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. Author manuscript; available in PMC: 2011 Jan 15.
Published in final edited form as: Cancer Res. 2010 Jun 15;70(14):5931–5941. doi: 10.1158/0008-5472.CAN-10-0694

Src family kinase inhibitor saracatinib (AZD0530) impairs oxaliplatin uptake in colorectal cancer cells and blocks organic cation transporters

Christopher J Morrow 1,5, Mohammad Ghattas 2, Christopher Smith 3, Heinz Bönisch 4, Richard A Bryce 2, D Mark Hickinson 3, Tim P Green 3,*, Caroline Dive 1,2,5
PMCID: PMC2906706  EMSID: UKMS30922  PMID: 20551056

Abstract

Elevated Src Family Kinase (SFK) activity is associated with tumour invasion and metastasis. The SFK inhibitor saracatinib (AZD0530) is currently in Phase II trials in patients including those with colorectal cancer (CRC), where links between SFK activity and poor prognosis are particularly striking. Saracatinib is likely to be used clinically in combination regimens, specifically with 5FU and oxaliplatin in CRC. The aim of this study was to determine the impact of saracatinib on oxaliplatin and 5FU efficacy in CRC cells. Saracatinib did not modulate 5FU efficacy but antagonized oxaliplatin in a schedule specific manner through reduced oxaliplatin uptake via an SFK independent mechanism. Saracatinib resembles the pharmacophore of known organic cation transporter (OCT) inhibitors and reduced oxaliplatin efficacy maximally in cells over-expressing OCT2. These data suggest that oxaliplatin uptake in CRC is attenuated by saracatinib via inhibition of OCT2, a potential consideration for the clinical development of this SFK inhibitor.

Keywords: saracatinib/AZD0530, Src Family Kinase, Colorectal Cancer, Oxaliplatin Uptake, Organic Cation Transporters

Introduction

The gene encoding the non-receptor protein tyrosine kinase c-Src was the first proto-oncogene to be described when identified as the cellular homologue of v-Src, the transforming factor of the Rous sarcoma retrovirus (1). c-Src is one of nine members of the Src Family Kinases (SFK) and, along with two other members Fyn and Yes, is ubiquitously expressed (2). Of the nine SFKs, c-Src is the most comprehensively studied regarding its role in cancer. Increased levels of c-Src activity occur in several solid tumour types, including colorectal (CRC), breast and lung cancers (3). In CRC, c-Src activity is elevated in more than 70% of tumours and increases with tumour progression (4-7) where high c-Src activity correlated with poor clinical prognosis (8).

A major role of c-Src activity in cancer is promotion of migration and invasion leading to a more aggressive and metastatic cancer phenotype (9). Currently there are four SFK inhibitors in oncology clinical trials, including saracatinib (AZD0530), a potent, orally available SFK inhibitor (10). Consistent with the studies outlined above, SFK inhibition by saracatinib has varying and cell context dependent effects on cell proliferation in vitro and in vivo but consistently inhibits migration, invasion and metastasis across a range of cancer types (11). In common with other targeted agents, saracatinib is likely to be used clinically in combination with other chemotherapeutic agents; indeed, preclinical studies have demonstrated that saracatinib enhanced the anti-migratory effects of the EGFR inhibitor gefitinib in endocrine resistant breast cancer cell lines (12) and restored tamoxifen sensitivity to resistant breast cancer cell lines (12, 13).

In a Phase II trial in patients with previously treated metastatic colorectal cancer, saracatinib alone had negligible effect on overall survival (14). Thus, combining saracatinib with standard of care agents may present an alternative strategy to target SFK in CRC. Therefore, the aim of this study was to investigate the affect of saracatinib on oxaliplatin and 5FU responses in CRC cell lines.

Materials and Methods

Cell culture

HCT116 cells (ATCC) were cultured in McCoys-5A (Gibco), WiDr (ATCC) in RPMI (Gibco) and parental HEK293 (ATCC) and HEK293 over-expressing OCT isoforms (15) in DMEM:F12 (Gibco), all supplemented with 10% fetal bovine serum (Biowest) in a humidified atmosphere at 37°C and 5% CO2. OCT over-expressing cells were also supplemented with 800μg/ml G418 (Gibco). All cell lines were authenticated using the Ampflstr system (Applied Biosystems) during the study. Oxaliplatin (Alexis Biochemicals), cisplatin (Sigma), 5FU (Sigma), saracatinib and PP2 (4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine – Calbiochem) were diluted to the indicated concentration in media from stock solutions.

