Ascites interferes with the activity of lurbinectedin and trabectedin: Potential role of their binding to alpha 1-acid glycoprotein

E. Erba a,1, M. Romano a,1, M. Gobbi b, M. Zucchetti a, M. Ferrari a, C. Matteo a, N. Panini a, B. Colmegna a, G. Caratti a, L. Porcu a, R. Fruscio c, M.V. Perlangeli d, D. Mezzanzanica e, D. Lorusso f, F. Raspagliesi f, M. D’Incalci a

Abstract

Trabectedin and its analogue lurbinectedin are effective drugs used in the treatment of ovarian cancer. Since the presence of ascites is a frequent event in advanced ovarian cancer we asked the question whether ascites could modify the activity of these compounds against ovarian cancer cells. The cytotoxicity induced by trabectedin or lurbinectedin against A2780, OVCAR-5 cell lines or primary culture of human ovarian cancer cells was compared by performing treatment in regular medium or in ascites taken from either nude mice or ovarian cancer patients.
Ascites completely abolished the activity of lurbinectedin at up to 10 nM (in regular medium corresponds to the IC90), strongly reduced that of trabectedin, inhibited the cellular uptake of lurbinectedin and, to a lesser extent, that of trabectedin. Since a1-acid glycoprotein (AGP) is present in ascites at relatively high concentrations, we tested if the binding of the drugs to this protein could be responsible for the reduction of their activity. Adding AGP to the medium at concentration range of those found in ascites, we reproduced the anticytotoxic effect of ascites. Erythromycin partially restored the activity of the drugs, presumably by displacing them from AGP. Equilibrium dialysis experiments showed that both drugs bind AGP, but the affinity of binding of lurbinectedin was much greater than that of trabectedin. KD values are 8 ± 1.7 and 87 ± 14 nM for lurbinectedin and trabectedin, respectively.
The studies intimate the possibility that AGP present in ascites might reduce the activity of lurbinectedin and to a lesser extent of trabectedin against ovarian cancer cells present in ascites. AGP plasma levels could influence the distribution of these drugs and thus they should be monitored in patients receiving these compounds.

Keywords:
Lurbinectedin
Trabectedin Ascites
a1-Acid glycoprotein
Ovarian cancer

1. Introduction

Lurbinectedin (PM01183) is a trabectedin analogue under clinical development for the therapy of several malignancies including ovarian, breast and lung cancer [1–3]. The chemical structure of lurbinectedin is similar to that of trabectedin. It contains the same pentacyclic skeleton of the fused tetrahydroisoquinoline rings, but it differs by the presence of a tetrahydro beta-carboline that replaces the additional tetrahydroisoquinoline of trabectedin. (Fig. 1).
The mode of action of lurbinectedin is similar to that of trabectedin. Both drugs bind to the DNA minor groove forming adducts at the N2 position of guanines with similar effects on the DNA repair and transcription machineries [4,5]. Both drugs are more cytotoxic against cells that are deficient in Homologous Recombination Repair and less cytotoxic against cells deficient in Nucleotide Excision Repair [6–8]. Both drugs affect the regulation of transcription by displacing some oncogenic transcription factors from their target promoters [9,10] and at high concentrations they cause degradation of RNA polymerase II [11]. The in vivo antitumor activity of both trabectedin or lurbinectedin seems to be due not only to their direct effect on cancer cells but also to their ability to reduce the number of tumor associated macrophages and to inhibit the production of several pro-tumoral inflammatory and angiogenic factors [12–14]. The clinical pharmacokinetic parameters of lurbinectedin differ from those of trabectedin. In particular the apparent volume of distribution of lurbinectedin is 4 times lower than that of trabectedin [15–19].
We previously reported that in Igrov-1 ovarian cancer cells the uptake and cytotoxicity of trabectedin was reduced by increasing the amount of foetal bovine serum (FBS) but not by human serum or by human serum albumin (HSA) suggesting that some proteins different from albumin, present in FBS, tightly bind to the drug reducing the amount of free drug available to enter the cells and to exert its action [20]. This observation prompted us to investigate whether a protein rich-pathological fluid – present in the peritoneal cavity of many ovarian cancer patients such as ascites[21] influenced the cytotoxicity of trabectedin or lurbinectedin. The results show a dramatic inhibitory effect of ascitic fluid on the cytotoxicity of lurbinectedin and to a lesser extent of trabectedin and provide evidence that a1-acid glycoprotein (AGP) is likely to be implicated in this effect.

