The Preliminary Experiment Of The Effect Of P-glycoprotein On¹⁸F-FDG And^99mTc-MIBI Uptake

Part Ⅰ Verification of P-glycoprotein functional and detection of someprotein related to18F-FDG uptake expressed in cell linesObjective The expression of P-glycoprotein (P-gp), Glut-1and HK-II in Bcap37/MDR1and Bcap37cells were detected using Western Blot. The function of P-gp was evaluatedusing Rhodamine123.Methods The proteins for Western Blot assay were extracted in Bcap37/MDR1andBcap37cells. For the function evaluation, Bcap37/MDR1and Bcap37cells were seeded insix-well plates. Those cells were incubated using serum-free RPMI1640mediumcontaining5μmoL/L Rhodamine123with or without100μmol/L verapamil (VER) for1hat37℃. Then the cells were washed with ice-cold phosphate buffered saline (PBS) forthree times and analyzed by the fluorescent microscopy.Results P-gp and Glut-1were strongly expressed in Bcap37/MDR1cells, but weaklyexpressed in Bcap37cells. HK-II was expressed strongly both in Bcap37/MDR1andBcap37cells. Bcap37cells exhibited bright fluorescent staining in the presence ofRhodamine123. In contrast, Bcap37/MDR1cells showed only very weak fluorescence.Additionally, inhibition of the P-gp function by adding VER resulted in a marked increaseof the fluorescence in Bcap37/MDR1cells. There was no significant difference in thefluorescence of Bcap37cells in the presence or absence of VER.Conclusions Functional P-gp was strongly expressed in Bcap37/MDR1cells.Bcap37/MDR1and Bcap37cells can be used to evaluate the relationship between P-gp and18F-FDG and99mTc-MIBI uptake.Part Ⅱ The uptake of99mTc-MIBI in Bcap37and Bcap37/MDR1cells Objective To evaluate the uptake kinetics change of99mTc-MIBI in Bcap37andBcap37/MDR1cells using the classical inhibitors of P-gp: VER or GF120918.Methods Bcap37and Bcap37/MDR1were plated in six-well plates3days prior to theexperiments at a density of1×106/well. On the day of experiment,74KBq/mL99mTc-MIBI,74KBq/mL99mTc-MIBI+100μmoL/L VER or74KBq/mL99mTc-MIBI+50μmoL/LGF120918was added to each well. After an incubation period of10min,30min,60minand120min (37℃,5%CO2), the medium was removed and immediately washed threetimes with1mL ice-cold PBS. The cells were collected from the wells usingtrypsin-ethylenediaminetetraacetic acid (EDTA) treatment. The radioactivity of99mTc-MIBI was immediately determined using a gamma counter. The uptake of99mTc-MIBI was expressed at overall99mTc-MIBI radioactivity in Bcap37orBcap37/MDR1cells to the overall radioactivity added to the cells. Three independentexperiments were performed.Results The uptake of99mTc-MIBI had no significantly difference with or without VER orGF120918in Bcap37cells. VER or GF120918significantly increased the uptake of99mTc-MIBI in Bcap37/MDR1cells after an incubation period of30min,60min and120min (P<0.05). The uptake of99mTc-MIBI was significantly higher in Bcap37cells than inBcap37/MDR1cells after incubation for30min,60min and120min (P<0.05). The uptakeof99mTc-MIBI in Bcap37/MDR1was a little lower than that in Bcap37cells in thepresence of VER or GF120918.Conclusions The difference in the uptake of99mTc-MIBI between Bcap37andBcap37/MDR1reflected the expression level of P-gp. The uptake kinetics of99mTc-MIBImay be used to evaluate the function of P-gp. The inhibitors of P-gp (VER and GF120918)can significantly increase the uptake of99mTc-MIBI in Bcap37/MDR1cells. The change ofthe uptake of99mTc-MIBI in multidrug resistant cell lines can be used to evaluate theinhibitor efficiency of P-gp inhibitors.Part Ⅲ The uptake of18F-FDG in Bcap37and Bcap37/MDR1cellsObjective To evaluate the uptake kinetics change of18F-FDG in Bcap37and Bcap37/MDR1cells in the presence or absence of VER or