SNS-032

Interactions of cyclin‑dependent kinase inhibitors AT‑7519, flavopiridol and SNS‑032 with ABCB1, ABCG2 and ABCC1 transporters and their potential to overcome multidrug resistance in vitro

Daniela Cihalova1 · Frantisek Staud1 · Martina Ceckova1

Received: 28 February 2015 / Accepted: 6 May 2015
© Springer-Verlag Berlin Heidelberg 2015

Abstract

Purpose ATP-binding cassette (ABC) transporters play an important role in multidrug resistance (MDR) toward anticancer drugs. Here, we evaluated interactions of cyclin- dependent kinase inhibitors (CDKi) AT-7519, flavopiridol and SNS-032 with the following ABC transporters in vitro: P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and multidrug resistance-associated protein 1 (ABCC1).
Methods Inhibitory potency of studied CDKi to the transporters was evaluated by accumulation assays using fluorescent substrates and MDCKII cells overexpressing human ABCB1, ABCG2 or ABCC1. Resistance of trans- porter-expressing cells to the CDKi was evaluated by XTT proliferation assay. Observed interactions of CDKi were verified by ATPase assay in ABC transporter-expressing Sf9 membrane vesicles. Combination index analysis was additionally performed in ABC transporter-expressing can- cer cell lines, HepG2 and T47D.
Results Flavopiridol showed a significant inhibitory potency toward ABCG2 and ABCC1. SNS-032 also decreased ABCG2-mediated efflux, while AT-7519 failed to inhibit ABCB1, ABCG2 or ABCC1. Both flavopiridol and SNS-032 showed synergistic antiproliferative effects in
combination with relevant ABC transporter substrates such as daunorubicin and topotecan in cancer cells. ABCB1 was found to confer significant resistance to AT-7519 and SNS-032, but not to flavopiridol. In contrast, ABCG2 and ABCC1 conferred resistance to flavopiridol, but not to AT-7519 and SNS-032.

Conclusion Our data provide detailed information on interactions of flavopiridol, SNS-032 and AT-7519 with ABC transporters, which may help elucidate the pharma- cokinetic behavior and toxicity of these compounds. More- over, we show the ability of flavopiridol and SNS-032, but not AT-7519, to overcome ABC transporter-mediated MDR.

Keywords Cyclin-dependent kinase inhibitor · Multidrug resistance · ABC transporter · AT-7519 · Flavopiridol · SNS-032

Introduction

Multidrug resistance (MDR) is one of the major causes of failure in cancer chemotherapy. This phenomenon occurs when cancer cells become resistant to different anticancer drugs and are able to survive treatment by a multitude of structurally and functionally unrelated chemotherapeutics. Overexpression of ATP-binding cassette (ABC) trans- porters, such as ABCB1 (P-glycoprotein, P-gp), ABCG2 (breast cancer resistance protein, BCRP) and ABCC1 (mul- tidrug resistance-associated protein 1, MRP1), in tumor cells is one of the most important mechanisms of MDR as these efflux transporters recognize different chemothera- peutic agents and transport them out of the cell, thereby decreasing their intracellular accumulation [1, 2]. These transporters have become attractive molecular targets, and great effort has been made to find their inhibitors (MDR modulators) in order to increase bioavailability after oral administration, to enhance tissue penetration of transported drugs and to overcome drug resistance in cancer cells [3–5]. Several generations of ABC transporter modulators have failed to fulfill their expectations in the clinical area so far [6, 7]. However, MDR research has recently been directed to examining modulatory effects of compounds that were not originally designed for reversing multidrug resistance, such as tyrosine kinase inhibitors (TKI) [8]. TKI have been shown to block or antagonize ABC transporters in vitro and in vivo, and preclinical data indicate that TKI are effective in overcoming MDR when used with standard anticancer drugs [9, 10]. Small molecule kinase inhibitors may therefore represent a new approach in ABC transporter modulation.