2. Materials and methods

2.1. Ascites samples

2.1.1. Patients

Ascites was collected in the operating theatre during the laparotomy carried out as primary treatment of epithelial ovarian cancer or for relapsed disease from patients hospitalized at the S Gerardo Hospital in Monza or at Istituto Tumori, Milan. Samples of ascites, obtained from 32 ovarian cancer patients, were collected in heparinized bottles and the cells were separated by centrifugation. Then the supernatant was stored at 20 C before use (range of storage time at 20 C 1–6 months). The study was approved by the Ethics Committees of the S. Gerardo Hospital and Istituto Tumori with patients’ written consent. All procedures were carried out in accordance with the ethical standards of the Declaration of Helsinki.

2.1.2. Animal model

Ascites was collected after sacrifice from 5 female Ncr-nu/nu mice orthotopically transplanted with HOC8 ovarian cancer cells derived from a malignant pleural effusion removed from a previously untreated patient [22]. Procedures involving animals and their care were conducted in conformity with the following laws, regulations, and policies governing the care and use of laboratory animals: Italian Governing Law (D.lgs 26/2014; Authorization n.19/2008-A issued March 6, 2008 by Ministry of Health); Mario Negri Institutional Regulations and Policies providing internal authorization for persons conducting animal experiments (Quality Management System Certificate – UNI EN ISO 9001:2008 – Reg. N 6121); the NIH Guide for the Care and Use of Laboratory Animals (2011 edition) and EU directives and guidelines (EEC Council Directive 2010/63/UE) and in line with Guidelines for the welfare and use of animals in cancer research [23]. Animal experiments have been reviewed and approved by the IRFMN Animal Care and Use Committee (IACUC) that includes members ‘‘ad hoc” for ethical issues. Animals were housed in the Institute’s Animal Care Facilities, which meet international standards; they are regularly checked by a certified veterinarian who is responsible for health monitoring, animal welfare supervision, experimental protocols and procedures revision.

2.2. Drugs

Trabectedin and lurbinectedin (purity: 100%) were kindly provided by Pharma Mar, S.A. (Colmenar Viejo, Spain), stocked in DMSO (Sigma Aldrich, St Louis, MO, USA) at a concentration of 1 mM, stored at 20 C and were diluted in RPMI-1640 medium just before use. AGP, erythromycin, carboplatin and paclitaxel were purchased from Sigma Aldrich and were diluted in RPMI-1640 medium (Thermo Scientific, Waltham, MA, USA) just before use.

2.3. Cells

2.3.1. Cell lines

Human ovarian carcinoma A2780 and OVCAR-5 cells were grown in RPMI-1640, 10% FBS and 2 mM L-glutamine (Thermo Scientific) in T 25 cm2 flasks (Eppendorf, Hamburg, Germany) and maintained at 37 C in a humidified atmosphere at 5% CO2.

2.3.2. Human ovarian cancer primary cultures

The ascitic fluids were collected in heparinized bottles and the cells were separated by centrifugation. A first gradient with 100% of Ficoll-Hypaque (d = 1.077; Sigma Aldrich, St Louis, MO, USA) was performed (600 g for 20 min) to remove RBC contamination and debris. In cases of gross lymphocyte and granulocyte contamination, a second discontinuous gradient (75% Ficoll- Hypaque, layered on 100% Ficoll-Hypaque) was performed. After these steps, in one case, tumor cells were freed of macrophages by adhesion on plastic culture dishes. Final cell suspension was seeded at 70,000 cells cm2 in 6-well multiwell tissue culture plates (Eppendorf).

2.4. Proliferation assay

The evaluation of the cytotoxicity induced by trabectedin or lurbinectedin on cells in the different experimental conditions was assessed by WST-1 cell proliferation assay (Roche, Basel, Switzerland) [24]. The cells were seeded at the concentrations of 5000 cells/ml in 96 –wells tissue culture plates; at 48 h after seeding the cells, in exponentially growing phase, were treated with trabectedin or lurbinectedin for 24 h in medium plus 10% FBS, regular medium, or in 100% ascites obtained from xenografts or from patients affected by ovarian cancer. In the cases of ascites obtained from human patients, in each experiment the ascitic fluid was obtained only from one patient. After 24 h the drug-containing medium was removed, the cells were washed with PBS and fresh medium plus 10% FBS, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin was added for 72 h after drug-washout, when the effect of treatment was evaluated.