Western blotting

Cell lysis and western blotting were carried out as previously described (16). The following primary antibodies were used; anti-pY576FAK, anti-pY861FAK (Biosource), anti-FAK (Santa-Cruz), anti-c-Src (Upstate), anti-Yes, anti-Fyn (Wako) and anti-actin (Sigma).

SRB assay

Cells were plated in 96-well plates and treated as described, fixed for 1h with ice cold 10% trichloroacetic acid and left to dry. Cells were then stained with 0.4% Sulforodamine B (SRB) for 15min, washed with 1% acetic acid, the dye solubilized with 1.5M Tris.HCl (pH 8.8) and the absorbance at 540nm determined.

Comet-X assay

After treatment, cells were irradiated with 45Gy from an X-ray source and processed for comet-X assay as previously described (17) where 100 nuclei on two slides were measured for each treatment.

Platinum concentration determination

After treatment, cells were either processed with a blood and cell culture DNeasy blood and tissue kit (Qiagen, following manufacturer's instructions) to generate samples to determine DNA-associated platinum concentration, or lysed in cell lysis buffer (Cell Signaling supplemented with protease inhibitor cocktail, Sigma) to determine soluble and protein-associated platinum levels. DNA concentration was determined on a Nanodrop-2000 (Thermo Scientific) and protein concentration was determined by BCA assay (Perbio) following manufacturers' instructions. Samples were hydrolyzed by incubating 90μl of sample with 10μl 10M NaOH at 60°C for one hour and then adding 900μl 1M nitric acid and incubating at 70°C overnight. Platinum concentration was measured on an ELAN DRc ICP-MS (Perkin Elmer SCIEX).

RNA interference

HCT116 cells were transfected with c-Src or non-targeting SMARTpool siRNA (Dharmacon) at 100nM using the DharmaFECT 2 siRNA transfection reagent according to the manufacturer's instructions. After 24h, siRNA was replaced with full growth media.

Wound-healing assay

Scratches were made in confluent HCT116 cells monolayers grown in ImageLock 24-well plates using a Woundmaker (Essen), media was removed and replaced with media containing the specified drugs. Cells were placed in an Incucyte (Essen) and the wound imaged every two hours for 48h. The % wound closure after 48h was determined using Incucyte scratch wound assay software (Essen).

ASP+ uptake assay

106 HEK293 cells over-expressing OCT isoforms were washed with warm HCO3-free Ringer's like solution (RLS - 130mM NaCl, 4mM KCl, 1mM CaCl2, 1mM MgSO4, 1mM NaH2PO4, 20mM HEPES, 18mM glucose pH7.4) and incubated in RLS containing the indicated concentration of saracatinib for 10min at 37°C. 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP+ - Sigma) was added to a final concentration of 1μM and the cells were incubated for a further 30min at 37°C. Cells were washed twice in ice cold RLS and solubilized in isopropanol. Fluorescence was measured after 485nm λex at 610nm on a fluostar optima plate reader (BMG Labtech)

Molecular modeling

The OCT pharmacophore was generated as follows: tetrapentylammonium ion (TPA) was overlaid on Decynium-22 using the Omega software package (OpenEye Scientific Software) and low energy conformations of tetrapentylammonium ion and Decynium-22 were generated. ROCS (Rapid Overlay of Chemical Structures) (18) were then used to overlay TPA conformations on the lowest energy Decynium-22 conformer, based only on shape (Shape-Tanimoto scoring function). The best 1000 TPA conformers from this filter were then compared to Decynium-22 using shape and electrostatics, using the ET-Combo scoring function from EON. Finally, using this overlaid Decynium-22/TPA pair, a four-point pharmacophore was generated using MOE (Chemical Computing Group, Montreal). Subsequently, a pharmacophore search was performed for conformations of a set of 20 known non-transported OCT2 inhibitors (19) and for saracatinib. The MOE Wash function was used in combination with ACD/I-Labs (Advanced Chemistry Development Inc, Toronto) to assign protonation states and tautomers of the library of compounds, prior to conformer enumeration by Omega.

Statistics

Student's t-test, 2-way-ANOVA and IC50 calculations were performed using Prism (Graphpad).