Another novel group of kinase inhibitors is targeted toward cyclin-dependent kinases (CDKs), which are ser- ine/threonine protein kinases regulating the progression of the cell through the cell cycle and RNA transcription. CDKs are regulated by positive phosphorylation by CDK- activating kinase [11], as well as by negative phosphoryla- tion by endogenous Cip/Kip or INK inhibitors [12, 13]. Because of their critical role in cell cycle progression, cel- lular transcription and apoptosis, CDKs are major targets for deregulation in different human tumors [14, 15]. This fact has led to the development of CDK inhibitors (CDKi) as an effective method for controlling tumor growth and hence a potential therapeutic tool for cancer treatment [16, 17]. Small molecule CDKi have been shown to be highly effective against the activity of several CDKs, caus- ing significant cell cycle arrest and apoptosis in many cancers; some of these compounds have entered clinical trials, and among the most advanced are AT-7519, fla- vopiridol (alvocidib) and SNS-032 (BMS-387032). These CDKi have been tested for several indications, including solid and hematological malignancies, either as single agents or in combination with other chemotherapeutics. AT-7519 is currently undergoing phase II clinical trials for the treatment of leukemia and lymphoma (clinicaltrials. gov, NCT01627054 and NCT01652144, respectively). Fla- vopiridol has shown clinical activity in chronic lympho- cytic leukemia [18] and ovarian carcinoma [19] in phase II clinical trials, and SNS-032 is reported to be in phase I development in metastatic refractory solid tumors and B cell malignancies [20, 21].

These new CDKi clearly offer a possibility of improved therapy for cancer patients. However, data on their drug– drug interactions are insufficient, although crucial for a thorough understanding of their pharmacokinetic behavior or the behavior of other simultaneously administered com- pounds. Currently, only a few studies have examined the effect of these three CDKi in cell lines or animal models that overexpress ABCB1, ABCG2 or ABCC1 transporters. In our previous study, we demonstrated the inhibitory effect of several CDKi, including flavopiridol and SNS-032, toward the ABCB1 transporter [22]. In the present work, we aimed to comprehensively investigate the in vitro effect of three promising CDKi, AT-7519, flavopiridol or SNS- 032, on the efflux activity of not only ABCB1, but also ABCG2 and ABCC1 transporters in MDCKII cells and to determine whether the inhibiting compounds can potentiate the efficacy of other conventional antineoplastic drugs in cancer cells through these interactions. Moreover, we also evaluated the causative role of the ABC transporters in cel- lular resistance to the CDKi.

Materials and methods

Materials

AT-7519 was obtained from Axon Medchem (Groningen, the Netherlands). Flavopiridol and SNS-032 were sup- plied by SelleckChem (Houston, TX, USA). Daunorubicin (DNR), mitoxantrone (MIT), XTT sodium salt (XTT), phenazine methosulfate (PMS) and ABCC1 inhibitor MK-571 were purchased from Sigma-Aldrich (St. Louis, MO, USA). ABCB1 inhibitor LY335979 was obtained from Toronto Research Chemicals (North York, ON, Can- ada) and ABCG2 inhibitor Ko143 was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Cell cul- ture reagents were supplied by Sigma-Aldrich (St. Louis, MO, USA) and Gibco BRL Life Technologies (Rockville, MD, USA). ABCB1, ABCG2 and ABCC1 PREDEASY™ ATPase kits (SB MDR1/P-gp, SB BCRP HAM Sf9 and SB MRP1, respectively) were purchased from Solvo Biotech- nology (Szeged, Hungary).

Cell culture

Madin–Darby canine kidney (MDCKII) cell lines trans- duced with the human transporters ABCB1 (MDCKII- ABCB1), ABCG2 (MDCKII-ABCG2) or ABCC1 (MDCKII-ABCC1) which stably express ABCB1, ABCG2 or ABCC1 transporter, respectively, and the MDCKII parent cell line were obtained from Prof. Piet Borst and Dr. Alfred Schinkel (The Netherlands Cancer Institute, Amsterdam, the Netherlands). The stable expression of ABC transporters was verified by RT-PCR (see Sect. 3.4). The cell lines were grown in complete Dulbecco’s modi- fied Eagle’s medium (DMEM) supplemented with 10 % FBS. For in vitro drug combination studies, we also used human liver carcinoma HepG2 and human ductal breast carcinoma T47D cell lines. HepG2 cells were purchased from American Type Culture Collection (LGC Promochem, Teddington, Middlesex, UK) and were grown in minimal essential medium supplemented with 1 mM sodium pyru- vate, 0.1 mM non-essential amino acids and 10 % FBS. T47D cells, obtained from the European Collection of Cell Cultures (PHE, Salisbury, Wiltshire, UK), were cultured in DMEM without phenol red, supplemented with 10 % FBS and 2 mM L-glutamine. Dimethyl sulfoxide was applied as a solvent in concentrations not exceeding 0.5 % (1 % in ATPase assays).