2.5. Flow cytometric cell cycle analysis

Cell cycle perturbations induced by 24 h trabectedin and lurbinectedin treatment in presence of medium plus 10% FBS or in 100% ascites on A2780 cells were evaluated by standard flow cytometric methods. Cells in exponentially growing phases were treated with different concentrations of trabectedin or lurbinectedin. Control and treated cells were counted by using Coulter Counter (ZM, Beckman Coulter, Brea, CA, USA) after 24 h treatment and every 24 h after drug washout and fixed in 70% ethanol before DNA staining. Fixed cells were washed with PBS plus 5% FBS and stained overnight with 1 ml of 12.5 mg/ml propidium iodide and 12.5 ml of RNAse 1 mg/ml (Calbiochem Merck Millipore). Flow cytometric analysis was performed on 10,000 events by using a Gallios flow cytometer instrument (Beckman Coulter) [24].

2.6. AGP detection in ascites

The AGP concentrations present in the ascites derived from patients hospitalized at the S Gerardo Hospital Monza and at Istituto Tumori Milan were determined by a immunoturbidimetric assay using Tina-quant a1-acid glycoprotein Gen.2 kit (Roche Diagnostics S.p.A., Monza, Italy).

2.7. HPLC-MS/MS method for the determination of trabectedin or lurbinectedin

The total concentration of trabectedin in medium and in A2780 cell extract was determined by a previously published, validated method based on liquid chromatography/tandem mass spectrometry technique [25]. To quantify the unknown medium samples, a calibration curve of six points (range: 0.1–80.00 ng/mL) was prepared in control medium assessing the precision and the accuracy of the run and analysing at the same time a set of freshly prepared quality controls samples. The LOQ of the method was 0.1 ng/ml.
The total concentration of lurbinectedin in medium and in cell extract was determined according to liquid chromatography/tandem mass spectrometry (LC–MS/MS) method coupled with an electrospray ionization (ESI) source [26]. After the addition of the deuterated internal standard (IS, [2H4] lurbinectedin) to 100 mL of medium, the sample was extracted with tert-butyl-methyl ether (TBME). The recovered TBME was evaporated under a nitrogen gas and then reconstituted with 100 ll of mobile phase. The chromatographic module was coupled with a Triple Quadrupole API4000 (AB SCIEX, USA) mass spectrometer as detector. The chromatographic separation of the analytes was conducted on a Sunfire C18 column (Water, USA), 3.5 lm (150 mm 2.1 mm,) using a gradient mobile phase, pumped at a flow rate of 0.20 mL/min. The run time of elution of the analytes was 6 min. The detection was obtained by MS/MS, monitoring the transitions 767.5 ? 495 m/z for lurbinectedin and 771.7 ? 277.2 m/z for the deuterated IS. To assay study samples, a calibration curve of seven points (range: 0.5–80.0 ng/mL) was prepared in control medium assessing the precision and the accuracy of the run analysing at the same time a set of freshly prepared quality controls samples.

2.8. Equilibrium dialysis

Protein-binding assays were performed by equilibrium dialysis. For these studies we used the Thermo Scientific Pierce RED Device (Catalog number: 90006), consisting of disposable inserts made of two side-by-side chambers separated by a vertical cylinder of dialysis cellulose membrane with a cut-off of 8.0 kDa, validated for minimal nonspecific binding.
For these studies, AGP was present in just one of the two chambers (the red one) at the concentrations of 0, 3, 10, 30 or 60 mM (range 0.1–2.46 mg/ml) whereas the small molecules, trabectedin or lurbinectedin, were initially present in both the chambers (the red and the black one) at a concentration of 100 nM, in RPMI medium containing 40 mg/mL BSA. The physiological concentration of BSA was required to avoid precipitation of the small molecules. Each experimental condition was in triplicate.
Samples were left to equilibrate for 12 h at 37 C with slow rocking. The drug concentrations were then measured in both the red and black chambers by HPLC-MS (25). These values were then used to calculate the percentage of drug bound to AGP, for each AGP concentration, according to the formula: ðð½drugred ½drugblackÞ=ð½drugred þ ½drugblackÞÞ 100
These values were then plotted against the corresponding AGP concentrations and the obtained saturation curve (see Fig x) fitted with the one-site binding, hyperbola equation, using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla California USA), to obtain binding affinity values (i.e Kd values).

