 nm,and 100 nm)were applied to OCI-MY5 and KSM28PE(EV and OE),and an enlarged and flattened shape,a reminiscence of senescence phenotype were appeared.SA-?-Gal staining showed that a dose dependent positive staining increase was observed in OCI-MY5(EV)and KSM28PE(EV),compared with OCI-MY5(OE)()and KSM28PE(OE)().These results were also demonstrated in the Bortezomib treated cell,exclude DOX.The p16/pRB and p53/p21 axes are two major senescence-triggering pathways in response to stresses[].To elucidate the mechanism which ckslb block MLN4924,Bortezomib,and DOX-induced resistance in senescence,we determined firstly the activation status of these pathways in all drugs-treated cells.It is found a dose dependent increase expression of p16 and p53 protein by Bortezomib and DOX.But the results show p16 and p53,except for p21,have no alternation with MLN4924.This result is consistence with JIA's study.Ckslb block MLN4924 induced senescence through upgradetion of p21.But p16 and ckslb block Bortezomib treat cell,respectively.(Fig.),indicated ckslb block MLN4924 and Bortezomib induce senescence through p21 only,respectively. |
PDF Full Text Request