| Multiple myeloma is a lethal hematopoietic malignant tumor which is easy to relapse,and the median survival is only about five years after recurrence.Although autologous stem cell transplantation,the advances in chemotherapy,and introduction of novel drugs such as proteasome inhibitors have significantly improved response and survival rates in patients with MM,a vast majority of patients with MM eventually relapse,which is a difficult to solve the tough problem.Cancer stem cells(CSCs)that show the features of self-renewal,multi-directional differentiation,and multidrug resistance,are believed to be the major cause of tumor recurrence.The MM treatment failure has demonstrated that the current agents are ineffective on eradicating MM CSCs.The high drug efflux capacity of MM CSCs is likely to be the major cause of drug resistance in myeloma.ATP-binding cassette(ABC)transporters represent one family of transmembrane proteins including ABCG2,which are expressed in normal tissues.ABCG2 protein may use the energy from ATP hydrolysis to efflux cytotoxic compounds such as doxorubicin and mitoxantrone,across the cellular membrane,which is believed to be one of the major causes of multidrug resistance in cancer therapy.In MM,CSCs show stronger activity of the ABCG2 transporter when compared to non-CSCs,thus,high expression of ABCG2 is an important marker of MM CSCs.Microbubble(MB)is formed by membrane shell packaging gas,and is mostly composed with lipid or biodegradable polymers.As a novel drug delivery carrier,MB has many advantages,which include high security,high drug loading capability by MBs through various methods.The biological effect of ultrasound on MBs can enhance the capability of drug delivery.Microbubble-ultrasound interaction makes passive target true,particularly,targeting the tumor forwardly if MBs are conjugated with tumor-specific antibody.Therefore,MBs in combination with ultrasound exposure is promising for targeted treatment of tumors.Epirubicin(EPI)is one of anthracycline drugs used commonly in therapy of MM,breast,sarcoma and many other malignant tumors.The dosage forms of common EPI can not target tumors,and distribute both in tumor and normal tissue when injected venously.EPI has various adverse effects including digestive tract toxicity,myelosuppression,function lesion of liver and kidney and so on.Cardiotoxicity is the dose-limiting toxicity of EPI and influences its clinical application dramatically.In order to solve the relapse,drug resistance of MM,and the toxicity of EPI,in this study,we isolated MM CD138-CD34-CSCs from human MM cell lines by immune magnetic activated cells sorting method,and then prepared EPI-loaded lipid MBs conjugated with anti-ABCG2 monoclonal antibody(mAb),which can target directly the ABCG2 that high expressed in MM CSCs.EPI was released from the targeting MBs when ultrasound exposure was applied on tumors in order to improve the EPI concentration in tumor site and reduce the adverse reaction of chemotherapy.To achieve the goal,EPI-loaded lipid MBs conjugated with anti-AB CG2 mAb were used to treat the MM bearing NOD/SCID(nonobese diabetic/severecombined immunodeficient)mouse models to evaluate the therapeutic effect on MM and analyze the related mechanism.ObjectiveTo investigate the effects of epirubicin-loaded MBs conjugated with anti-ABCG2 mAb targeting CSCs combined with therapeutic ultrasound on MM CD138-CD34-CSCs,analyze the related mechanisms,and provide a new method for enhancing therapeutic effect on MM patients and decreasing the recurrence rate in clinical trials.Methods and Contents1.Isolation and identification of MM CSCs.The MM CSCs were sorted from the human MM cell line RPMI8226 by using an immune magnetic activated cell sorting method,based on the cell surface molecular markers CD138-and CD34-phenotypes.The characteristics of CSCs were confirmed repeatedly in vitro and in vivo assays according to the methods previously used.2.Preparation and characterization of targeted drug-loaded MBs.Anti-ABCG2 mAb was conjugated with EPI-loaded MBs by using biotin-avidin method to prepare targeted drug-loaded MBs.The conjugated antibody,physical and chemical properties of MBs such as particle size,zeta potential,and drug-loading rate were detected,respectively.The targeted capability of the drug-loaded MBs binding to MM CSCs as well as the release of EPI under the action of ultrasound was also examined.3.The inhibiting and killing assays of EPI-MBs+mAb+US on MM CSCs in vitro.The experiment was divided into seven groups including PBS(Control),EPI-loaded MBs(EPI-MBs),EPI-loaded MBs conjugated with anti-ABCG2 mAb(EPI-MBs+mAb),EPI,EPI combined with ultrasound(EPI+US),EPI-loaded MBs combined with ultrasound(EPI-MBs+US),EPI-loaded microbubbles conjugated with anti-ABCG2 mAb combined with ultrasound(EPI-MBs+mAb+US)groups.The inhibition and apoptosis effects of various treatments on MM CSCs were respectively tested.The related mechanisms were explored by observing the changes of mitochondrial membrane potential,cell cycle,and the levels of apoptosis related proteins such as Casepase-3,Casepase-8 and Casepase-9.The ultrastructure of MM CSCs after different treatments was also detected by TEM.4.Examination of the effects of EPI-MBs+mAb+US on MM bearing mouse model established by subcutaneous injection.1×106 