The Flow Cytometrical Evaluation of P-gp Pump Function on Leukemic Cells with Calcein-AM and Its Significance.
Authors: Tang Yongmin , Ning Botao , Lan Zhijian , Yang Shilong , Shi Shuwen , Song Hua , Shen Hongqiang , Qian Baiqin 
Published: April 2020

Abstract
Multi-drug resistance (MDR) remains the major obstacles for the successful treatment of patients with unfavorable hematopoietic malignancies. The mechanism of MDR is very sophisticated and not well understood. One of the major mechanisms for MDR is the pump function of the MDR1 gene product P-glycoprotein (P-gp). However a practical assay to accurately determine the pump function of P-gp is still lacking. In this study, an assay based on a fluorescent dye (Calcein acetoxymethyl esteror, Calcein-AM) and multi-parameter flow cytometry capable of accurately determining the pump function was established. By using the assay, the effectiveness of two reversal agents, Verapamil (VER) and cyclosporin A (CsA) could be evaluated.
Methods: A pair of leukemia cell lines, K562 and its vincristine (VCR) resistant counterpart K562/VCR were used in the study. IC50s of VCR cytotoxicity on both K562 and K562/VCR were carried out after 72 hrs of incubation under a clinical achievable level of VCR. The cells were treated with VER, CsA and VER+CsA 15 minutes prior to the addition of VCR and the reversal folds calculated. A multi-parameter flow cytometry was used to analyze the pump function of the P-gp on K562/VCR stained with Calcein-AM in either its uptake inhibition or its efflux promotion. All data were analyzed on SPSS 10.0 software.
Results: The positive rate of P-gp on K562/VCR cell membrane was 61.69% with no expression of multi-drug resistance associated protein (MRP) and both P-gp and MRP were negative on K562 cells. The resistance factor (RF) of K562/VCR to VCR was 1,287 folds more than its parental cell line K562. Although CsA, VER and CsA+VER could increase the sensitivity of K562/VCR to VCR significantly, all the reversal activities were only minimal. When Calcein-AM was used to evaluate the function of the P-gp pump on K562/VCR cells, the fluorescent intensity in K562 and K562/VCR cell lines was found to be brighter during the first 24 hrs and almost faded away at 48 hrs. The fluorescent intensity of the dye in K562 was significantly greater than that in K562 /VCR cells at each time points of 0, 12, 24, 36, 48, 72 and 120 hrs (48 hrs and 120hs P &lt;0.05, the remainder P &lt; 0.01). Uptake tests showed that although CsA, VER and CsA + VER could also increase the uptake of Calcein-AM in K562 cells, the uptake of the dye in K562/VCR was more significant at 120 min (P &lt; 0.01). The efflux inhibition of Calcein-AM in K562 cells was not significant while the inhibition became more evident in K562/VCR cells by CsA, VER either alone or in combination, among which the biggest increment of fluorescent intensity was 1.68, 1.22 and 1.39 folds, respectively.
Conclusions: With the increment of P-gp expression on the K562/VCR cells, the accumulation of Calcein-AM is reduced and the efflux is increased. CsA, VER and CsA+VER can increase the uptake of Calcein-AM in K562/VCR while reduce the efflux of the dye from the cells. However, no significance has been found statistically between the CsA+VER and CsA or VER. CsA, VER and CsA+VER can increase K562 to uptake the Calcein-AM into the cytoplasm, but they did not affect the efflux of the dye. As compared with the use of CsA or VER alone, the combination of the two is not shown to generate higher reversal activity on MDR positive cells significantly. The assay can be used to evaluate the MDR reversal agent(s) and might be used to facilitate the discovery of new reversal agents in our future work.