RNA isolation and RT‑PCR for ABCB1, ABCG2 and ABCC1 expression in cell lines

Total RNA was isolated from confluent monolayers of ABC transporter-expressing MDCKII, MDCKII parent, HepG2 and T47D cells using the TRI Reagent (Sigma- Aldrich, St. Louis, MO, USA) according to the manufac- turer’s instructions. Each cell line has been sampled at least in triplicate. RNA was dissolved in DEPC-treated water, and concentration and purity of each sample were deter- mined spectrophotometrically from A260/A280 measure- ments (NanoDrop, Thermo Scientific, Wilmington, DE, USA). Integrity of RNA was also checked by agarose gel electrophoresis. cDNA was prepared from 1 µg extracted total RNA by MMLV reverse transcriptase using oligo(dT) VN nucleotides (gb Reverse Transcription Kit, Generi Bio- tech, Hradec Kralove, Czech Republic). PCR analysis was performed on QuantStudio 6 (Life Technologies). cDNA (40 ng) was amplified using 2 × Probe Master Mix (Generi Biotech, Hradec Kralove, Czech Republic) and predesigned PCR assays for ABC transporters: ABCB1, ABCG2 and ABCC1 (hABCB1_Q2, hABCG2_Q2 and hABCC1_Q2, Generi Biotech, Hradec Kralove, Czech Republic) and ref- erence genes: HPRT and β2-microglobulin (hHPRT_Q2 and hB2M_Q2, Generi Biotech, Hradec Kralove, Czech Republic). The temperature profile was 95 °C for 3 min and 35 repeats of a cycle consisting of 95 °C for 10 s and 60 °C for 20 s. Ct values for particular samples were noted, and PCR products were additionally separated by agarose gel electrophoresis and visualized by UV (Bio-Rad Laborato- ries). Mean Ct values of HPRT and β2-microglobulin were used as normalizing controls for each sample of carcinoma cells to calculate ΔCt values. Normalizing genes have been chosen based on previous experiments showing no statis- tically significant difference in Ct values for HPRT and β2-microglobulin between the HepG2 and T47D cells.

XTT cell proliferation assays

A total of 1 × 104 MDCKII-ABCB1, MDCKII-ABCG2, MDCKII-ABCC1 or MDCKII parent, 2 × 104 HepG2 or 1.5 × 104 T47D cells were grown in 96-well culture plates and incubated for 24 h. Individual CDKi diluted with growth medium were added to the exponentially growing cells, and the resulting mixtures were incubated for 72 h at 37 °C, 5 % CO2. Cell viability was assessed using the XTT assay as follows: Cells were incubated with 0.167 mg/mL XTT with 4 µM PMS in Opti-MEM for 2 h. The absorb- ance of the soluble formazan released was measured at 470 nm on a microplate reader (Tecan, Männedorf, Swit- zerland). The median effective antiproliferative concen- trations (EC50) of the compounds were calculated using GraphPad Prism 6.00. To determine the influence of ABC transporters on growth inhibition, resistance factor (RF) was calculated by dividing the EC50 value of ABC trans- porter-overexpressing cell line by the EC50 value of the respective parental cell line; RF therefore represents a fold increase in resistance caused by the presence of a particu- lar ABC transporter [23]. As an indirect method to assess whether the tested CDKi are substrates of ABCB1, ABCG2 or ABCC1, cell proliferation assays were repeated with the addition of model inhibitors of the three transporters (1 µM LY335979, 1 µM Ko143 and 25 µM MK-571 for ABCB1,ABCG2 and ABCC1, respectively) to abolish the potential influence of ABC transporter on the resistance.

DNR and MIT accumulation assays

The effect of CDKi on the intracellular DNR accumula- tion in MDCKII-ABCB1 and MDCKII-ABCC1, and MIT accumulation in MDCKII-ABCG2 cells was examined by flow cytometry (Accuri C6, Accuri, Ann Arbor, USA). Parental MDCKII cells were analyzed as a control using both substrates separately. Cells were seeded at a density of 1.5 × 105 on a 12-well plate 24 h before experiment and treated with five concentrations of CDKi, solvent (0.5 % DMSO, control) or Opti-MEM (untreated control) for 30 min at 37 °C, 5 % CO2. DNR or MIT was then added to a final concentration of 2 or 1 µM, respectively, and the cells were incubated under the same conditions for a further 60 min. Accumulation was stopped by cooling the samples on ice and washing twice with ice-cold PBS. The cells were detached with 10× trypsin–EDTA and resus- pended in PBS with 2 % FBS. The intracellular accumu- lation of individual cells was measured with an excitation/ emission filter of 488/585 nm for DNR and 488/670 nm for MIT and recorded as histograms. As positive controls for ABCB1, ABCG2 and ABCC1 inhibition, 1 μM LY335979, 1 µM Ko143 and 50 µM MK-571 were used, respectively. Viable cells were gated based on forward and side scatter plots. The median fluorescence intensity (MFI) of 10,000 measured cells was used to compare the fluorescence resulting from each of the treatments. Relative values were identified by dividing the MFI of each measurement by that of untreated control cells.

