| Background:Despite significant advances over the past two decades in the treatment of Multiple Myeloma(MM),which was first fully documented in 1844,MM remains an incurable hematologic tumor with malignant plasma cell proliferation and the secondary leading hematologic malignancy to non-Hodking’s lymphoma.In recent years,with the increasing incidence of MM in the aging population,the median age of onset is 64.Common symptoms include osteolytic lesions,anemia,kidney damage and infection,which seriously affect the life quality of patients,however the exact cause and pathogenesis of MM remains unknown.In the last two decades,many new therapies have emerged,such as proteasome inhibitors,monoclonal antibodies,immunoregulatory drugs,and therapies including chimeric antigen receptor T cells(CAR-T).Most of the clinical treatment programs are combined,and the drug combined with dexamethasone(Dexa)is widely used in the treatment of MM.More than 50 years ago,Dexa began to be used in clinical practice.But so far,Dexa resistance has become a unavoidable problem in patients.However,Dexa plays an important role in the treatment of multiple myeloma,which is anti-inflammatory and rapidly kills tumor cells in the lymphatic system.Dexa-resistant patients are closely associated with poor prognosis,and the survival time of these patients is often significantly shortened.Therefore,new drugs are urgently needed for MM treatment to overcome drug resistance.It has been reported that artemisinin(ART)and its derivatives can play the role of chemotherapeutic sensitization when used in combination with other anti-tumor drugs,so as to overcome tumor resistance,but no studies reported in MM.Objective and significance:ART and its derivative,Dihydroartemisinin(DHA),have been extensively studied in anti-tumor studies,but little study in MM,and there is no comparison between ART and DHA in anti-tumor studies.Therefore,solving the problem of what effect ART and its derivative DHA have on MM,and how to and when use the drugs,will provide alternative choices for the clinical treatment of MM.Therefore,ART-related drugs,which can overcome drug resistance and reduce damage to normal cells at the same time,become apotential drug that needs to be tested in MMResearch methods:Based on the 5TMM3VT myeloma model mice and ARP1 and H929 MM cells,we compared the different therapeutic effects of ART and DHA.The effects of ART and DHA on cell cycle,ROS,iron divalent,oxygen consumption and related pathways at the same dose were detected by flow cytometry,oxygen consumption experiment and Western blot,respectively.Subsequently,our group investigated whether DHA can overcome drug resistance.We first applied DHA to a pair of sensitive and drug resistant cell lines 8226.WT and 8226.DR to bortezomib and a pair of sensitive and drug resistant cell lines MM.1S and MM.1R to Dexa,respectively.Subsequently,we used MTT experiment to verify the combination drug index and the effect of the drug combination on the patient’s primary CD138+cells.The changes of apoptosis,ROS,mitochondrial membrane potential and related pathways were detected by flow cytometry,microscopic imaging,western blot and other methods.Finally,we tested the effect of the combination on tumor growth in mice before and after the use of xenotransplantation in mice,further revealing that DHA is a potential drug for the treatment of MM.Results:In vivo results showed that both ART and DHA significantly improved the survival rate of 5TMM3VT myeloma model mice compared with the control group.Compared with the ART group,the survival rate and survival time of mice treated with DHA were significantly increased.The results of MTT and apoptosis experiments in vitro showed that DHA could significantly inhibit the growth and induce the apoptosis of MM cells.Cell cycle test results showed that the G2/M phased proportion of MM cells treated with DHA was significantly lower than that of the ART group and control group.Flow cytometry showed that compared with the ART group,DHA produced more ROS in MM cells,and also increased the level of divalent iron ions in cells under the action of DHA.The results of O2R showed that DHA increased the oxygen consumption of MM cells.Western blot results showed that compared with the ART group,the DHA group was more likely to cause the increase of cleaved caspase 3 and the decrease of Bcl-2 and Bad.MTT results showed that DHA could not inhibit the growth of bortezomib resistant strains,but could inhibit the growth of Dexa-resistant strains.When Dexa and DHA combined with Dexa were applied to Dexa resistant and sensitive strains,the combined drug index(CI)was<1.After the action of DHA combined with Dexa on the primary CD138+cells of patients,the inhibition effect of 7.5 μM Dexa and 120 μM DHA combined was stronger than that of the treatment alone.The results of flow cytometry on apoptosis and ROS showed that compared with the treatment of DHA and Dexa alone,the proportion of cells with early and late apoptosis was significantly increased after the combined treatment,and the ROS production was significantly increased.The results of mitochondrial membrane potential showed that the number of cells emitting red fluorescent cells decreased while the number of cells emitting green fluorescent cells increased after the combined treatment.Combined with flow analysis,the mitochondrial membrane potential significantly decreased after the combined treatment.Western blot results showed that cytoplasmic Cyt c was significantly increased and mitochondrial Cyt c was significantly decreased after the combined treatment.Expression of cleaved Caspase-3 was increased when combined with the drug.The expression level of Bcl-2 increased after Dexa was given for 48,72 and 96 h,and the expression level of Bcl-2 decreased with the increase of Dexa concentration at 24 h.Dexa combined with DHA could restore Dexa-upregulated Bcl-2 at both low concentration(3.75 μM)and high concentration(60 μM)after acting on cells for 48 h.The results of xenografted mouse experiments showed that there was no significant difference in the tumor volume of mice after MM.1S injection before and after the combination of drugs,but the tumor mass became lighter after the combination of drugs.However,both tumor volume and tumor mass were decreased after MM.1R injection.Conclusion:In vitro and in vivo experiments showed that DHA was superior to ART in prolonging survival rate of mice,inhibiting growth of myeloma cells and promoting apoptosis of myeloma cells.It may be due to the differences in mitochondrial pathway apoptosis induced by ROS.The effect of DHA on ROS level,oxygen consumption level and iron divalent ion level,as well as Caspase 3,Bcl-2 and Bad was much greater than that of ART.DHA combined with Dexa can overcome Dexa resistance with synergistic effect,which can inhibit the growth of myeloma cells in vivo and in vitro compared with the treatment alone.DHA can increase the ROS level produced by Dexa,reduce mitochondrial membrane potential,cause more outflow of Cyt c and inhibit the up-regulation of Bcl-2,thus inducing more apoptosis of myeloma cells.The results of this study provide theoretical basis for clinical use of DHA as a potential drug for the treatment of multiple myeloma. |
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