Comparison Of The Kinetics Of ⁹⁹Tc^m-MIBI In Tumor Cells With Or Without P-glycoprotein-mediated Multidrug-resistance

ObjectiveNowadays chemotherapy is an important methods to malignant tumors. But some patients appear resistance to some drugs before or after induction of chemotherapy that may affect the treatment. Research indicated that multidrug resistance (MDR) is one of the reasons that lead to chemotherapy failure. It influences the effect of chemotherapy and prognosis severely. The mechanism of MDR is complicated, among it the excessive expression of P-glycoprotein (Pgp) coded by multidrug resistance genel (mdr1) may play an important role. MDR reversal agents are drugs that overcome the resistance of tumor cells to chemotherapy drugs to aim directly at different drug-resistance mechanism, accordingly improve the effect of chemotherapy. But it is restricted in clinic due to the toxic followed dosage increase. We compare the uptake and washout of ⁹⁹Tc^m-MIBI in tumor cells with or without Pgp expression and the change of Pgp function induced by MDR reversal agents since ⁹⁹Tc^m-MIBI is confirmed to be the substrate of Pgp. Aim at finding simple but effect methods to estimate the MDR of tumor cells to provide useful message to problems encountered in clinical chemotherapy.MethodsHuman drug-sensitive mylogeneous leukemia cell line K562 and its resistant subline K562/D, originally isolated from the K562 by successive stepwise selections in adriamycin were cultured in RPMI 1640 medium/10% FCS in humidified atmosphere with 5% carbon dioxide at 37℃. Pgp was detected by western bloting. We observed the uptake and washout of ⁹⁹Tc^m- MIBI in each cell line with or without MDR reversal agents.Cellular ⁹⁹Tc^m-MIBI accumulation: cells (2×10⁶) from K562 and its resistant subline were incubated with ⁹⁹Tc^m-MIBI(8MBq) and different modulator in RPMI 1640 medium/10% FCS, the cells were washed with 5ml of ice-cold phosphate-buffered saline(PBS) followed by centrifugation (5min, 180g, 4℃) at different time intervals. The wash step as described above was repeated three times. The cellular ⁹⁹Tc^m-MIBI was measured with aγ-counter. For efflux studies: 2×10⁶ cells from these cell lines were incubated in 5ml of RPMI 1640 medium/10% FCS for 30min at 37℃with 8MBq ⁹⁹Tc^m-MIBI as described above. Thereafter the cells were washed with RPMI 1640 medium/10% FCS. After 0,10,20 or 30min, the efflux of ⁹⁹Tc^m-MIBI were terminated by adding ice-cold PBS followed by centrifugation (5min, 180g, 4℃) and measurement of the cellular ⁹⁹Tc^m-MIBI.Grouping:The cellular ⁹⁹Tc^m-MIBI uptake was expressed at overall concentration of ⁹⁹Tc^m-MIBI accumulated in the cells to the overall concentration of ⁹⁹Tc^m-MIBI concentration added to the cells. Six independent experiments were performed, each in duplicate. Statistical significance was determined with the Student's t test and q test. Only P-value<0.05 were considered to be significant.ResultsWestern bloting indicated expression of human Pgp in K562/D cells by presence of a large band corresponding to a molecular mass of 170Kda. Pgp was not detectable in K562 cells.The uptake of ⁹⁹Tc^m-MIBI increased almost linearly during the first 30min,then plateaus in K562 cells and K562/D cells. The uptake rate is (1.559±0.529 and 0.107±0.036 for sensitive and resistant K562 cells, respectively. ⁹⁹Tc^m-MIBI accumulation is 15-times lower in resistant cells than in the parent cells. As for efflux washout is rapid processes during the first 10min for both cell lines, at 30min the ⁹⁹Tc^m-MIBI decreased to 36.8 % and 34.1 % of the total accumulation in K562 and K562/D.Different concentration of verapamil(2.5～10μg/ml) and cyclosporin A(0.1～0.4μg/ml) significantly increased the ⁹⁹Tc^m-MIBI uptake of K562 resistant subline, while the uptake of K562 cell line expressing nondetectable Pgp was not affected. The increase of ⁹⁹Tc^m-MIBI uptake in K562/D cell lines accompanied with the concentration of modulator. But there was no significant difference between different concentration of verapamil or cyclosporin A.combination of low dose verapamil (2.5μg/ml) and cycolsporin A (0.1μg/ml) had similar effect on ⁹⁹Tc^m-MIBI accumulation with higher dose of inhibitor lonely.ConclutionsThe different uptake of ⁹⁹Tc^m-MIBI between K562 cells and K562/D cells can reflect the Pgp level, so we can use kinetics of active accumulation of ⁹⁹Tc^m-MIBI to evaluate Pgp function. Various concentrations of verapamil and cyclosporin A increase ⁹⁹Tc^m-MIBI uptake in K562/D cells, indirectly reflect the effect of the MDR reversal agents. Combination of lower dosages of modulators may play same reverse effect with less side effects, this provide new useful message to clinical chemotherapy. ⁹⁹Tc^m-MIBI imaging could be a good noninvasive technique for the in vivo diagnosis of the presence of the Pgp protein. This approach may ultimately be used to guide chemotherapeuticprotocols, assist clinical trials or screening of new Pgp reversing agents.