Results

Saracatinib reduced the efficacy of oxaliplatin, but not cisplatin, in a schedule-dependent manner

As saracatinib is likely to be used to treat patients with metastatic CRC in combination with other standard of care drugs, the impact of saracatinib was assessed in two CRC cell lines treated with oxaliplatin or fluorouracil (5FU). The cell lines chosen for this study, HCT116 and WiDr, have low and medium levels of c-Src activity respectively (20, 21). Saracatinib treatment for 24h reduced the levels of phosphorylated FAK, a target of SFKs, in a concentration dependent manner (Figure 1A) demonstrating that saracatinib inhibited SFK activity in these cellular contexts. It is interesting to note that even low concentrations of saracatinib lead to increased levels of total FAK, although the mechanism by which this occurs is unclear. Saracatinib had little effect on the proliferation of HCT116 or WiDr cells; a 6 day treatment of 1μM saracatinib had minimal effect on either cell line (Figure 1B), consistent with previously published data (11). In order to better mimic clinical exposure, cells were treated for 1h with oxaliplatin, or 6 days with 5FU, both of which caused a concentration-dependent reduction in cell population (Figure 1B and Supplemental Figure S1). The addition of saracatinib had no effect on 5FU efficacy in either cell line (Supplemental Figure S1). However, if saracatinib and oxaliplatin were added simultaneously and saracatinib replenished after oxaliplatin removal there was a significant decrease in oxaliplatin efficacy in both cell lines (Figure 1B – p<0.001 for oxaliplatin vs. oxaliplatin and saracatinib in HCT116 and WiDr according to 2-way-ANOVA). The negative affect of saracatinib on oxaliplatin was schedule-dependent; if saracatinib was added to cells after the 1h oxaliplatin exposure there was no affect on oxaliplatin efficacy (Figure 1C). Furthermore, concomitant saracatinib exposure did not affect cisplatin (Figure 1D) or carboplatin (Supplemental Figure S2) efficacy suggesting saracatinib does not reduce the efficacy of all platinating agents, but interacts with oxaliplatin specifically.

Figure 1. Saracatinib reduced oxaliplatin efficacy in a schedule-dependent manner.

Figure 1

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(A) HCT116 (left) and WiDr (right) cells were exposed to the indicated concentrations of saracatinib for 24 hours and the effect on total FAK or FAK phosphorylation on tyrosine 576 or 861 was assessed by western blotting. Result is representative of three independent experiments. (B-D) HCT116 (left) and WiDr (right) cells were exposed to the indicated concentrations of oxaliplatin (B and C) or cisplatin (D) for one hour and/or saracatinib for six days (B-D). Where two drugs were used in combination (white squares) saracatinib was either added at the same time as oxaliplatin/cisplatin and more saracatinib added after the removal of oxaliplatin/cisplatin (B and D) or saracatinib was only added after the removal of oxaliplatin (C). Six days after the removal of oxaliplatin/cisplatin cells were fixed, stained with SRB and the absorbance relative to untreated (UnT) cells determined as an approximation of cell population. Graphs show mean of three independent experiments carried out in triplicate +/− S.E.M.

Saracatinib reduced oxaliplatin-induced DNA-crosslinks

The mechanism of action of oxaliplatin is thought to be predominantly via DNA damage induced by DNA-platinum-DNA interstrand crosslinks (22). Therefore, the affect of saracatinib on oxaliplatin-induced DNA crosslinks was investigated using the comet-X assay (23). Cells were treated with oxaliplatin or cisplatin for 1h in the presence (and sara) or absence (then sara) of saracatinib and then grown in the absence of the platinum agent, with saracatinib where indicated, for a further 8h to allow time for DNA interstrand crosslinks to form (24). In the comet-X assay, reduced DNA in the comet tail is indicative of increased DNA interstrand crosslinking. The exposure of HCT116 or WiDr to oxaliplatin or cisplatin caused a significant reduction in the amount of DNA in the comet tail, while the presence of saracatinib during the 1h oxaliplatin exposure caused a significant increase in the comet tail relative to oxaliplatin only (Figure 2A). Adding saracatinib after oxaliplatin exposure did not alter comet tail size, nor did addition of saracatinib during cisplatin exposure. This suggests that saracatinib can cause a reduction in the amount of oxaliplatin-induced DNA interstrand crosslinks but only when present at the time of oxaliplatin treatment.