CD138-CD34-CSCs were injected subcutaneously into NOD/SCID mice.The target accumulation of EPI-MBs+mAb in tumor was observed by pathological histology and ultrasonic imaging after certain EPI-MBs and EPI-MBs+mAb were injected venously into NOD/SCID mice bearing MM.The experiment was divided into four groups including Control,EPI-MBs+mAb,EPI,and EPI-MBs+mAb+US groups(3 mice/group).The effects of different treatments were observed,and the related mechanisms were evaluated by pathological assays such as hematoxylin and eosin(HE)staining,immunohistochemisty(IHC),TUNEL,and Western blot which was applied to analyze the expression of Casepase-3,Caspase-8,Caspase-9,Bax,Bcl-2,and topoisomerase ??.The experiment was repeated twice.5.Examination of the effects of EPI-MBs+mAb+US on MM bearing mouse model established by tail vein injection.5×106 MM CD138-CD34-CSCs were injected into NOD/SCID mice via tail vein.About 21 days later,the bone mineral density,renal damage and urine protein of the mice bearing MM were respectively measured to confirm whether the MM model could be success.EPI-MBs+mAb labeled with CY7.5 and EPI-MBs were developed and injected venously into mice.The targeted accumulation of EPI-MBs+mAb in tumor was assessed by in vivo under a fluorescence imaging.The experiment groups and treatment selection were as above-mentioned.The therapeutic effects were observed and the mechanisms were explored by cytological technology,pathological assays and imaging analyses,respectively.Results1.The results of RT-PCR,WB and FCM showed that the expression level of ABCG2 in MM CD138-CD34-cells(MM CSCs)was higher than that of MM non-CD138-CD34-CSCs(P<0.05 or P<0.01).The activities of proliferation,clone formation,migration and invasion as well as the tumorigenecity in the MM CD138-CD34-CSCs were all significantly higher than that in the MM non-CD138-CD34-CSCs(P<0.05 or P<0.01).2.Characterization of EPI-MBs+mAb revealed the characteristic of good dispersion,uniform particle sizes,high stability and high drug-loading rate.In addition,EPI-MBs+mAb targeted to MM CSCs and released EPI to cells in vitro under ultrasonic intervention.3.The influence of various agents incubated with MM CSCs was evaluated in vitro.Cell proliferation was inhibited in EPI group but no inhibition effect in the EPI-MBs and the EPI-MBs+mAb groups.EPI-MBs+mAb+US group,however,showed the most significant inhibitory effect compared with other groups.The apoptosis effect detected by FCM among the groups was consistent with the inhibitive effect on cellular proliferation.Western blot results revealed that the EPI-MBs+mAb+US induced the expression of cleaved Caspase-3 and cleaved Caspase-9 except but no influence on expression of Caspase-8.4.The tumors were touched in NOD/SCID mice 18 days after subcutaneous injection of 1 X 106 MM CSCs.EPI-MBs+mAb targeted to the subcutaneous MM site quite well,and remained in it for a long time,which could be detected by ultrasoud imaging.Compared with control group,EPI,EPI-MBs+mAb and EPI-MBs+mAb+US groups inhibited the tumor volumes and prolonged the mouse survival in different extent,particularly the EPI-MBs+mAb+US group,which indicated the best effect on inhibition of MM growth.The results of IHC and Western blot showed that EPI-MBs+mAb+US enhanced the expression of cleaved Caspase-3,9 and Bax,meanwhile,inhibited the expression of Bcl-2,PCNA and topoisomerase ?? obviously,which was significantly increased compared with EPI group(P<0.05),and the EPI-MBs+mAb group(P<0.05)respectively.The results of HE staining and TUNEL showed that the EPI-MBs+mAb+US group may significantly induce stronger apoptosis of MM CSCs than those of other groups(P<0.05 or P<0.01).5.Lytic bone lesions,renal damage and proteinuria were found in the mice injected with MM CSCs via tail vein in 18 days compared with that of normal group,which was statistically significant(P<0.001).The results suggested that MM bearing mouse model was established successfully.It was found that the specific binding of EPI-MB+mAb to MM in bone morrow tissues of spine and limbs in mice mediated by anti-ABCG2 mAb compared with EPI-MBs without anti-ABCG2 mAb under in vivo fluorescence imaging.All the treatment groups had reduced bone and kidney damages,and anemia to different degrees,and there are significant differences between the EPI-MBs+mAb+US and the EPI-MBs+mAb groups(P<0.05),between the EPI-MBs+mAb+US and the EPI groups(P<0.05),and between the EPI-MBs+mAb+US and the model groups(P<0.01).Conclusions1.EPI-MBs+mAb+US could target MM CSCs directly by the antibody specific blocking ABCG2 pump activity partly,and released the EPI in combination with ultrasound exposure,which resulted in permanent inhibition of MM growth.The mechanisms of anti-MM efficacy may be associated with inhibition of MM CSC proliferation and increase of apoptosis.Furthermore,activation of the complement dependent cytotoxicity,and antibody-dependent cell-mediated cytotoxicity may contribute to the cytotoxic effects on tumor in vivo MM bearing mice.2.These data support our hypothesis that with the use of EPI-MBs+mAb could target well to MM CSCs in MM bearing NOD/SCID mice,and developed significant anti-MM effects combined with ultrasound exposure,which may provide a better strategy to eliminate MM CSCs and cure MM. |
PDF Full Text Request