Preclinical report 917

Spongean alkaloids protect rat kidney cells against cisplatin-induced cytotoxicity Florian Funka,*, Katharina Kru¨gera,*, Christian Henningera, Wim Wa¨tjena, Peter Prokschb, Ju¨rgen Thomalec and Gerhard Fritza Nephrotoxicity is the major dose-limiting adverse effect of cisplatin (CisPt) and results from CisPt-induced damage of tubular cells. Nephroprotective strategies are preferential to improve supportive care in cancer. We investigated a subset of purified substances originating from various plants or from marine sponges as to their potency to protect rat renal tubular cells (NRK-52E) against the cytotoxic and genotoxic effects of cisplatin. Cotreatment with a substance pool containing five purified substances originating from marine sponges increased the viability of NRK-52E cells following cisplatin treatment. Cytoprotection was accompanied by a reduced level of DNA damage as indicated by a lower amount of S139 phosphorylated histone H2AX (cH2AX) 24 h after treatment. Cytoprotection and genoprotection by the sponge substance pool did not comprise the anthracycline derivative doxorubicin. The spongean alkaloid aaptamine was identified as major bioactive compound that mediates cisplatin resistance. Aeroplysinin-1 was less cytoprotective than aaptamine. Notably, aaptamine preferentially conferred resistance to cisplatin, but not to oxaliplatin. Cytoprotection by aaptamine was also observed in rat glomerular endothelial cells, but not in RT-112 bladder cancer cells. Protection by aaptamine does not rest on

Introduction

a reduced formation of DNA damage caused by cisplatin treatment. Aaptamine and aeroplysinin-1 affected cisplatin-stimulated DDR as reflected on the level of S15-phosphorlyated p53 and S345-phosphorylated checkpoint kinase-1. Summarizing, the spongean alkaloid aaptamine alleviates cisplatin-induced damage in tubular and glomerular rat kidney cells. Therefore, we hypothesize that aaptamine might be useful to widen the therapeutic window of a cisplatin-based therapeutic regimen. Antic 2014 Wolters Kluwer Health | Cancer Drugs 25:917–929  Lippincott Williams & Wilkins. Anti-Cancer Drugs 2014, 25:917–929 Keywords: aaptamine, cisplatin, natural compounds, nephrotoxicity, normal tissue damage a

Institute of Toxicology, Heinrich Heine University, Moorenstrasse 5, Du¨sseldorf, Institute of Pharmaceutical Biology and Biotechnology, Universita¨tsstrasse 1, Du¨sseldorf and cInstitute for Cell Biology, University Duisburg-Essen, Hufeland Strasse 55, Essen, Germany

b

Correspondence to Gerhard Fritz, Institute of Toxicology, Heinrich Heine University Du¨sseldorf, Moorenstrasse 5, 40225 Du¨sseldorf, Germany Tel: + 49-211-8113022; fax: + 49-211-8113013; e-mail: [email protected] *Florian Funk and Katharina Kru¨ger contributed equally to this article. Received 4 November 2013 Revised form accepted 19 March 2014

Nephrotoxicity is the most serious therapy-limiting adverse effect in the course of cisplatin-based anticancer

therapy. It is believed that tubular cells are particularly sensitive to cisplatin damage [2,8]. Apart from hydration and stimulation of diuresis, clinically approved nephroprotective therapeutic options are limited. With respect to tumor cells, numerous mechanisms have been identified that contribute toward cisplatin resistance in vitro and in vivo, including transport, DNA repair as well as DNA damage response (DDR) and apoptosis [9,10]. By contrast, key players that determine the susceptibility of normal cells to cisplatin and other platinum compounds are not well characterized. Previously, the formation of reactive oxygen species (ROS) [11], ATR-Chk2 signaling [12], and mechanisms of transport [13,14] have been suggested to play pivotal roles in the outstanding sensitivity of the kidney to cisplatin-induced injury. Recently, prevention of ROS formation [15,16] and targeting of Nrf2 signaling [17] have been proposed as nephroprotective strategies. Nevertheless, nephroprotective concepts for supportive care in cancer are rare and therefore preferable.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.anti-cancerdrugs.com).

Natural compounds (NC) are frequently analyzed with respect to their antitumor potency in vitro and in vivo,

Normal tissue damage induced by anticancer drugs limits their clinical applicability. Cisplatin belongs to the group of platinum-based anticancer drugs, which are used for the treatment of manifold malignancies, including lung, cervical, and bladder cancer [1]. The clinically most relevant doselimiting adverse effect of cisplatin is nephrotoxicity [2]. Upon uptake into the cell by different types of drug transporters, the chloride ligands of cisplatin are replaced by water, resulting in an electrophilic complex that induces various types of DNA adducts by an SN2-like mechanism independent of liver metabolism. Apart from monoadducts and GG-interstrand cross-links, DNA intrastrand cross-links (GG and AG) are most abundant [3–6]. The platin-induced DNA adducts formed block transcription and replication, thereby inducing cell death [5,7].

c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4973 

DOI: 10.1097/CAD.0000000000000119

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918 Anti-Cancer Drugs 2014, Vol 25 No 8

both as monotherapy and in combination with clinically approved anticancer drugs [18,19]. Induction of tumor cell death by NC is favored by multiple mechanisms including the formation of ROS [20], inhibition of microtubular function [21], or inhibition of topoisomerases [22]. Moreover, anti-invasive effects of NC have also been reported [23]. In addition, plant ingredients are often discussed for chemoprevention [24,25] and cardioprotection [26]. Nevertheless, aspects of possible cytoprotective capacities of NC against anticancer drug induced normal tissue damage have rarely been addressed to date. In the present study, we therefore investigated the influence of various NC, isolated either from different plants or from marine sponges, on the cytotoxic and genotoxic effects caused by the anticancer drug cisplatin. As an in-vitro cell model for our screening analyses, we used the rat kidney tubular cell line NRK-52E.