ABCB1, ABCG2 and ABCC1 ATPase assays

Membrane preparations overexpressing ABC transport- ers show vanadate-sensitive ATPase activity that is modu- lated by interacting compounds. In the activation assay, transported substrates can stimulate baseline vanadate- sensitive ATPase activity, whereas in the inhibition assay, which is carried out in the presence of a known activa- tor of the transporter, inhibitors may reduce the maxi- mally stimulated vanadate-sensitive ATPase activity [24]. ATPase activity was measured by assessing the amount of phosphate liberated from ATP by the ABCB1, ABCG2 or ABCC1 transporters using the PREDEASY™ ATPase Kit for a corresponding transporter according to the manufac- turer’s instructions. For this purpose, Sf9 cell membranes (4 µg protein per well) were mixed with each of the test compounds in solutions, with concentrations ranging from 0.14 to 300 µM, and then incubated at 37 °C for 10 min in the presence or absence of 1.2 mM sodium orthovana- date. ATPase reaction was started by adding 10 mM ATP magnesium salt to the reaction mixture and stopped 10 min later, and the absorbance at 590 nm was measured after a further 30-min incubation (Tecan, Männedorf, Switzer- land). ATPase activity in each sample was determined as the difference in liberated amounts of phosphate measured in the presence and absence of 1.2 mM sodium orthova- nadate. Phosphate standards were prepared in each plate; verapamil, sulfasalazine and N-ethylmaleimide-glutathione (NEM-SG) served as positive controls for ABCB1, ABCG2 and ABCC1 stimulation, respectively, as provided by the manufacturer. Results are expressed as vanadate-sensitive ATPase activities.

Drug combination studies

The combination index (CI) method of Chou–Talalay, based on the median-effect equation, was used to calculate combined drug effects; this approach offers quantitative definition for additive, synergistic and antagonistic effects (CI values of 0.9–1.1, <0.9 and >1.1, respectively) [25]. Data generated from the CI method were used to quantify dose-reduction indices (DRI) for pairs of the tested drugs. DRI represents the fold change of a focal effect when individual agents are used simultaneously relative to their separate effects, and their activity is synergistic if DRI > 1. CDKi exhibiting inhibitory activity on ABC transporters (flavopiridol and SNS-032) were combined with DNR or topotecan (TOP), commonly used anticancer drugs and ABC transporter substrates. The XTT cytotoxicity assay was used to measure the cell viability in the tested cell lines, i.e., HepG2 and T47D, in the presence of the CDKi and DNR/TOP both alone and in combination, at constant concentration ratios, ranging from 0.1 to 2 multiples of the respective, predetermined EC50 values. Data acquired from these drug combination experiments were analyzed using CompuSyn version 3.0.1 software (ComboSyn Inc., Para- mus, NJ, USA).