2.9. Statistical analysis

For the WST-1 cell proliferation assay the arithmetic mean and the s.e. of number of viable cells were calculated. The ratio of viable cells between treated and control cells was computed; on log scale, an estimate of the s.e. of this ratio was obtained by the Delta method as:where lT and lC are the sample means of the treated and control viable cells, respectively, and rT and rC are the sample s.e. of the treated and control viable cells, respectively. Using the same analytical approach to estimate its s.e., the ratio of viable cells between treated and control cells in ascites was divided by the ratio of viable cells between treated and control cells in medium. A randomeffects model was used to estimate mean and 95% CI of the percentage of viable cells.

3. Results

3.1. Influence of ascites on the cytotoxicity of trabectedin and lurbinectedin

Initial experiments were performed testing the ascites taken from the peritoneal cavity of mice orthotopically transplanted with a human ovarian cancer xenograft HOC 8. Results of a representative experiment are illustrated in Fig. 2. The cytotoxic effect induced in A2780 and OVCAR-5 cells by 24 h treatment with trabectedin or lurbinectedin was evaluated 72 h after drug-washout. The treatment was performed in medium plus 10% FBS (regular medium) or in 100% ascites obtained from HOC 8 xenograft bearing mice. When treatment was performed in regular medium both trabectedin and lurbinectedin exerted cytotoxicity in A2780 and OVCAR-5 cells in a dose–dependent manner with IC50 values of approximately 2–3 nM and 1 nM for A2780 and OVCAR-5 cells respectively. When the drugs were dissolved in ascites the cytotoxicity of the two drugs was clearly diminished. Lurbinectedin in the range of concentrations from 0.5 to 10 nM was totally inactive, whereas trabectedin was inactive up to the concentration of 5 nM, and at 10 nM it caused approximately 40% and 90% inhibition of cell growth on A2780 and OVCAR-5 cells respectively.
Since the effects of the ascites on the cytotoxicity of trabectedin and lurbinectedin were similar in both A2780 and in OVCAR-5, subsequent experiments were made only on A2780 cell line.Further experiments were performed using human ascites. A representative experiment is illustrated in Fig. 3 that shows a comparison between the cytotoxic effects of trabectedin or lurbinectedin dissolved in regular medium or in ascites. No cytotoxic effect of lurbinectedin was observed even at the concentration of 10 nM when the treatment was performed in ascites, whereas the drug caused dramatic cytotoxicity when the treatment was performed in regular medium. The cytotoxic effect induced by trabectedin was also markedly decreased when treatment was performed in ascites. As shown for lurbinectedin, exposure of cells for 24 h to 10 nM trabectedin induced strong cytotoxicity when the treatment was performed in regular medium, while the drug was less active in the presence of ascites. Table 1 summarizes the data obtained by comparing the cytotoxic effects induced by trabectedin or lurbinectedin using the two treatment protocols, i.e regular medium or ascites derived from a large number of different ovarian cancer patients. Trabectedin and lurbinectedin induced a similar cytotoxic effect when treatment was performed in regular medium. Instead, when the treatment was performed in ascites, the cytotoxicity of lurbinectedin was completely abolished, and it was reduced in case of trabectedin. The difference in influence of ascites on the cytotoxicity of lurbinectedin and trabectedin is clearly indicated by the ratio ascites/medium values (table 1).

3.2. Influence of ascites on drug uptake

A2780 cells were exposed to 10 nM of trabectedin or lurbinectedin for 4 h and the intracellular amounts of drug, as adjudged by a HPLC-MS/MC assay, were compared. As shown in Fig. 5 the intracellular amounts of trabectedin were 0.2 (0.27 mmoli) or 0.1 (0.135 mmoli)/ng in 106 cells exposed to the drug in medium or ascites, respectively. The amount of lurbinectedin recovered from cells with medium was 0.8 ng/106 cells, and the drug was undetectable in cells in the presence of ascites. These data explain why lurbinectedin was inactive when the treatment was performed in ascites. The reduction of the cytotoxicity of trabectedin when the treatment was performed in ascites is consistent with a reduction of drug uptake.

