Transport of classical and novel taxanes in human cancer cells and in tumors of mammary gland

Introduction

The classical taxanes (paclitaxel, docetaxel) have been successfully used in the therapy of various cancers, mainly breast and ovarian cancers. However, multidrug resistance (MDR) of tumor cells represents a problem in the chemotherapy employing taxanes. MDR is a significant cause of the failure of chemotherapy in tumors with inherent or acquired resistance due to enhanced expression of ABC transporters, especially P-glycoprotein (encoded by ABCB1). Novel taxanes (e.g. SBT-1216, SBT-12854) represent a new generation of taxoids particularly more effective in resistant cancer cells.

The aim of this study was to investigate differences in transport of taxanes in our established experimental model of carcinoma cell lines MDA-MB-435 (highly sensitive) and NCI/ADR-RES (highly resistant) to classical taxanes. In addition, the transport of paclitaxel was studied directly in the slices of tumors of mammary gland.

Matherials and Methods

Cell Growth and Survival Human sensitive (MDA-MB-435) and adriamycin-resistant (NCI/ADR-RES) cancer cell lines were maintained in a culture medium at 37°C in a humidified atmosphere of 5% CO₂ in air. Cell growth and survival were evaluated after 96 h of incubation in the culture medium without taxane (control) and with various concentrations of taxanes using MTT assay.

Transport of Taxanes Cell lines were seeded at 5 x 10⁵ cells/ 1 ml of medium into wells of a 24-well plastic plate. For the uptake assay, cells were exposed to fresh medium or medium containing [¹⁴C]paclitaxel in DMSO or novel taxanes. After incubation for 15–240 min, medium containing the drug was quickly aspirated and the adherent cells were washed three times with icecold phosphate buffered saline (PBS). The cells were released by 2x 400 ml of trypsin and EDTA and then ejected into a glass tube containing ethyl acetate (for high performance liquid chromatography [HPLC] analysis), or into a scintillation vial ([¹⁴C]paclitaxel for liquid scintillation) using Bray solution. Sodium dodecyl sulfate (200 µl) was added up to 2% final concentration for lysis of cells and release of the drug. The efflux measurements were performed after 2-h loading with 100 nM [¹⁴C]paclitaxel. Cells were then washed twice with PBS, placed in fresh medium and analyzed after 10, 20, 30, 60, and 90 min. Releasing of cells was the same as described above.

Uptake by tumor slices Tumor slices of mammary gland (1x1.5 mm) were incubated in culture medium (D-MEM). After incubation for 15-60 min, the slices were homogenized and ejected into a glass tube containing ethyl acetate (2x 3.5 ml). The extracts were dissolved in amonnium chloride and extracted by SPE CN-columns. The uptake was analyzed by HPLC using acetonitrile/methanol/ammonium acetate buffer (4:1:5) mobile phase.

RT-PCR Analysis Approximately 10⁶ cells per sample were harvested by low-speed centrifugation and washed twice with PBS. The cells were then lysed in 500 µl of lysis buffer (4 M guanidium isothiocyanate, 0.5% sarcosyl, 25 mM sodium citrate, pH=7.0) on ice. Lysates were frozen at –70°C for further analysis. Total RNA (1 µg) was reversely transcribed by 120 U of MMLV (Moloney murine leukemia virus) reverse transcriptase (Invitrogen, Carlsbad, CA, USA) per sample according to the manufacturer’s instructions using random hexamer primers. Prepared cDNA (one-tenth of prepared amount, 1 µl) was subjected to polymerase chain reaction (PCR) for detection of ten ABC transporter family transcripts. PCR products were analyzed by electrophoresis in 2% agarose gel. The amount of the selected PCR product was densitometrically determined using AIDA software. The calibration curve was calculated from several DNA fragments of known mass/quantity.

Conclusion

We have demonstrated significantly higher resistance (100-fold) to paclitaxel in P-glycoprotein highly expressing NCI/ADRRES cells in comparison to P-glycoprotein non-expressing MDA-MB-435 cells. The different accumulation is to a certain extent due both to the P-glycoprotein-dependent outward transport of the taxane and related lower uptake (approximately 4 to 5-fold). Gene expression study showed that, both cell lines also significantly differ in the expression of at least two other ABC transporters (ABCB5, ABCC2) which can be involved in the transport of taxanes. Our experimental model thus proved to be interesting also in terms of the significantly different expression of ABC transporter genes.

Novel taxanes (SBT-1216 and SBT-12854) were accumulated and simultaneously more efficient in resistant cancer cells. They seem to be potential candidates for therapy of classical taxane-resistant tumors.

Our experiments with the transport of paclitaxel in human breast tumor slices also showed differences in the uptake between individual patients and we presently plan to investigate the transport of novel taxanes in tumor slices.

This work was supported by Grants IGA 9426-3 and GACR 305/07/P347.