A Fluorescent Cell-Based Technique for Monitoring Efflux of MRP4

Background: Overexpression of efflux pumps is the drug resistance and adaptation mechanism employed by some eukaryotes and bacteria to transport endogenous and chemotherapeutic compounds from the intracellular to the extracellular environment. Aim: The study aimed at establishing a fluorescent cell-based assay to monitor the efflux activities of an ABC-transporter, multi-drug resistance protein 4 (MRP4). Methods: DH5α competent E. coli cells were transformed with pcDNA-MRP4 by the heat-shock process. The presence of the MRP4 gene was analyzed by the digestion of plasmid using EcoRI and analyzed on a 1% agarose gel. HEK 293 cells were transfected with purified pcDNA-MRP4 under optimized conditions using a Polyethylenimine (PEI) protocol. The level of MRP4 in the HEK 293 cells was characterized by western blotting analysis using M4I-10 anti-MRP4 and anti-Rat IgG (whole molecule)-Alkaline phosphatase antibodies. The fluorescent uptake study was performed by the incubation of 0.02 mM 8-[fluo-cAMP] with the MRP4-transfected and control HEK 293 cells for 1 h. The level of fluorescence was analyzed using fluorescence microscopy and spectrometer. Results: The agarose gel analysis showed a plasmid of 9.4 kb and restriction product of 5 kb, which correspond with the pcDNA and MRP4 sizes respectively. The western blot results of the transfection showed 4 μg pcDNA-MRP4 and the N/P ratio of 9 was the optimized condition to transfect our HEK 293 cells as it showed the broadest band. In the efflux studies, the fluorescence images of the MRP4-transfected HEK 293 cells were very low compared to the untransfected control. The level of fluorescence accumulation was significantly (P ≤ 0.0001) higher 228.6 ± 13.1 RFU in the untransfected cells than the MRP4-transfected cells 8.6 ± 1.8 RFU. Conclusion: The higher levels of fluorescence detected in the control in both the fluorescent microscopy and spectrophotometer showed that MRP4-transfected cells had effluxed the 8-[fluo-cAMP] substrate out of the cell. This method could be employed in the detection of MRP4 functions in bacteria and cancer cells.


Introduction
The development of some novel drugs has been hampered due to the expression and upregulation of efflux pumps and secretory systems in some eukaryotic organisms and bacteria [1]. Efflux pumps are an energy-driven system used by both eukaryotes and some bacteria to transport molecules against the concentration gradient. This mechanism is both beneficial and deleterious. Some endogenous molecules in humans and microbes are translocated via this mechanism to sites that they are needed. During drug administration, cells that harbor these efflux pumps remove the drug from within the cell via these pumps rendering the drug inactive. In humans, a high level of expression of MRP4 mRNA has been found in kidney and prostate and low levels have been shown in blood cells, neurons, lungs, adrenal gland, testis, and ovary [2] [3] [4]. These efflux pumps have also been described in bacteria such as Staphylococcus aureus and Escherichia coli (MsbA) [5] [6].
Cytotoxicity studies on MRP4-transfected cells showed broad substrate specificity as it can transport a range of endogenous and exogenous compounds [10].
The detection of the interactions between chemotherapeutic compounds and MRP4 is crucial for the development of the drugs to target cells or microorganisms that express this transmembrane transporter. These interactions provide information on bioavailability, drug-drug, and drug-biomolecular interactions.
According to Lechner et al. [11], this will provide a more in-depth understanding of the pharmacokinetics of drugs in cells expressing MRP4.
This study aimed at developing a fluorescent cell-based assay to measure MRP-mediated efflux that could potentially be utilized for the screening of MRP4 inhibitors. 8-[Fluo-cAMP] was used as the substrate for the transporter because MRP4 is an active transporter of cyclic nucleotides monophosphates. 8-[fluo-cAMP] is a fluorescein-modified analog of the parent cAMP linked at the eighth position of the molecule via a six-atom spacer [12]. The study transfected human  [13]. The importance of this study is that there has been no previous record of a fluorescent cell-based investigation of MRP4. Previous researches detected MRP4 function by analyzing fluorescence in MRP4-containing vesicles and lysed cells [12] [14].

Transformation of E. coli with pcDNA-MRP4
The vector, pcDNA-MRP4, encodes the transporter gene MRP4 and ampicillinresistance gene. DH5α competent E. coli cells were transformed with pcDNA-MRP4 by the heat-shock process. The heat-shocked E. coli cells were incubated in LB broth and plated on LB-ampicillin plates and incubated at 37˚C overnight.
The transformed pcDNA-MRP4 E. coli cells were cultured by incubated individual colonies of the transformed cells in LB-ampicillin broth overnight. were analyzed on 1% agarose gel.