GF120918, and to elucidate therelationship of18F-FDG uptake and P-gp expression at the cellular level.Methods Bcap37and Bcap37/MDR1cells were plated at a density of1×106/well insix-well plates3days prior to the experiments. On the day of experiment,37KBq/mL18F-FDG,37KBq/mL18F-FDG+100μmol/L VER or37KBq/mL18F-FDG+50μmoL/LGF120918was added to each well. After an incubation period of10min,30min,60minand120min (37℃,5%CO2), the medium was removed and the cells were washed threetimes with1mL ice-cold PBS immediately. The cells were collected from the wells bytrypsin-EDTA treatment. The radioactivity of18F-FDG was determined immediately usinga gamma counter. The uptake of18F-FDG was expressed at overall18F-FDG radioactivityin Bcap37or Bcap37/MDR1cells to the overall radioactivity added to the cells. Threeindependent experiments were performed.Results18F-FDG uptake was higher in Bcap37/MDR1cells than that in Bcap37cells afterincubation of10min. The uptake rate of18F-FDG was1.88%±0.19%in Bcap37/MDR1cells and1.37%±0.18%in Bcap37cells (P<0.05). In contrast,18F-FDG uptake wassignificantly higher in Bcap37cells than that in Bcap37/MDR1cells after60min and120min incubation. The uptake rate of18F-FDG was2.29%±0.23%,2.34%±0.15%inBcap37/MDR1cells and1.47%±0.14%,1.53%±0.22%in Bcap37cells (P<0.05).18F-FDG uptake was significantly higher in the presence of VER or GF120918inBcap37/MDR1cells than that in the absence of VER or GF120918after60min and120min of incubation (P<0.05), the uptake rate of18F-FDG in the presence of VER orGF120918was2.45%±0.21%,2.46%±0.25%and2.50%±0.24%,2.48%±0.27%.VER or GF120918did not influence the uptake of18F-FDG in Bcap37cells.Conclusions18F-FDG was a substrate of P-gp in cellular experiments. P-gp may act as anefflux pump to reduce18F-FDG uptake in Bcap37/MDR1cells. The kinetics of the uptakeof18F-FDG can be used to evaluate the function of P-gp.18F-FDG may have potentialvalue for detecting P-gp in vivo.Part Ⅳ The uptake of99mTc-MIBI in Bcap37and Bcap37/MDR1tumors Objective Planar scintigraphic images of99mTc-MIBI were acquired in BALB/c severecombined immune deficiency (SCID) mice carrying Bcap37or Bcap37/MDR1tumor. Itwas to be evaluated whether99mTc-MIBI imaging could be used to reflect the function ofP-gp in vivo.Methods Bcap37or Bcap37/MDR1cells (1×107cells/mL diluted using RPMI1640culture media,0.2mL) were injected into the subcutaneous tissue of the right axillary fossain SCID mice. The animals underwent99mTc-MIBI SPECT imagine when the solid tumorshad grown to a size of1~1.5cm (after3weeks for Bcap37cells and4weeks forBcap37/MDR1cells). Volumes of200μL99mTc-MIBI, dissolved in0.9%NaCl to a finalconcentration of37MBq/200μL, was injected into mice via the tail vein using a1mLLuer syringe. Five mice carried Bcap37tumors, and five carried Bcap37/MDR1tumors.Image acquisition was performed using a three-head gamma camera equipped with alow-energy high-resolution parallel-hole collimator. Planar scintigraphic images wereacquired at10min and60min after the injection of99mTc-MIBI. The VER-treated animalswere co-injected with0.5mg/Kg VER and37MBq99mTc-MIBI intravenously as a bolus.Image acquisition was performed at10min and60min after injection of99mTc-MIBI. Afterthe scintigraphy were completed, the animals were sacrificed by cervical dislocation.Tissue specimens of tumor and liver were taken from the animals for electron microscopy.Mitochondria was compared between tumor cells and liver cells using electron microscopy.Results Planar scintigraphic images of SCID mice showed that the physiologic distributionof99mTc-MIBI uptake was in the liver, intestine, and urinary bladder. In addition, theinjection site at the tail was visible in several animals. There