Direct assessment of P-glycoprotein efflux to determine tumor response to chemotherapy

Abstract

Multidrug resistance is a major impediment to the success of cancer chemotherapy. The overproduced P-glycoprotein that extrudes anticancer drugs from cells, is the most common mechanism detected in multidrug-resistant cancers. Direct measurement of cellular efflux of tumors in vivo, rather than estimation of MDR1 mRNA and P-glycoprotein levels in samples stored or embedded, can functionally characterize the mechanism of drug resistance and determine the choice of anticancer drugs for cancer patients. Herewith, we introduce a new approach to directly determine P-glycoprotein efflux of tumors. Employing Flutax-2 (Oregon green-488 paclitaxel) and fluorescence spectrophotometry, this method has successfully measured cellular transportability including efflux and accumulation in diverse cancer cell lines, tumors and other tissues with high reproducibility. With this method, we have quantitatively determined cellular efflux that is correlated with P-glycoprotein levels and the reversal effects of agents in cell lines of breast, ovarian, cervical and colon cancers, and in tumor-bearing mice. It has sensitively detected these alterations of P-glycoprotein efflux in approximately 5mg tumor or other tissues with high confidence. This direct and quick functional assessment has a potential to determine drug resistance in different types of cancers after surgical resection. Further validation of this method in clinic settings for the diagnosis of drug resistance purpose is needed.

Fig. 1

Quantitation of cellular accumulation and efflux using Flutax-2. (A) Cellular accumulation and extrusion. Human NCI/ADR-RES cells were incubated with increasing concentrations of Flutax-2 for 2 hr. Cellular Flutax-2 was measured to evaluate cellular accumulation (dashed line). After additional 2 hr incubation, the extruded Fluta-2 in medium was measured to evaluate cellular extrusion (solid line). (B) Detection of cellular efflux after P-gp inhibition. After verapamil pre-treatments (2 hr), NCI/ADR-RES cells were incubated with Flutax-2 (500 nM) for the accumulation and extrusion. *, p<0.001 compared with vehicle control in accumulation; **, p<0.001 compared with vehicle control in efflux; efflux#, efflux (efflux/accumulation) is presented as percents in solid circles and line.

Fig. 2

Examination of cellular accumulation and efflux in diverse cancer cell lines. (A) P-gp protein levels. Equal amount of detergent-soluble cell proteins (50 μg/lane) were resolved using SDS-PAGE and were immunoblotted with anti-P-gp antibody. The levels of P-gp were presented in the ratios of P-gp/GAPDH densities. *, p<0.001 compared with drug-sensitive counterpart. (B) Cellular accumulation and efflux of cancer cells. Cells were incubated with Flutax-2 (0.5 μM) for 2 hr to measure the accumulation. After washing, the cells were incubated in fresh medium for additional 2 hr to measure the efflux. *, p<0.001 compared with drug-sensitive counterpart. (C) P-gp in cells after MBO-asGCS treatment. NCI/ADR-RES cells were treated with MBO-asGCS for 6 days. Equal amount of detergent-soluble proteins (50 μg/lane) were resolved and were immunoblotted with anti-P-gp antibody. *, p<0.001 compared with vehicle control. (D) The accumulation and efflux of cells after MBO-asGCS treatment. After MBO-asGCS treatments, NCI/ADR-RES cells were incubated with Flutax-2 (0.50 μM) for accumulation and efflux assay. *, p<0.001 compared with vehicle control.

Fig. 3

Accumulation and extrusion of Flutax-2 in tissues. (A) Flutax-2 accumulation in cell suspension of tissues. Cell suspensions of NCI/ADR-RES tumor, small intestine and liver of athymic mice were incubated with Flutax-2 (1 μM) in medium containing collagenase IV. The smear slides from each incubation condition were observed under microscope (left side, bright field; right side, fluorescence filed). (B) Accumulation and extrusion of Flutax-2 in cell suspensions of tumor, small intestine and liver. Increasing amounts of cell suspensions were incubated with Flutax-2 (1 μM) in medium containing collagenase. After 2 hr, cellular Flutax-2 was measured to evaluate accumulation (dashed line). The extrusion of the suspensions was measured after 2 hr additional incubation (solid line).

Fig. 4

Kinetics of Flutax-2 accumulation and extrusion in tumors. Cell suspensions of NCI/ADR-RES tumors were incubated with Flutax-2 (1 μM) in medium containing collagenase. After 2 hr incubation, cellular Flutax-2 was measured to evaluate accumulation (dashed line). The efflux of tumor cell suspension was measured after 2 hr additional incubation (solid line). The parameters of kinetics were evaluated and presented in Table 1.

Fig. 5

Alterations of P-gp in tumors of mice after treatments in vivo. After MBO-asGCS treatments, NCI/ADR-RES and SW620AD tumors were removed and used to extract proteins. Equal amount of detergent-soluble proteins (50 μg/lane) were resolved using SDS-PAGE and were immunoblotted with anti-P-gp antibody. P-gp levels were presented in the ratios of P-gp/GAPDH densities. *, p<0.001 compared with saline treatments.

Fig. 6

Cellular transportability of tumor after storage. After resection, NCI/ADR-RES tumor tissues were stored in 4°C for indicated periods of time. The cell suspensions of tumors were incubated with Flutax-2 (1μM) in RPMI-1640 medium containing collagenase to measure cellular accumulation and extrusion. *, p<0.001 compared with fresh tumors. Efflux#, efflux (efflux/accumulation) is presented as percents in solid circles and line. Data represent mean ± SD of triplicates.