3.3. Influence of AGP on drug cytotoxicity

Both trabectedin and lurbinectedin undergo binding to albumin, but the concentrations of albumin did not affect the cytotoxicity of either of the two drugs (data not shown). We hypothesized that a potential protein candidate present in ascites which might be responsible for the drug binding is AGP. This hypothesis was based on the reported presence of this protein of acute phase in ascites. We have found that in our ascites samples the concentration of AGP was within the range of 0.58 to 1.84 mg/ml (14.0– 44.9 mM).
Since it is known that erythromycin binds AGP with high affinity, we tested its ability to restore trabectedin and lurbinectedin cytotoxicity when the treatment was performed in ascites. Erythromycin (200 mM) was able to increase the cytotoxicity of trabectedin, for lurbinectedin the effect was evident only at the high drug concentration (Fig. 6, panel A). Fig. 6, panel B, shows the influence of the treatment combination of AGP and erythromycin on the cytotoxicity of trabectedin or lurbinectedin when the treatment was performed in regular medium. The cytotoxicity of trabectedin or lurbinectedin was increased when erythromycin was present, suggesting that it was able to displace the AGP bound to the drugs. The effect was greater for trabectedin than for lurbinectedin. For lurbinectedin the reversion by erythromycin of the inhibitory effect of AGP was evident only at the high drug concentration.

3.4. Binding affinities of trabectedin and lurbinectedin to AGP

From this a KD of 7 lM (K.a = 1.4 105 M1) can be calculated for the binding between trabectedin and AGP. A similar calculation allowed to estimate a KD of 1 lM (Ka = 1 106 M1) for the binding between lurbinectedin and AGP. These KD values must be considered only as estimates since they were obtained in an indirect manner. Nevertheless these studies suggest that both drugs interact with AGP, and that the binding affinity of lurbinectedin is about one order of magnitude higher than that of trabectedin.
Initial attempts to directly determine the affinity of the two drugs for AGP were carried out using Surface Plasmon Resonance. However, we did not succeed in immobilizing AGP at sufficient levels due to its very low isoelectric point. We therefore carried out equilibrium dialysis experiments, which confirmed that lurbinectedin binds to AGP more avidly than trabectedin (Fig. 7, panel B). The drug concentration used for these studies (100 nM) was negligible in comparison with the AGP concentrations (10–60 lM), thus allowing estimation of the KD values by fitting the corresponding saturation curves. It should be noted that the range of AGP concentrations are consistent with plasmatic AGP concentrations, ranging from 3 lM (0.1 mg/ml) (in healthy controls) to a maximum of 60 lM (2.49 mg/ml) as reported in cancer patients. Notably, these studies were carried out in the presence of physiological concentrations of BSA. Under these conditions the estimated KD values were 8.0 ± 1.7 nM (Ka = 1.25⁄108 M1) for lurbinectedin and 87 ± 14 nM (Ka = 1.15⁄107 M1) for trabectedin. These values might be overestimates due to the concomitant presence of albumin competing for binding, i.e. the affinities for AGP might be higher. Nevertheless the results confirm that the affinity of lurbinectedin to AGP is about one order of magnitude higher than that of trabectedin.

3.5. Influence of ascites on the human ovarian cancer’s primary culture

The effects of ascites on the cytotoxicity of trabectedin and lurbinectedin were also investigated on the human ovarian cancer cells obtained from one of the ascites used in this study. As shown in Fig. 8 the presence of ascites completely abolished the cytotoxicity of lurbinectedin at up to 10 nM and strongly reduced that of trabectedin, as observed on A2780 and OVACAR-5 cell lines.