Figure 2. Saracatinib inhibits oxaliplatin uptake.

Figure 2

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(A-C) HCT116 (left) and WiDr (right) cells were not treated (white bars) or exposed to 120μM (HCT116) or 80μM (WiDr) oxaliplatin (black bars) or 90μM (HCT116) or 60μM (WiDr) cisplatin (grey bars) for one hour in the presence (ox/cis and sara) or absence (ox/cis then sara) of 1μM saracatinib. Cells were either harvested (B and C – 0 Hours) or fresh media with or without 1μM saracatinib (as indicated) was added and cells were harvested 8h later (B and C – 8 Hours and A). (A) Cells were processed and analyzed for comet-X assay as described in materials and methods. (B and C) Genomic DNA was extracted as described in materials and methods (B) or cells were lysed in cell lysis buffer (C) and the level of platinum in the samples determined by ICP-MS. Platinum concentration was normalized to the equivalent oxaliplatin or cisplatin only treatment from the corresponding time-point. Graphs show mean of three independent experiments +/− S.E.M. * p<0.05, ** p<0.01, *** p<0.001 according to two-tailed unpaired t-test.

To formally test if the reduction in oxaliplatin-induced DNA interstrand crosslinks caused by saracatinib was due to reduced platinum-DNA adducts, the level of DNA-associated platinum was measured using inductively coupled plasma mass spectrometry (ICPMS). Genomic DNA was isolated from cells treated with oxaliplatin or cisplatin in the presence or absence of saracatinib, either immediately after the removal of the platinum, or 8h after platinum removal. Results shown in Figure 2B are relative to the corresponding oxaliplatin or cisplatin only treatment. The presence of saracatinib during the 1h oxaliplatin exposure (ox and sara) reduced the amount of DNA-platinum adducts by ~50% immediately after and 8h after oxaliplatin removal (Figure 2B). If saracatinib was added after removal of oxaliplatin (ox then sara), it had no affect on DNA-platinum adduct level. Regardless of the schedule, saracatinib had no affect on DNA-platinum adducts in cisplatin-exposed cells. This confirmed that saracatinib reduced oxaliplatin-induced DNA-platinum adduct levels if present during the oxaliplatin exposure.

Saracatinib reduced uptake of oxaliplatin

There are at least two possible explanations for the change in oxaliplatin-induced DNA-platinum adducts caused by saracatinib; either saracatinib causes an increase in the rate of removal of oxaliplatin-induced DNA-platinum adducts (if present during treatment), or saracatinib reduced the total level of oxaliplatin in the cell, either by inhibiting uptake or increasing efflux. Due to the fact that the relative level of DNA-platinum adducts did not change over time, the reduced oxaliplatin uptake explanation seemed the more plausible. Therefore, the level of soluble/protein-adducted platinum was determined from cells treated with the same drug combinations as in Figure 2B. The level of soluble/protein-adducted platinum was reduced to 60-80% compared to oxaliplatin only when saracatinib was present during the 1h oxaliplatin exposure (ox and sara) and not in any of the other experimental conditions (Figure 2C). This strongly suggested that the presence of saracatinib reduced the uptake of oxaliplatin into HCT116 and WiDr CRC cells.

c-Src RNAi did not phenocopy the affect of saracatinib on oxaliplatin efficacy

The affect of c-Src RNAi on oxaliplatin efficacy was assessed to determine whether the interaction between saracatinib and oxaliplatin was due to inhibition of c-Src by saracatinib. Transfection of siRNAs targeting c-Src mRNA into HCT116 cells consistently reduced the level of c-Src (Fyn levels were also reduced while levels of Yes were unchanged – Figure 3A) and also reduced phosphorylated FAK levels, demonstrating reduced c-Src activity. However, c-Src RNAi did not affect oxaliplatin efficacy (Figure 3B – p>0.05 according to 2-way-ANOVA) suggesting that c-Src activity is not necessary for oxaliplatin efficacy or uptake. Therefore, the observed interaction between saracatinib and oxaliplatin is unlikely to be due to inhibition of c-Src activity.

Figure 3. Effect of saracatinib on oxaliplatin is not due to inhibition of SFK activity.

