Elsevier

Biochemical Pharmacology

Volume 78, Issue 11, 1 December 2009, Pages 1366-1373
Biochemical Pharmacology

Involvement of drug transporters in the synergistic action of FOLFOX combination chemotherapy

https://doi.org/10.1016/j.bcp.2009.07.006Get rights and content

Abstract

FOLFOX is a cytostatic drug combination for adjuvant treatment of colorectal cancer (CRC) consisting of 5-fluorouracil (5-FU), leucovorin, and oxaliplatin. The mechanism of synergistic interaction of these drugs is poorly understood and little is known concerning the role of drug transporters and the impact of oxaliplatin metabolites oxalate and dichloro-diaminocyclohexane platinum. We therefore investigated the influence of FOLFOX components on drug transporter expression by quantitative real-time polymerase chain reaction and on the efficacy of each FOLFOX component by proliferation assay in the CRC model cell line LS180. Control experiments with transporter over-expressing cell lines were used to assess the significance of important transporters for the cytostatic activity of FOLFOX components. Moreover, we assessed the pharmacological contribution of the oxalato-ligand to the effect of oxaliplatin. FOLFOX components led to several alterations in expression of drug transporters. For instance, 5-FU significantly suppressed ATP7B and human organic cation transporter 2 and increased multidrug resistance-associated protein (MRP) 2 mRNA expression (5.8-fold). This was accompanied by a significant sensitisation to oxaliplatin. Over-expression of certain ABC-transporters (BCRP/ABCG2, MRP2/ABCC2 or MRP3/ABCC3) was demonstrated to be beneficial for the efficacy of oxaliplatin. The results obtained indicate that both down- and up-regulations of drug transporters could favour synergistic action of this drug combination. Moreover, oxaliplatin metabolite oxalate seems to positively modulate oxaliplatin's action as elucidated by median effect analysis. In conclusion, we propose as one mechanism for FOLFOX synergism the 5-FU mediated suppression of ATP7B, the over-expression of glutathione exporters such as MRP2/ABCC2 and the decrease of glutathione levels by oxalate.

Introduction

The addition of oxaliplatin to standard chemotherapy with 5-fluorouracil (5-FU) and folinic acid (leucovorin) (FOLFOX regimen) has substantially improved the outcome of patients with colorectal cancer (CRC) [1]. The mode of synergistic interaction between these compounds, however, is not well investigated. Moreover, also the potential role of drug transporters limiting access of chemotherapeutic agents and co-factors to CRC cells has been addressed [2], [3], but their role in the FOLFOX regimen has not been thoroughly investigated.

The ATP-binding cassette (ABC-) transporter superfamily contains several family members that may confer intrinsic or acquired multidrug resistance (MDR) by extruding anticancer agents or their metabolites from cells [4] and suppression of such transporters may lead to sensitisation to cytostatic agents [5]. Indeed, BCRP/ABCG2 and several members of the multidrug resistance-associated protein (MRP/ABCC) family have been identified as folate export transporters [6] and the activity of both, MRP5/ABCC5 and MRP8/ABCC11 have been linked to MDR to 5-FU [7], [8].

In contrast, transport processes may also promote antineoplastic action. The distribution of Pt containing antineoplastic agents is regulated by copper homeostasis components. As an example, human copper transporter 1 (hCTR1/SLC31A1) mediates the cellular uptake of copper, cisplatin, and oxaliplatin [9]. In addition, the two P-type ATPases ATP7A and ATP7B are also associated with transport and resistance to oxaliplatin, either by promoting efflux out of the cell or by sequestration into subcellular compartments [10]. Human organic cation transporter 2 (hOCT2/SLC22A2) was also shown to mediate oxaliplatin uptake [11]. Moreover, glutathione, which inactivates cisplatin and oxaliplatin by conjugation [12], [13], is exported by several MRPs/ABCCs, either alone, as co-substrate, or in its conjugated form. Thus, enhanced MRP/ABCC expression can lead to decreased cellular glutathione content [14].

The aim of this in vitro study was to scrutinise the mode of synergistic action of FOLFOX components by evaluation of their effects on both expression of drug transporters and cytostatic properties and to assess the pharmacological contribution of the oxalato ligand to oxaliplatin's effect, because it is well known that oxalate has profound effects on redox status [15], production of radical oxygen species [16], and cytotoxic properties of Pt drugs [17].

Section snippets

Materials

Culture media, foetal calf serum (FCS), medium supplements, antibiotics, glutamine, phosphate buffered saline (PBS), and 5-chloromethylfluorescein diacetate (CMFDA) were purchased from Invitrogen (Karlsruhe, Germany). Acetylcystein, dimethyl sulfoxide (DMSO), 2-(N-morpholino)ethanesulfonic acid (MES), aprotinin, dichloro-diaminocyclohexane (DACH)-platinum (Cl2-DACH-Pt), DACH, leucovorin, 5-FU, and verapamil hydrochloride were purchased from Sigma-Aldrich (Taufkirchen, Germany) and bromphenol

Differential effects of FOLFOX components on resistance/sensitivity of LS180 cells

After incubation with oxaliplatin or 5-FU for 72 h, LS180 cells were significantly more sensitive to oxaliplatin. IC50 values were approximately 50% of vehicle treated cells. The effect of 5-FU preincubation on 5-FU activity was even greater but did not reach statistical significance. In contrast, leucovorin did not influence the vulnerability of the cells to any of the drugs applied (Fig. 1).

Differential effects of FOLFOX components on mRNA expression of drug transporters

With the exception of hOCT2/SLC22A2, which was suppressed during leucovorin treatment, leucovorin had no

Discussion

Multidrug resistance increasingly requires anti-cancer combination therapy. The aim of this in vitro study was to elucidate the transcriptional alterations of drug transporters’ mRNA expressions and functional alterations following the exposure of LS180 cells to individual components of the FOLFOX regimen. Functionally, preincubation with oxaliplatin or 5-FU both sensitised LS180 cells to oxaliplatin. Incubation with oxaliplatin was accompanied by a decrease of mRNA and protein expression of

Acknowledgement

Dirk Theile was funded by grant #107173 from the “German Cancer Aid”.

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    These authors contributed equally to this work.

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