Statistical analysis

Data are presented as mean ± SD of at least three inde- pendent experiments. Statistical significance was deter- mined using two-tailed unpaired Student’s t test or one- way ANOVA implemented in GraphPad Prism 6.00, and P < 0.05 is considered significant. Results Role of ABCB1, ABCG2 and ABCC1 in chemoresistance to CDKi To determine the possible role of ABC transporters in causing cellular resistance to AT-7519, flavopiridol and SNS-032, we examined the antiproliferative effect of the CDKi in ABCB1-, ABCG2- and ABCC1-overexpressing MDCKII and control MDCKII parent cell lines using XTT assay. Chemotherapeutic agents, DNR and TOP, were also included in the XTT assays to verify the role of particu- lar ABC transporters in cellular resistance to these drugs and to justify further employment of these ABC trans- porter substrates in combination studies (see Sects. 2.7 and 3.5). Mean EC50 values and RF are shown in Table 1. MDCKII-ABCB1 cells were significantly more resistant to both AT-7519 and SNS-032, with RF values of 17 and 13, respectively, compared to the parental cell line. In contrast, neither ABCG2 nor ABCC1 was able to confer resistance to AT-7519 or SNS-032 over the tested concentration range in the respective overexpressing MDCKII cell lines. On the other hand, we observed that MDCKII-ABCG2 and MDCKII-ABCC1 cells were significantly more resistant to flavopiridol, with RF values of 2.3 and 1.4, respectively, compared to the drug-sensitive parental cell line, while no effect of ABCB1 on flavopiridol resistance was observed in MDCKII-ABCB1 cells. To confirm the causative role of ABC transporters in the resistance to studied CDKi, cell proliferation assays were repeated for a combination of CDKi with model inhibi- tors of ABC transporters in transporter-overexpressing MDCKII cells. Indeed, we have observed a reversal of the resistance to AT-7519 and SNS-032 in the presence of 1 µM LY335979 in MDCKII-ABCB1, and to flavopiri- dol in the presence of 1 µM Ko143 or 25 µM MK-571 in MDCKII-ABCG2 and MDCKII-ABCC1 cells, respectively (Table 1). Inhibitory effect of CDKi on the transporter‑mediated efflux of DNR and MIT DNR, a fluorescent substrate of ABCB1 and ABCC1, was used to determine the effect of CDKi on ABCB1- and ABCC1-mediated efflux. AT-7519 revealed no significant effect on intracellular accumulation of DNR in MDCKII- ABCB1 or MDCKII-ABCC1 cells (Fig. 1a, b). Employing MDCKII-ABCC1 cells, flavopiridol treatment enhanced the intracellular accumulation of DNR in a dose-dependent manner, showing significant inhibitory potency at 1 µM concentration. At concentrations of 10 μM and higher, fla- vopiridol caused the maximal transporter inhibitory effect (2.3-, 2.6- and 2.6-fold increase in the presence of 10, 30 and 50 µM flavopiridol, respectively), which was compa- rable to the activity of a model inhibitor, MK-571 (2.4-fold increase in DNR accumulation). No significant effect was observed in the MDCKII parental cell line. SNS-032 did not show significant effect on the DNR accumulation in MDCKII-ABCC1 cells over the tested concentration range (1–50 µM) (Fig. 1b). The fluorescent ABCG2 substrate MIT was used to investigate the influence of studied CDKi on ABCG2- mediated efflux. The intracellular accumulation of MIT in MDCKII-ABCG2 cells was significantly increased by fla- vopiridol and SNS-032, while no effect on MIT accumula- tion was observed in the control parental cell line. In the presence of 10, 30 and 50 μM flavopiridol, the intracellular accumulation of MIT was enhanced to 3.5-, 4.2- and 4.2- fold, respectively, while 50 μM SNS-032 increased the intracellular accumulation of MIT in MDCKII-ABCG2 cells 1.9-fold. In contrast, AT-7519 did not significantly modify the intracellular accumulation of MIT in MDCKII- ABCG2, indicating a lack of inhibitory potency to the ABCG2 transporter (Fig. 1c). ATPase assay To further characterize interactions of CDKi with ABC transporters, we tested the modulatory effects of the drugs on vanadate-sensitive ATPase activity in isolated insect Sf9 cell membranes overexpressing human ABCB1, ABCG2 or ABCC1. In the ATPase inhibition study, flavopiridol and SNS-032 lowered the sulfasalazine-stimulated vanadate- sensitive ATPase activity of ABCG2 in a dose-dependent manner at a concentration of 300 µM, while AT-7519 did not lower the stimulated activity at all. Similarly, AT-7519 did not show any decrease in verapamil-stimulated ATPase in ABCB1 membrane preparations. Flavopiridol (300 µM) significantly reduced the NEM-SG-stimulated vanadate- sensitive ATPase activity of ABCC1, while AT-7519 and SNS-032 showed no effect. In the ATPase activation assays, only flavopiridol (but not AT-7519 and SNS-032) signifi- cantly increased the baseline vanadate-sensitive ATPase activity of ABCG2 at concentrations from 10 to 100 µM, while no other effects of CDKi on baseline ABCB1 or ABCC1 ATPase activity were observed (Fig. 2). Fig. 1 Effects of AT-7519, flavopiridol and SNS-032 on the intracel- lular