Figure 3

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(A and B) HCT116 cells were transfected with non-targeted siRNA or siRNA targeting c-Src. (A) 48h later the level of c-Src, Yes, Fyn, FAK or FAK phosphorylated on tyrosine 576 or 861 were determined by western blotting. Actin was used as a loading control. (B) 24h after transfection cells were plated and 24h later exposed for one hour to the indicated concentration of oxaliplatin. Three days later cells were processed as in Figure 1. (C) HCT116 cells were exposed to the indicated concentrations of saracatinib or PP2 for one hour and the effect on total FAK or FAK phosphorylation on tyrosine 861 was assessed by western blotting. (D) HCT116 cells were exposed to the indicated concentrations of oxaliplatin and/or saracatinib (top) or PP2 (bottom) for one hour. Three days later cells were processed and in Figure 1. All western blots are representative of three independent experiments. Graphs show mean of three independent experiments carried out in triplicate +/− S.E.M.

The SFK inhibitor PP2 did not phenocopy the affect of saracatinib on oxaliplatin efficacy

As well as inhibiting c-Src, saracatinib also inhibits other members of the SFK family (11). Therefore, to determine whether the interaction observed between saracatinib and oxaliplatin could be attributed to SFK inhibition, the affect on oxaliplatin efficacy of a second SFK inhibitor, PP2, was investigated. Unlike saracatinib treatment, three day exposure of HCT116 cells to PP2 lead to a reduction in cell number (data not shown), presumably due to inhibition of a non-SFK kinase by PP2 but not by saracatinib (25) which makes direct comparison between saracatinib/oxaliplatin and PP2/oxaliplatin combinations difficult over prolonged periods. Thus the experimental design was altered to examine the effect of PP2 or saracatinib only during the 1h oxaliplatin exposure. Both saracatinib and PP2 inhibited SFK activity within 1h, as assessed by western blotting for levels of phosphorylated FAK (Figure 3C). When saracatinib was added to cells only during the 1h oxaliplatin exposure oxaliplatin efficacy was reduced (Figure 3D top panel – p<0.001 for oxaliplatin vs. oxaliplatin and saracatinib according to 2-way-ANOVA) as predicted by the hypothesis that saracatinib inhibits oxaliplatin uptake. However, the addition of PP2 during a 1h oxaliplatin exposure had no effect on the efficacy of oxaliplatin (Figure 3D bottom panel – p=0.98 for oxaliplatin vs. oxaliplatin and PP2 according to 2-way-ANOVA). Overall these data comparing two SFK inhibitors in conditions were SFKs are inhibited by both suggest that the affect of saracatinib on oxaliplatin uptake is independent of inhibition of SFK activity.

Oxaliplatin did not affect the potency of saracatinib

As saracatinib inhibited oxaliplatin uptake it is possible that both drugs enter the cell via the same transporter and that reciprocally, the presence of oxaliplatin may also inhibit the uptake of saracatinib. To test this hypothesis, the affect of oxaliplatin on biomarkers of SFK inhibition in saracatinib treated cells was investigated. As the predominant effect of saracatinib in a range of cancer cell types (including CRC, breast and lung) was to inhibit migration (11) combinations of saracatinib and oxaliplatin were assessed regarding their anti-migratory properties. 1μM saracatinib alone caused a 74% reduction in the migration of HCT116 cells, 3μM oxaliplatin (a concentration whose efficacy was antagonized by saracatinib in constant challenge experiments – data not shown) had no effect, while 10μM oxaliplatin caused a 34% reduction in migration (Figure 4A - p<0.05 according to Student's t-test). When 3μM oxaliplatin was combined with 1μM saracatinib, it did not alter the ability of saracatinib to inhibit migration, whilst there was a further reduction in migration when 1μM saracatinib and 10μM oxaliplatin were combined (p<0.05 according to Student's t-test). The affect of oxaliplatin on saracatinib-mediated reduction in phosphorylated FAK was also assessed where HCT116 cells were treated for 2h with varying concentrations saracatinib and oxaliplatin. A 2h time point was chosen as oxaliplatin can provoke an adaptive increase in SFK activity, but it takes longer than 2h for this to occur ((26) and confirmed by data not shown). Irrespective of the presence of oxaliplatin, saracatinib reduced FAK phosphorylation over the same concentration range (Figure 4B). These data suggest that oxaliplatin does not impair the ability of saracatinib to inhibit SFK activity and thus that it is unlikely that saracatinib uptake is inhibited by oxaliplatin.

