Role Of NF-κB In Chemotherapy Resistance Mechanism And Combination Therapy In Anaplastic Thyroid Cancer

Nuclear factorκB (NF-κB)plays a crucial role in cell survival,angiogenesis andcarcinogenesis.In normal cells NF-κB is strictly regulated,whereas in cancer cells it isconstitutively activated to high level.And more importantly,NF-κB activation in cancercells is proven by many researchers as one of the major culprits of their resistance tochemotherapy.Taxanes (including docetaxel and paclitaxel)break the equilibrium ofmicrotubule polymerization by preventing tubulin depolymerization during mitosis,impairing cell mitosis and proliferation in tumors.Taxanes have been used for severalmalignant tumors in clinic.In fact,paclitaxel has been confirmed effective in ATC cellstoo.However,it has been reported that taxanes can induce NF-κB activation in differenttypes of malignant cells which can attenuate anti-tumor activities of the drugs and createresistance to chemotherapy.NF-κB inhibitors have been considered as an appealing andtarget-oriented approach to deal with the chemoresistant issue.And in fact,a number ofNF-κB inhibitors have been reported as effective.Dehydroxymethylepoxyquinomicin(DHMEQ),a NF-κB inhibitor designed from the structure of an antibioticepoxyquinomicin C,can inhibit NF-κB nuclear translocation.It has been tested inanaplastic thyroid cancer (ATC)cells to an effective,non-toxic anti-cancer agent in vitroand in vivo.From the published literature,no research investigating docetaxel's effect onATC cells using animal model or reporting combination of NF-κB inhibitor andchemotherapeutic agent in ATC cells in vitro and in vivo has been found.In this study acombined regiment was tested to determine if DHMEQ can optimize the therapeuticeffects of taxanes in ATC cells.Objectives:We sought to establish the NF-κB activation theory in chemoresistancemechanism of ATC cells.In order to prove this molecular mechanism,we used DHMEQ,a novel NF-κB inhibitor,in combination with taxanes to treat ATC cells.Apoptosisassessment experiments were carried out to determine whether NF-κB inhibitor canenhance taxanes' anti-tumor activities in ATC cells in vitro and in vivo.Materials and Methods:Cell survival assay was performed firstly to assessapoptosis and to determine drugs' proper concentrations to use in vitro.The agents' effectson nuclear NF-κB's signaling were assessed by DNA-binding assay and the nuclear NF-κB protein changes were detected by Western blot.Changes of key apoptotic factorswere determined by Western blot.Flow cytometry with the annexin V/propidium iodidestaining were used to analyze the induction of apoptosis for further confirmation.For invivo study,FRO xenograft nu/nu mice models were created.For pilot in vivo experiment,the proper dosage of docetaxel was determined.For formal in vivo experiment,docetaxeltreated mice were i.p.injected with 5 mg/kg once a week;DHMEQ treated mice were i.p.injected with 6mg/kg each day for two weeks;combined treatment was given with bothdrugs,and control group mice received vehicle injections only.Tumor dimensions weremonitored for 32 days in total.One day after treatment,TUNEL apoptotic staining wasperformed on excised tumor tissues,and apoptotic index was calculated accordingly.Results:①Cytotoxic effects of taxanes and/or DHMEQ on cells were shown in cellsurvival assay with significant higher suppressive effect of combination treatment(P＜0.01),although survival rates of ATC cells showed inverse relationship to the dosageof any drug.And,for in vitro experiments,concentrations of taxanes and DHMEQ weredetermined as 4nM and 10μg/ml respectively.②Effects of taxanes and DHMEQ onNF-κB in ATC cells were tested in DNA-binding assay.Cells were treated with docetaxelfor 24 hours,during the time course the binding ability of nuclear p65 increased.However,if first incubated the cells with DHMEQ for 1 hour and then added docetaxel,the level ofp65 was suppressed (P＜0.01),especially during the first 8 hours.Drugs-treated ATC cellswere examined for nuclear NF-κB protein by Western blot.Results showed the amount ofNF-κB increased in taxane group only but decreased in DHMEQ and combinationtreatment groups.③Caspase 3 and PARP were examined by Western blot to determine thedegree of apoptosis.After 24 hours of treatment,any drug could induce cleaved PARP andactivate caspase 3.After combined treatment,PARP cleavages and caspase 3 increasedmuch further.During the 24-hour time course treatment with docetaxel,their levelsincreased as time went by.However,if first incubated with DHMEQ for 1 hour and thenadded docetaxel,the cleaved PARP and caspase 3 appeared much earlier and increasedeven more.④Basal levels of XIAP and survivin were found to be high,taxanes increasedtheir levels further,but when treated with DHMEQ,or DHMEQ plus taxane,their levelsreduced.These indicated both constitutive levels and taxanes-induced activation of theseanti-apoptotic factors can be suppressed by DHMEQ.⑤In flow cytometry,five groups of FRO cells were harvested,they were control group,treatment with docetaxel for 16 hoursgroup,treatment with DHMEQ for 16 hours group,combined treatment group andexposure to 10 J/m~2 of ultraviolet group (as positive control for apoptosis).For each group,4×10~5 cells were double stained with FITC-conjugated annexin V and propidium iodideand then cells were analyzed by flow cytometry.Results showed that although either drugcan induce apoptosis,combined treatment increased apoptosis further,as the positivepercentages of annexin V and propidium iodide jumped to 11.91% and 10.7%,respectively.These created even more apoptosis than positive control group.⑥In FROxenograft models,it was showed that,although any drug was able to delay tumor growth,the combined treatment of DHMEQ and docetaxel for two weeks was much moreeffective (P＜0.01).The TUNEL apoptotic staining and apoptotic index also showeddrug-treatment induced many positively stained cells,while the combined regimenttremendously increased positively stained cells (P＜0.01).Conclusions:1.This research demonstrated for the first time that taxanes induced NF-κB signalingpathway in ATC cells,but by inhibiting the nuclear translocation of NF-κB,DHMEQcould effectively suppress this phenomenon.2.In vitro experiment showed either taxanes or DHMEQ could induce apoptosis,yetcombined treatment synergistically enhanced apoptosis.3.For the first time,the treatment of docetaxel and combination of DHMEQ anddocetaxel were tried on ATC-cell xenograft in vivo models.Nude mice experimentindicated that combined treatment was synergistically more effective than any drug tosuppress tumor growth.4.The above experiments proved the NF-κB activation mechanism exits in theprocess of chemoresistance of ATC cells.Based on this molecular mechanism,inhibitionof NF-rB by DHMEQ can lower the threshold of apoptosis in ATC cells and increase theirsensitivity to chemotherapeuticals.Combination of NF-κB inhibitors emerges as anattractive therapeutic strategy to enhance the response of taxanes in ATCs.