accumulation of DNR in MDCKII-ABCB1 cells (a), DNR in MDCKII-ABCC1 cells (b) and MIT in MDCKII-ABCG2 cells (c). Results are presented as fold changes in fluorescence intensity com- pared to untreated control cells. LY335979 (1 µM), MK-571 (50 μM) and Ko143 (1 μM) are used as model inhibitors for ABCB1, ABCC1 and ABCG2 inhibition, respectively. Data represent mean ± SD of three independent experiments performed in duplicate. *P < 0.05;**P < 0.01; ***P < 0.001 determined by one-way ANOVA followed by Dunnet’s test. Expression of ABCB1, ABCG2 and ABCC1 in cell lines RT-PCR was performed to verify the expression of indi- vidual transporters in the carcinoma cell lines employed in subsequent combination studies. MDCKII stably express- ing ABCB1, ABCG2 or ABCC1 were used as positive controls, while MDCKII parent cells served as negative control. We confirmed that hepatocarcinoma HepG2 cells express all of the studied transporters, while the expres- sion of ABCB1 in breast carcinoma T47D cell line was too low and not detectable using agarose gel electrophoresis of PCR products (Fig. 3a). Based on the observed differences between normal- ized Ct values reached for ABC transporters in HepG2 and T47D cells (Fig. 3b) and comparison of particular gene expressions calculated as 2ΔCt, we could estimate ABCB1 and ABCG2 expression being approximately 800- and 18-fold higher, respectively, in HepG2 than in T47D cells, while ABCC1 mRNA expression is approximately fivefold higher in T47D cell line compared to HepG2. Drug combination studies Combination studies were performed to assess the abil- ity of the CDKi, which showed inhibitory effect on ABC transporters in the accumulation studies, to sen- sitize cancer cells to selected cytotoxic substrates of the relevant ABC transporters. The growth inhibitory effect of flavopiridol and SNS-032 treatment alone was there- fore assessed and compared to combination treatment with DNR (ABCB1 and ABCC1 substrate) and/or TOP (ABCB1, ABCG2 and ABCC1 substrate) using the com- bination index method of Chou–Talalay. In all cases, com- bination treatments yielded greater growth inhibition than DNR/TOP alone. CI values are shown in Fig. 4, and the results are summarized in Table 2. Combinations in which synergism was observed (CI < 0.9) were used to calculate DRI. Fig. 2 Effect of CDKi on the ATPase activity of ABCB1-Sf9 (a), ABCC1-Sf9 (b) and ABCG2-Sf9 (c) membrane preparations. Vana- date-sensitive activity in the presence of flavopiridol, AT-7519 or SNS-032 in activation (filled circle) and inhibition (empty square) experiments. Lower dotted line represents baseline vanadate-sensi- tive ATPase and upper dotted line represents activated ATPase in all graphs. Presented data are mean ± SD representative of three experi- ments performed in duplicate. Statistical significance of differences between control and CDKi-treated samples in activation assays (†P < 0.05) and inhibition assays (*P < 0.05; **P < 0.01) is deter- mined using unpaired two-tailed t test. Fig. 3 mRNA expression of human ABCB1, ABCG2 and ABCC1 in human ABC transporter-overexpressing MDCKII, parent MDCKII, HepG2 and T47D cell lines. Amplicon sizes: ABCB1 (122 bp), ABCG2 (165 bp), ABCC1 (118 bp) (a); data are representative of three similar independent experiments. ΔCt values for human ABCB1, ABCG2 and ABCC1 in HepG2 (white columns) and T47D (black columns) normalized to mean Ct values for HPRT and β2- microglobulin in both cell lines are shown; data represent mean ± SD of three independent samples (b) Fig. 4 Cytotoxic effect of flavopiridol and daunorubicin (a, d), fla- vopiridol and topotecan (b, e) and SNS-032 and topotecan (c, f) in HepG2 (a, b, c) and T47D (d, e, f) cells. The r values from the median-effect plots calculated by CompuSyn for single drugs and their combinations fall in the range of 0.95–0.99. Combination index analysis of flavopiridol combined with DNR (g) or TOP (h) and SNS-032 combined with TOP (i) in HepG2 (filled square) and T47D (empty circle) cell lines. Lines represent computer-simulated CI plots in HepG2 (full line) and T47D (dashed line) cell lines. The concentration ratio was based on the EC50 ratio of individual drugs (Table 1). CI values <0.9, 0.9–1.1 and >1.1 indicate synergism, additivity and antagonism, respectively. Drug effect levels (%) are calculated from the cell viability values and correspond to the proportional amount of cells affected by the drug combination; 0 % means no antiprolif- erative effect, and 100 % means absolute antiproliferative effect. Data are presented as mean ± SD of three independent experiments per- formed in triplicate Flavopiridol inhibited all three studied transporters and was therefore combined with both DNR and TOP. The combination of DNR with flavopiridol showed synergis- tic cytotoxicity at drug effect levels >70 % in HepG2 and >75 % in T47D cell line. When combined with TOP, the synergistic effect was also reached at drug effect levels >75 % in HepG2 and >80 % in T47D cells.