Figure 4. Oxaliplatin does not affect the efficacy of saracatinib.

Figure 4

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(A) HCT116 cells were exposed to the indicated drugs and the affect on migration assessed in a wound healing assay as described in materials and methods. Data is normalized to untreated cells and is the mean of three independent experiments +/− S.E.M. * p<0.05 according to two-tailed unpaired t-test. (B) HCT116 cells were exposed to the indicated concentrations of oxaliplatin and saracatinib for two hours and harvested. The level of FAK and FAK phosphorylated on tyrosine 861 was determined by western blotting. Result is representative of three independent experiments.

Saracatinib inhibited uptake by Organic Cation Transporters

The observation that saracatinib inhibited the uptake of oxaliplatin but not cisplatin implies that saracatinib inhibits a transporter that distinguishes between these two platinum drugs. Members of the organic cation transporter (OCT) family of transporters can influx oxaliplatin more effectively than cisplatin (27, 28), while OCT3 expression has been shown to correlate with oxaliplatin sensitivity in CRC (29). OCT1, OCT2 and OCT3 are expressed in HCT116 and WiDr cells (30, 31) and therefore the affect of saracatinib on uptake via OCT1, OCT2 and OCT3 was assessed. HEK293 cells which over-express OCT1, OCT2 or OCT3 (15) were incubated in saracatinib for 10min before Asp+, a fluorescent substrate of OCTs, was added to the cells. The affect of saracatinib on Asp+ uptake was then determined. Saracatinib was able to inhibit uptake of Asp+ in all three cell lines (IC50 against OCT1=27.1μM, OCT2=0.46μM, OCT3=10.3μM), although it was over an order of magnitude more potent against cells over-expressing OCT2 (Figure 5A – Asp+ uptake in parental HEK293 cells was negligible). Thus saracatinib inhibited uptake via OCT2 at concentrations that inhibit oxaliplatin uptake. Furthermore, PP2 had no significant effect on Asp+ uptake in cells overexpressing OCT2 (Supplemental Figure S3 – p>0.05 PP2 vs. DMSO according to 2-way-ANOVA), consistent with PP2 not causing oxaliplatin resistance.

Figure 5. Saracatinib inhibits uptake through OCT2.

Figure 5

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(A) HEK293 cells overexpressing the indicated OCT isoforms were incubated in the presence of the indicated concentration of saracatinib for 10min and then 1μM Asp+ and saracatinib for 30min at 37°C. The level of Asp+ uptake was measured and normalized to the Asp+ uptake in cells not exposed to saracatinib (100%) or Asp+ (0%). (B and C) Parental HEK293 and HEK293 overexpressing the indicated isoforms of OCT were exposed to the indicated concentrations of oxaliplatin and saracatinib for three days and processed as in Figure 1 (B-D). All graphs show mean of three independent experiments carried out in triplicate +/− S.E.M.

To test further the causal relationship between OCT transporter function and saracatinib-mediated antagonism of oxaliplatin, the affect of combining oxaliplatin and saracatinib was determined in OCT over-expressing cells. All the HEK293 cells, including parental cells, exhibited a concentration response to oxaliplatin in the absence of saracatinib; however, cells over-expressing OCT2 were >20-fold more sensitive to oxaliplatin than any of the other cells (Figure 5B – oxaliplatin IC50 OCT2=14nM, Parental=320nM, OCT1=520nM, OCT3=910nM). Furthermore, while the addition of saracatinib reduced the oxaliplatin efficacy in all cell lines this was most pronounced in OCT2 over-expressing cells (Figure 5C). Specifically, 1μM saracatinib caused the IC50 of oxaliplatin to increase 1.7, 1.6 and 2.4 fold in parental, OCT1 and OCT3 cells respectively and 5.8 fold in OCT2 over-expressing cells. Taken together these data implicate saracatinib-mediated modulation of OCT2 as the mechanism underlying reduced uptake of oxaliplatin in CRC cell lines

Saracatinib fits the pharmacophore of known OCT inhibitors

In order to explore the molecular basis for saracatinib (Figure 6A) as an OCT2 inhibitor, a pharmacophore was derived identifying key molecular features required for recognition by OCT2. Using two potent OCT2 inhibitors, Decynium-22 and tetrapentylammonium ion (Supplemental Table S1), a four-point pharmacophore was generated, consisting of a central feature satisfied by a cationic or hydrogen bond donor group, surrounded by three hydrophobes in an approximately T shaped configuration (Figure 6B). This pharmacophore was then able to identify 19 compounds from a set of 20 known non-transported OCT inhibitors (19) (Supplemental Table S1). Interestingly, the pharmacophore derived here bears considerable similarity in orientation and nature to a previously reported four-point OCT1 pharmacophore (32). However, their central pharmacophore point differs in requiring solely a cationic group, which would lead to exclusion of a small number of compounds from the OCT inhibitor set considered here.