3.6. Influence of ascites on the cytotoxicity of carboplatin and paclitaxel

Carboplatin and paclitaxel are two anticancer drugs widely used in the treatment of ovarian cancers. It seemed very interesting to study whether the presence of the ascites could alter the cytotoxic effect of these drugs too. As shown in Fig. 9 the cytotoxic effect caused by carboplatin (panel C) was similar when the treatment was performed in the presence of ascites or in regular medium. Instead, for paclitaxel, when the treatment was performed in ascites, the cytotoxicity of paclitaxel (panel D) was strongly reduced with IC50 values of approximately 25 nM compared to 2.5 nM in regular medium.

4. Discussion

The present study shows that the cytotoxicity of lurbinectedin is strongly reduced by ascites. Since a similar effect was observed when culture medium was complemented with AGP it seems plausible to suggest that this protein is a major factor responsible for the observed inhibition of cytotoxicity by ascites, although it cannot be excluded that other proteins are also involved. It is highly conceivable that the binding of the drugs to AGP and/or other proteins present in ascites reduces or prevents the cellular drug uptake.
The much higher affinity for AGP of lurbinectedin than of trabectedin explains the different degrees of inhibition of the cytotoxic effects of the two drugs. For trabectedin the reduction of cellular uptake with a consequent decrease of its cytotoxic effects, albeit significant, was less dramatic than that found for lurbinectedin. The binding was reversible and the drugs can obviously be displaced by erythromycin or perhaps other compounds which possess high affinity for AGP, thus restoring, at least in part, the cytotoxic activity of the drugs. These findings are inconsistent with notion that the drugs are metabolically inactivated by the ascites [27].
Since lurbinectedin is currently under investigation in advanced ovarian cancer patients it seems legitimate to ask the question if these findings are clinically relevant and should influence the ongoing development and future potential use of this drug. According to initial clinical data lurbinectedin seems to harbor promising potential for the therapy of ovarian cancer patients, even those with acquired resistance to platinum drugs [28].
An obvious potential implication of our findings concerns the activity of the two drugs against ovarian cancer cells floating in the ascites or against micro metastases present in the peritoneal cavity. It should be noted that ascites is a frequent event occurring in advanced ovarian cancer [21]. The cytotoxicity of the drugs present in this body compartment could be compromised – absent in the case of lurbinectedin and suboptimal for trabectedin. This probably applies also to paclitaxel as this drug binds to AGP with consequent reduction of its cytotoxic effect in presence of ascites [29,30]. This finding would require further studies to assess its clinical relevance.
No data on the concentrations of lurbinectedin or of trabectedin in the ascites of ovarian cancer patients who have received these drugs are available. It is therefore impossible to know if the drug concentrations used in our experiments are within the range of those present in the patients’ peritoneal cavity. However, this problem might be of limited importance as it seems likely that the major mechanism of delivery of drugs to the ovarian cancer mass occurs via the blood stream, and drug distribution by direct diffusion is negligible. In fact, even for cisplatin that has been widely used by intraperitoneal administration in ovarian cancer patients, penetration is limited to a few layers of cells nested in the peritoneal cavity, so that its antitumor activity is mainly related to its absorption and distribution to the neoplastic tissue through vascular delivery [31]. We proffer the opinion that the findings are of more importance in relation to the systemic pharmacokinetic behavior of the drugs, particularly for lurbinectedin and to a lesser extent for trabectedin. It seems possible that the relative low volume of distribution of lurbinectedin compared to that of trabectedin is related to the fact that the drug is present in blood highly bound to AGP thus partially limiting the rate of its tissue distribution. It may be propitious to note that AGP levels can be very variable in advanced cancer patients (27) a fact, which might constitute an important source of variability of the pharmacokinetics and toxicity of lurbinectedin. The finding reported here may offer an explanation for the finding that in spite of the very similar in vitro cytotoxic potencies of lurbinectedin and trabectedin (8), the MTD values in humans were 3–4 times higher for lurbinectedin than for trabectedin. One can surmise that both the antitumor and toxic effects of the drugs are probably related to the plasma levels of free drug that can distribute to tissues and not to total plasma drug concentrations that are 5 times higher for lurbinectedin than for trabectedin, when drug doses in the therapeutic range are compared.
Based on the data presented here the assessment of AGP plasma levels in patients under treatment with lurbinectedin seems to be indicated, and the influence of these levels on drug pharmacokinetics should be investigated.
Furthermore, it seems important to bear in mind potential interactions of lurbinectedin with drugs that bind to AGP with high affinity when co-administered.

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