SNS-032 was combined with TOP due to its inhibi- tory effect on ABCG2 only. This combination yielded synergistic activity at drug effect levels >55 % in HepG2 and >75 % in T47D.

Discussion

Multidrug resistance to various chemotherapeutic agents and drug interactions with anticancer drugs represent a con- siderable problem in the effectiveness and safety of cancer treatment [26–28]. These interactions may also potentially alter the pharmacokinetic and pharmacodynamic behav- ior of the compounds, as well as behavior of other simul- taneously administered drugs. As CDKi represent a novel group of anticancer agents [17], their interaction with major ABC transporters associated with MDR needs to be elucidated. We have previously reported on the potential of first-generation CDKi to interact with ABCB1 and ABCG2

transporters [29, 30]. Our recent work has demonstrated the inhibitory potency of several CDKi, including flavopiridol and SNS-032, on ABCB1-mediated efflux [22]; here, we aim to complete the interaction profile of flavopiridol and SNS-032 with the three major ABC transporters associated with MDR. We include also AT-7519 as a novel CDKi with perspective use in cancer therapy and evaluate the interac- tions of these drugs with ABCB1, ABCG2 and ABCC1 in detail.
Zhou et al. [31] demonstrated that mouse ABCG2 and ABCB1 limit the brain penetration of flavopiridol in mice with greater transport associated with ABCG2. Based on experiments in Caco-2 cell monolayers, it has recently been suggested that ABCB1 could be involved in transmembrane transport of flavopiridol [32]. Nevertheless, among the ABC transporters, mainly ABCG2 [33, 34] and to a lower extent ABCC1 [35] rather than ABCB1 seem to confer resistance to flavopiridol in cancer cells. In our assays, we directly addressed the contribution of ABC transporters to cellular resistance of particular CDKi and found that ABCB1 did not confer resistance to flavopiridol in MDCKII-ABCB1 cells, confirming our previous data based on ATPase assays [22]. MDCKII-ABCG2 cells were more resistant to fla- vopiridol compared to parental MDCKII cells, whereas ABCC1 function only slightly (but significantly) modified the cytotoxic effect of this drug.
SNS-032 has been revealed as a substrate of ABCB1 in pharmacokinetic studies in rats [36], and ABCB1 expres- sion has been suggested as an important SNS-032 resist- ance mechanism in neuroblastoma [37]. In our assays, we confirmed the major role of ABCB1 in SNS-032 resist- ance showing significantly increased viability in MDCKII- ABCB1 cell line compared to the parental MDCKII cells, with no such effect in ABCG2- or ABCC1-expressing cells.

Interestingly, AT-7519 follows the same resistance pattern as SNS-032; in our proliferation assays, MDCKII-ABCB1 cells show a significantly higher resistance to AT-7519 than MDCKII-ABCG2, MDCKII-ABCC1 and MDCKII parental cells. Our data therefore indicate that ABCB1 confers resistance to AT-7519 and SNS-032, but not to flavopiridol, while ABCG2 and ABCC1 are causative of chemoresist- ance to flavopiridol, but not to AT-7519 and SNS-032.

Besides being subjected to ABC transporter-mediated efflux, many drugs can act as “modulators” overcoming MDR by inhibition of the drug transporters [38]. It was reported previously that flavopiridol was able to inhibit ABCB1- and ABCC1-mediated efflux in ABCB1- or ABCC1-overexpressing cells, respectively, when applied at micromolar concentrations [22, 33, 35]. Previous stud- ies, including ours, also showed the ability of SNS-032 to inhibit the transport in ABCB1-expressing cells [22, 37].

Using accumulation assays in transporter-overexpress- ing MDCKII cells, we demonstrate here that flavopiridol can inhibit ABCG2-mediated efflux of MIT as well as ABCC1-mediated efflux of DNR even when applied at a low 1 µM concentration. Moreover, flavopiridol was able to achieve the maximal inhibition of ABCC1-mediated DNR efflux comparable to MK-571, a model ABCC1 inhibitor applied at 50 µM. These data indicate that flavopiridol is able to reverse not only ABCB1-mediated resistance, but also the resistance caused by ABCG2 and ABCC1 trans- porters. SNS-032 inhibited ABCG2-mediated transport of MIT but at high concentrations (50 μM) only. In contrast, AT-7519 had no effect on either ABCB1-, ABCG2- or ABCC1-mediated transport of the fluorescent substrates.