Seeding Cells into Six-Well Plates
The old medium was removed from the flask and cells were washed with PBS then cells were detached using trypsin-EDTA (at 37˚C). The old DMEM medium was replaced with a fresh medium and centrifuged at 10,000 rpm for 5 minutes at room temperature. The cell pellet was re-suspended in DMEM medium. The cell density was determined using hemocytometer and cells were seeded in a six-well plate at a concentration of 5 × 10 5 cells/ml and 2 ml per well. 25 kDa PEI was dissolved in distilled water to make the final concentrations of 10 mM while glucose was dissolved to make 5% of the distilled water. The dissolved PEI and glucose were filter sterilized. Because the efficiency of PEI transfection depends on the ratio of phosphate in the DNA to the nitrogen in the PEI (N/P ratio), the different transfection conditions were achieved by varying this ratio. The different amounts of PEI used were calculated as follows: 1 μg of DNA 3 nmol DNA phosphate = 1 μl of 10 mM PEI 10 nmol amine nitrogen = ( ) µg of DNA 3 N : P ratio µl 10 mM PEI 10

× × =
The above equation was used to obtain the DNA amounts and PEI used. The transfection mix was 10% of the medium used 200 µl/well; 100 µl of DNA/glucose mix and 100 µl of PEI/glucose mix.
The sterile PEI was mixed with the 5% sterile glucose and pcDNA-MRP4 was also mixed with the 5% sterile glucose. The PEI/glucose complex was added to the pcDNA-MRP4/glucose mixture at different N/P ratios (*different amounts of DNA were used in some instances) (N/P = 9 [4 µg DNA]; N/P = 12 [*4 µg DNA]; N/P = 12 [*2 µg DNA]; N/P = 15 [*4 µg DNA]; and N/P = 15* [2 µg DNA]) and incubated at room temperature for 30 minutes. N/P ratios of PEI/DNA complexes were the ratios of moles of the nitrogen groups of PEI to those of the phosphate of DNA. The PEI/DNA/glucose mixtures were added to the DMEM medium. The old medium was discarded from the cells and PEI/DNA/glucose/medium was added to the cells and the N/P ratios of the different wells were as mentioned above. A control was set up by adding only DMEM medium and no transfection mixture. The cells were incubated at 37˚C in a humidified 5% CO 2 atmosphere for 48 h. The amount of protein of each fraction was estimated using a Bio-Rad protein assay (Bio-Rad, Hercules, CA).

Western Blot Analysis and Immunoblotting with Anti-MRP4 in Rabbit
The western blotting for MRP4 detection was carried out using a whole-cell lysate. The samples were loaded on a 7% SDS-PAGE and run at 100 volts for 1 h.

Data Analysis
The western blot paper was photographed with a camera. Where necessary data was analyzed on GraphPad Prism 8.

(San Diego CA) using t-test and results
were considered to be statistically significant at P < 0.05.

MRP4 Expression in Transfected HEK 293
The levels of MRP4 in the transfected cell line during the optimization were T. P. Monsi et al.

8-[Fluo-cAMP] Efflux Studies
The efflux studies analyzed the fluorescence generated both by qualitative and quantitative methods. Figure 3 shows  (Figure 4(b)).

Discussion
The movement of biological molecules across the membranes plays an indispensable role in the maintenance of normal physiological activities in many bacteria and American Journal of Molecular Biology   A fluorescent cell-based approach offers a real-time, efficient, and rapid method of monitoring the efflux pump interactions. This method could further provide a suitable strategy for blocking its transport activities and potential drug-drug interactions indispensable in drug design [20].
The faint band seen in the undigested lane was more than 10 kb which could be another supercoiled plasmid present in the E. coli isolate while the 9.4 kb band represents the pcDNA-MRP4 plasmid (Figure 1(a) and Figure 1(b)). For lanes 3 and 4, the broadest bands represent the 5 kb which corresponds with the size reported by Barik et al. [21]. The other faint bands correspond to the ampicillin resistance gene and other associated regulatory genes. The purity of the digestion was high as there was no smear produced.
The western blot analysis showed the transfected MRP4 gene, using the PEI protocol, highly expressed the transporter in HEK 293 cells which could be con- Other studies carried out to monitor the function of MRP4 used MRP4 in vesicles [11] [14] to detect the efflux activities of the transporter but not the transfections of the gene. The transfection protocol adopted in the current investigation used PEI which is a 25 kDa cationic polymeric molecule that condenses the DNA into positively charged particles enabling its endocytosis into the cell [27].
The determination of the appropriate N/P ratio and DNA concentration is cru-

Conclusion
The study has established a fluorescent cell-based method for MRP4 interactions