Figure 6. Saracatinib fits a pharmacophore of known OCT inhibitors.

Figure 6

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(A) Structure of saracatinib. (B) Structure of the four-point pharmacophore containing two hydrophobic features (red), one hydrophobic or aromatic feature (orange) and a central cationic or hydrogen bond donor feature (purple). (C and D) Saracatinib mapped on to the pharmacophore model in low energy conformations.

Saracatinib was found to adopt low energy conformations which also satisfy all four features of this OCT pharmacophore. One such conformation of saracatinib satisfies the central polar feature via a cationic amine in the piperazine ring; ligand methylene and cyclic groups also satisfy the hydrophobicity requirements of the OCT pharmacophore (Figure 6C). The second distinct saracatinib conformation maps on to the polar feature of the pharmacophore via a NH group which links the benzodioxolane and quinazoline rings. For this conformation, two aromatic rings and one aliphatic six-membered ring satisfy the hydrophobicity requirements of the OCT pharmacophore (Figure 6D). Both these conformations lie within 0.2 kcal/mol of the lowest energy pose. Therefore, saracatinib possesses the shape and polarity necessary for OCT inhibition. Furthermore, PP2, which does not cause resistance to oxaliplatin or inhibit uptake via OCT2, does not satisfy the OCT pharmacophore.

Discussion

As cancer is a multi-lesion group of diseases which will usually require a combination therapy approach, it is imperative that drug interactions are understood in order to optimize the chances of treatment success. Drug resistance is a predominant obstacle for the treatment of cancer and can occur at a variety of loci proximal and distal to drug targets. Changes in drug transport in and out of cancer cells have been a recognized mechanism of drug resistance for a variety of anticancer drugs. This can occur at the biological level, where for instance drug efflux pumps are over expressed in tumour cells, or via competition of two drugs for the same uptake mechanism. A plethora of oncogenic signal transduction interrupting drugs have entered oncology clinical trials, and so far very few have achieved single agent status. As current standard of care therapies do show efficacy in most tumour types, combinations with conventional cytotoxic agents are common, not only following ethical considerations but also in a hope that efficacy can be increased by combination therapy. The rational for investigating the combination of saracatinib with CRC standard of care chemotherapeutics was to predict whether saracatinib might enhance or detract from the chemotherapeutic agent if the two were combined in the clinic – a likely scenario if saracatinib were used to treat patients with advanced metastatic CRC.

The main findings of this study are that the SFK inhibitor saracatinib can antagonize the effects of oxaliplatin in human CRC cells in vitro. This antagonism was highly schedule-dependent, the presence saracatinib during oxaliplatin treatment being essential for antagonism. Furthermore, saracatinib did not antagonize cisplatin or carboplatin efficacy, demonstrating oxaliplatin specificity not common to all DNA platinating agents. The oxaliplatin antagonism was due to reduced uptake of oxaliplatin in the presence of saracatinib and was independent of SFK inhibition. Finally, saracatinib was also shown to inhibit uptake of OCT substrates, including oxaliplatin, in cells engineered to over-express OCT2 consistent with a fit of saracatinib as an OCT inhibitor pharmacophore.

The data presented here differs from work reporting a CRC cell line-dependent synergy between the SFK inhibitor dasatinib and oxaliplatin (33). In this report the synergy is dependent on SFK activation after oxaliplatin exposure due to reactive oxygen species generation. While an increase in SFK activity is observed in both of the cell lines described in the present study after oxaliplatin exposure (data not shown) there is no observed synergy with saracatinib. However, it has been demonstrated that dasatinib is not a substrate for OCT (34) and therefore is unlikely to inhibit oxaliplatin uptake, potentially explaining these contrasting results. Another report has demonstrated that combining oxaliplatin and saracatinib in vivo had a beneficial effect in an orthotopic mouse model of liver metastasis1. Indeed, data presented in the present study (Figure 4A) shows that combining oxaliplatin and saracatinib has at least an additive effect on inhibiting cell migration. Therefore, it is possible that combining oxaliplatin and saracatinib may prove clinically beneficial in appropriate settings, which may be even more pronounced if scheduling regimens are utilized that avoid co-administration of the two drugs.