was no significant differenceof99mTc-MIBI uptake between the mice carrying Bcap37or those carrying Bcap37/MDR1tumors. The tumors were not visible in all mice. In addition, no significant differenceswere observed in the images obtained at10min and60min after injection. The tumorswere not visible in the mice treated with VER. Electron microscopy revealed that Bcap37and Bcap37/MDR1tumor cells showing almost no99mTc-MIBI uptake in vivo containedonly a few mitochondria, whereas hepatocytes, displaying high99mTc-MIBI uptake in vivo, were packed densely with mitochondria.Conclusions Neither Bcap37nor Bcap37/MDR1tumors could be detected in the SCIDmice with or without VER-treated. The cell lines, with high99mTc-MIBI uptake and richmitochondria, should be selected to evaluate the function of P-gp in vivo.Part Ⅴ The uptake of18F-FDG in the Bcap37and Bcap37/MDR1tumorsObjective To evaluate the relationship between18F-FDG uptake and P-gp expression usingBALB/c the SCID mice carrying Bcap37or Bcap37/MDR1tumors.Methods The mice were fasted for6h before the trace injection and maintained underisoflurane anesthesia during the injection and scanning periods. After anesthesia, theintravenous injection of0.2mL7.4MBq was completed as a bolus. The dynamic PETscans were carried out for90min. On the PET images, the tumors were covered with oneregion of interest (ROI), and the time-activity curves were obtained. The kinetics of18F-FDG uptake in mice carrying Bcap37or Bcap37/MDR1tumors in the presence orabsence of VER was compared. Ten mice, five with Bcap37tumors and five withBcap37/MDR1tumors, were used, and approximately7.4MBq/0.2mL18F-FDG with orwithout0.5mg/Kg VER was administered via the tail vein. The Micro-PET images wereacquired60min after injection. To quantify the uptake of18F-FDG by the tumors, ROIswere drawn over the tumors and SUVmean was obtained. After the micro-PET imaginewas completed, the animals were sacrificed by cervical dislocation. The tumors were cutand embedded in10%paraffin. P-gp, Glut-1and HK-II were detected usingimmunohistochemical stain.Results18F-FDG uptake in Bcap37tumors gradually increased to a peak level at10min to15min and then remained at a constant level. VER did not significantly alter the18F-FDGaccumulation curve in Bcap37tumors.18F-FDG uptake in Bcap37/MDR1tumorsgradually increased until9min and then decreased to a constant level at25min to30min.18F-FDG uptake in Bcap37/MDR1tumors treated with VER increased to a peak level at9min, decreased to a minimum level at15min and then increased again to a peak level atapproximately30min and remained constant level. Bcap37/MDR1tumors showed higher 18F-FDG accumulation than Bcap37tumors from0to9min. In static micro-PET imaging,the mean standardized uptake value (SUVmean) of18F-FDG was significantly higher inBcap37tumors than that in Bcap37/MDR1tumors (1.00±0.06,0.66±0.11,P<0.05). VERdid not influence18F-FDG uptake in Bcap37tumors (1.00±0.06,1.09±0.22,P>0.05).VER significantly increased the SUVmean in Bcap37/MDR1tumors (1.01±0.16,0.66±0.11, P<0.05). Bcap37/MDR1tumors showed strong P-gp and Glut-1immunoreaction,whereas Bcap37tumors showed almost complete absence of P-gp and Glut-1immunoreaction. Both Bcap37/MDR1and Bcap37tumors showed moderate HK-IIimmunoreaction.Conclusions It was indicated that18F-FDG was a substrate of P-gp in vivo experiments.18F-FDG combined with VER may be an effective noninvasive method for the diagnosis ofP-gp expression in tumors. In particular, the18F-FDG uptake kinetics curves hadcharacteristic shapes for the mice carrying Bcap37/MDR1tumors. It indicated that18F-FDG PET or PET/CT using the ListMode acquisition mode may have potential clinicalvalue for detecting P-gp in vivo.