The Experimental Study Of Reversal Effect Of Multidrug Resistance By High-Intensity Focused Ultrasound In K562/AO2 Cell Line

BACKGROUD :High-intensity focused ultrasound is a new technique in tumor therapy,and different dosages show various biological effects. Multidrug resistance (MDR) is a critical problem in cancer chemotherapy. Over-expression of P-glycoprotein (ABCB1, MDR1 gene product) on the plasma membrane is frequently observed in drug resistant cancer cells which leading to the failure of chemotherapy. A characteristic feature of P-glycoprotein is its broad substrate specificity. P-glycoprotein transports various hydrophobic compounds out of the cell by utilizing the energy of ATP hydrolysis. Such a diversity of P-glycoprotein substrates poses a significant problem for clinical treatment.P-glycoprotein also modulates drug absorption, bioavailability, tissue disposition , excretion and plays an important role in apoptosis , Multidrug resistance(MDR) of tumor cell results in abortive chemotherapy.Therefore, studying the mechanism of MDR and effectively reversing play a key role in tumor therapy.Traditional MDR reversal agents,the P-gp inhibitor drugs,show relatively lower efficiency but inevitable side effects.accordingly,it's necessary to develope a better trick that can overcome MDR..OBJECTIVE: This paper studys and evaluates the reversal effect of multidrug resistance by high-intensity focused ultrasound(HIFU) in K562/AO2,and primarily investigated the mechanism. Accordingly,we desiredly develop a promise approach in MDR tumor therapy.METHODS:①With fixed exposed duration or acoustic intensity,by the means of Trypan blue exclusion and MTT assay,the relationship between tumor cell livability and HIFU dosage that which includes defferent acoustic intensity and duration,was analyzed. ②Logarithmic growth K562 and K562/AO2 cell lines were respectively divided into four groups: contral;adriamycin singlely ; adriamycin combined with verapamil(VRP);adriamycin combined with HIFU administered. Cells proliferation efficiency is evaluated by assay of MTT ;By the way of FCM, intracellular adriamycin concentration,SPF and apoptosis ratio are tested; With the help of AnnexinⅤ/PI,apoptotic cells are observed by fluorescence microscopy.③The semiquantitative analysis of immunohistochemistry results of P-gp and PDCD5 were calculated by Image-pro plus.RESULT:①With the fixed exposed duratio(n5s), the tumor cell livability cut down when the ultrasound intensity ascends.with the fixed acoustic intensity(400/ cm2), when the duration increased, simultaneously ,the cell survival falls.The HIFU dosage of 400W/ cm2×5s does not influence cell livability;②HIFU and VRP show no significant influence in intracellular adriamycin concentration, cells proliferation and apoptosis ratio in K562 cell strain.but different results were detected in K562/AO2 cell strain,Compare with adriamycin singlely,HIFU and verapamil can significantly enhance intracellular adriamycin concentration,proliferation inhibition ration and apoptosis ratio.but no statistical difference was observed between group HIFU and VRP(P>0.05).③Compared with K562,K562/AO2 expresses higher level P-gp ,but lower level PDCD5. HIFU irradiation results in upregulation of PDCD5 and downregulation of P-gp in K562/AO2.CONCLSION : HIFU can effectively increase intracellular adriamycin concentration.And consequently,the adriamycin related cytotoxicity that showed in cellular proliferation inhibition and apoptosis ratio in K562/AO2 was sharply ascended..The mechanism of its reversal effect may realize through upregulation of PDCD5 and downregulation of P-gp.Thus,HIFU can reverse the resistance to adriamycin in K562/AO2 cells . Even though no significant difference was observed between HIFU and VRP,but we conclude that HIFU is a relatively ideal reversal agent because of its non-invasion,free toxicity and easy modulation.