There are two likely mechanisms by which saracatinib could inhibit uptake via OCT2; either saracatinib directly binds to and inhibits OCT2 or it inhibits a factor, such as a kinase, which is essential for OCT2 function. Several kinases have been implicated in regulating OCT2 activity including protein kinase A (PKA), protein kinase C (PKC) calmodulin-dependent kinase II (CaMKII) and the SFK member Lck (35, 36). Saracatinib can inhibit Lck activity (11), however PP2 also targets Lck (25) yet does not affect oxaliplatin efficacy, making it unlikely that saracatinib-inhibition of OCT2 activity is due to Lck inhibition. Furthermore, saracatinib did not inhibit serine/threonine protein kinases in a kinase screen (11), reducing the probability that saracatinib inhibits PKA, PKC or CaMKII. The fact that saracatinib fits a pharmacophore of known OCT inhibitors, many of which are also transported by OCT, suggests that saracatinib may bind to OCT2 and thus directly inhibit OCT2.

Previously, schedule dependent interactions of the VEGFR and EGFR protein kinase inhibitor vandetinib (Zactima) (37) and the EGFR inhibitor gefitinib (Iressa) (38) with oxaliplatin were attributed to mechanistic interactions between DNA damage and growth/survival signaling pathways. Oxaliplatin was shown to influence the sensitivity of cells to treatment with gefitinib as a result of increasing or decreasing EGFR phosphorylation (39), and has also been shown to activate SFKs via the generation if intracellular reactive oxygen species (33). In addition to a mechanism based interaction, vandetinib, gefitinib and saracatinib share an anilinoquinazoline backbone that fits the pharmacophore generated from known OCT inhibitors, suggesting a common mechanism by which these inhibitors may interact with oxaliplatin. Indeed, recent work has demonstrated that gefitinib can inhibit OCT1 and OCT2 function (40) while uptake of the Abl inhibitor imatinib is OCT dependent and down-regulation of OCTs in chronic myeloid leukaemia has been proposed as a mechanism for imatinib resistance (41). Interaction with OCT function may be a more generalized phenomenon that requires consideration when potential novel drug combinations are undertaken.

One of the major physiological roles of OCTs is the excretion of organic cations, including antibiotic, antiviral, antidiabetic and cancer chemotherapeutics, via the liver and kidney (19). There are several examples of drugs which inhibit OCT altering the PK-PD of other OCT substrates, such as the antiretroviral drugs lamivudine and zalcitabine which interact with each other (42) or the histamine H2-receptor antagonist cimetidine, which increases the plasma concentration of the antidiabetic drug metformin (43). Therefore, it is highly plausible that novel agents sharing the OCT pharmacophore could alter the PK-PD relationship of co-administered drugs and the data presented here for oxaliplatin and saracatinib suggests that this possibility should be anticipated and incorporated into schedule planning. In conclusion, this study has demonstrated that saracatinib inhibits uptake of oxaliplatin in CRC cells where uptake via OCT2 is strongly implicated as the underlying mechanism. Follow up studies where OCT2 is knocked down by RNA interference will allow this hypothesis to be tested, however this will first require the generation of specific antibodies to access efficient down-regulation of OCT2.

Supplementary Material

1

Acknowledgements

The authors would like to thank Tim Ward for advice on Comet-X assay analysis, Allan Jordan for insightful discussion regarding the OCT pharmacophore, Paul Elvin for comments on the manuscript and Elvin Wagenblast for technical assistance.

Funding This work was supported by Cancer Research UK core-funding to the Paterson Institute for Cancer Research (Grant number: C147)

Footnotes

Conflict of Interest C.S, D.M.H and T.P.G were employees of AstraZeneca at the time of the work

1

Work presented at AACR-NCI-EORTC Molecular Targets and Cancer Therapeutics Conference San Francisco 2007 – Abstract PR-11

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