Our previous studies showed that flavopiridol and SNS- 032 significantly reduced the activated ATPase of ABCB1 at concentrations higher than 100 µM, but neither of these two compounds was able to increase the baseline vanadate- sensitive ATPase in ABCB1 membrane vesicles [22]. In this study, we examined the effects of CDKi on the trans- porter-related ATPase activities in Sf9 membranes over- expressing human ABCB1 (AT-7519 only), ABCG2 or ABCC1. Flavopiridol decreased the activated ATPase of both ABCG2 and ABCC1 ATPase, confirming its interac- tion with both transporters. We also found that flavopiridol stimulated ABCG2 baseline ATPase activity, which pro- vides further indications that flavopiridol is a substrate of ABCG2. SNS-032 was able to significantly decrease the activated ABCG2 ATPase, albeit at concentrations higher than 100 μM only, while it had no effect on the baseline ABCG2 ATPase activity and neither did it affect ABCC1 ATPase-stimulated or baseline activity. These data confirm that SNS-032 interacts with ABCG2, but not with ABCC1, as observed in our accumulation assays as well as cyto- toxicity studies. In the case of AT-7519, the activation and inhibition mode of ATPase assays did not show any meas- urable effect on either ABCG2 or ABCC1 ATPase activities up to 300 μM concentration, providing first indications that AT-7519 does not interact with ABCG2 or ABCC1 trans- porters. Interestingly, AT-7519 failed to affect ABCB1- linked ATPase activity in this assay, which contradicts our cytotoxicity results. This phenomenon can be attributed to the fact that the value of resistance factor obtained by measuring cytotoxicity in MDCKII cells does not necessar- ily correlate with ABC transporter substrates and also to the fact that ATPase assays can provide false-negative results [39].

In undergoing clinical trials, CDKi are being evalu- ated not only as single agents but also in combination with other chemotherapeutics, with the aim of yielding syner- gistic activity [40–42], increasing their therapeutic effects [19, 43] and the survival of cancer patients [44]. We have previously suggested that ABC transporter inhibition may represent one of the mechanisms underlying the onset of synergistic effects of CDKi combined with conven- tional anticancer drugs [22, 29]. Therefore, here we also addressed whether ABC transporter-inhibiting compounds, flavopiridol and SNS-032, can potentiate the cytotoxic effects of anticancer agents in vitro. We applied CDKi in combination with topoisomerase inhibitors, DNR or TOP (commonly used anticancer drugs and ABC transporter substrates), to human cancer cell lines expressing ABC transporters [22, 45, 46], i.e., HepG2 and T47D, derived from hepatocyte carcinoma and ductal breast carcinoma, respectively. Flavopiridol inhibited all studied ABC trans- porters and was therefore combined with both DNR and TOP, while SNS-032 was combined with TOP due to its inhibitory potency toward ABCG2 only. We found that all studied CDKi inhibited the growth of both HepG2 and T47D cells in a dose-dependent manner and showed synergism when administered in combination with DNR or TOP. The combination of flavopiridol with daunoru- bicin exhibited a higher synergistic effect in the HepG2 cell line compared to T47D. As daunorubicin is a stronger substrate of ABCB1 than ABCC1, we believe that the lack- ing expression of ABCB1 in T47D cells may be the reason for higher CI values and thus lower synergistic effects in that cell line. Based on our inhibitory studies and resistance profile of TOP, flavopiridol could be expected to contribute to the synergistic effect mainly by inhibition of ABCG2- and ABCC1-mediated efflux of TOP. Both transporters are expressed at comparable levels in HepG2 and T47D cells, which might be the reason for the similar synergis- tic effect exhibited in both cell lines. SNS-032 in combina- tion with topotecan yielded synergistic effects in both cell lines as well, with a more prominent effect in the cell line with higher ABCG2 expression (HepG2). It is obvious that CDKi can function as modulators of apoptosis induced by other cytotoxic agents [47, 48] but, as we show here, they are also able to reverse MDR by inhibition of ABC trans- porters. CDKi may therefore represent a new class of ABC transporter modulators and, in combination with other anti- cancer agents, could also become a promising strategy to overcome resistance in ABC transporter-expressing tumors. In summary, we were able to determine and complete the interaction profiles of AT-7519, flavopiridol and SNS-032 with ABCB1, ABCG2 and ABCC1 multidrug transporters, which is an important part of their preclinical testing phase. Our results indicate that flavopiridol is able to reverse ABC transporter-mediated MDR and thus increase the intracel- lular concentrations of substrate chemotherapeutic drugs yielding synergistic antiproliferative effect. We also show here for the first time that ABCB1 can cause resistance to AT-7519 and SNS-032, but not to flavopiridol, while ABCG2 and ABCC1 confer resistance to flavopiridol, but not to AT-7519 and SNS-032. Based on these results, we conclude that all tested CDKi may play an important role in transporter-mediated interactions, pharmacokinetics, tissue distribution and drug resistance, and all therapies should be
adjusted accordingly.

Acknowledgments This work was supported by the Grant Agency of Charles University in Prague (Grant No. 700912/C/2012 and SVV/2015/260-185). The publication is co-financed by the European Social Fund and the state budget of the Czech Republic (Project No. CZ 1.07/2.2.00/28.0194, the title of the project FAFIS). The manu- script does not contain clinical studies or patient data.

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