Materials and methods Materials

Cisplatin, oxaliplatin, and the anthracycline derivative doxorubicin, which were obtained from the central pharmaceutical department of the university hospital Du ¨sseldorf, originate from Teva GmbH (Ulm, Germany) (cisplatin and oxaliplatin) or from Cell Pharm GmbH (Bad Vilbel, Germany) (doxorubicin), respectively. Rat kidney tubular NRK-52E and rat glomerular endothelial (RGE) cells originate from the DSMZ (Braunschweig, Germany). The antibody detecting Ser139-phosphorylated histone H2AX (gH2AX) was obtained from Millipore (Billerica, Massachusetts, USA), ERK2, p53, Bax, p21, caspase-7, PARP, and b-actin-specific antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, California, USA), p-JNK (Thr183/Tyr18), p-p38 (Thr180/Tyr182), p-Chk1 (S345) and p-p53 (S15) were from New England Biolabs (Frankfurt, Germany) and the antibody against cisplatin-induced GGintrastrand cross-links was kindly provided by J. Thomale (Essen, Germany) [27]. The following NC were provided by the Institute of Pharmaceutical Biology and Biotechnology of the Heinrich Heine University (Du ¨sseldorf, Germany): (i) a pool of five different plant substances belonging to the group of polyphenols [PP: 3,4-dihydroxy-5-methoxybenzoesa¨uremethylester (1 mmol/l); 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3,4-dihydro-2H-chromen-3-yl-3,4-dihydroxy-5-methoxybenzoate (1.5 mmol/l); 30 ,40 ,50 ,7-tetrahydroxyflavan-3-ol (4 mmol/l); 40 ,50 ,50 ,7-tetrahydroxyflavan-3-ol (4 mmol/l); galloyl-6-glucose-1-p-hydroxyphenylethanoid (0.2 mmol/l)] and (ii) a pool of five different purified substances [equimolar ratio (i.e. 2 mmol/l each)] from marine sponges [marine SP (genus Porifera)] [(–)-agelasin D, aeroplysinin-1, aaptamine, aerothionin, (+)-agelasidin C]. The purified compounds of the plant and SPs were chosen on the basis of their different chemical structures, which were considered as possible lead structures for novel bioactive compounds. Moreover, ethyl acetate extracts from 30 g of dried medicinal plants (C. xanthorrhiza, Salix spp., A. montana, G. glabra, C. officinalis, and H. procumbens) were also included in the study. Major

ingredients of the medicinal plants, pharmacological effects, and chemical structures of the individual substances are summarized in Supplementary Tables 2–4 (Supplemental digital contents 1, 2, 3, http://links.lww.com/ACD/A59, http:// links.lww.com/ACD/A57, http://links.lww.com/ACD/A62). Cell culture and drug treatments

Rat kidney proximal tubular cells (NRK-52E cell line) and human bladder cancer cells (RT-112) were grown in DMEM medium (containing L-glutamine), and RGE cells in RPMI medium (containing L-glutamine) (Invitrogen, Paisley, UK) supplemented with 10% (v/v) fetal calf serum (PAA Laboratories, Co¨lbe, Germany) at 371C in an atmosphere containing 5% (v/v) CO2. If not stated otherwise, treatment of the proliferating cells started 24 h after seeding. Pretreatment with different concentrations of the NC was performed for 24 h before the anticancer drugs (i.e. cisplatin, oxaliplatin, or doxorubicin) were added at the indicated concentrations. The doses of the NC used for cotreatment were subtoxic or only moderately cytotoxic on their own (i.e. reducing viability by B20–35%) (Supplementary Fig. S2, Supplemental digital content 4, http://links.lww.com/ACD/A58). If not stated otherwise, viability was analyzed after further incubation period of 48 h [fluorescence-activated cell sorting (FACS) analysis: 24–48 h] following addition of the anticancer drugs. Alterations in gH2AX protein levels were analyzed 16 and 20 h after pulse treatment with cisplatin (for 8 h) and doxorubicin (for 4 h), respectively. Isolation and origin of natural compounds

Pure compounds were obtained from a collection of one of the authors (P. Proksch) and had been isolated earlier either from plants or from marine sponges. The polyphenolic plant compounds used in this study (see Supplementary Table 3, Supplemental digital content 2, http://links.lww.com/ACD/A57) were all obtained from a leaf extract of the African medicinal plant Maytenus senegalensis. Briefly, air-dried leaves were ground and exhaustively extracted with ethyl acetate at room temperature (RT). The extract was dried using a rotary evaporator and then subjected to vacuum liquid chromatography on a silica gel. Fractions resulting from this separation were further purified by column chromatography on Sephadex LH-20 using methanol as a solvent. Final purification was achieved using semi-preparative HPLC [28]. All compounds were identified by one-dimensional and two-dimensional NMR spectroscopy and by mass spectrometry as well as by comparison with the literature. Isolation of the marine sponge-derived compounds (see Supplementary Table 4, Supplemental digital content 3, http://links.lww.com/ACD/A62) has been described earlier [29–31]. Substances were dissolved in DMSO. When treating cells, substances were diluted to ensure that the final DMSO concentration was 0.1% or less, (w/v) which is nontoxic. Extracts from medicinal plants (see Supplementary Table 2, Supplemental digital content 1, http://links.lww.com/ACD/A59) were obtained using ethyl acetate as an extraction solvent. In brief,

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Nephroprotection by the spongean alkaloids Funk et al. 919

30 g (dry weight) of commercially available plant material (Gahlke, Gittelde/Harz, Germany) was extracted with 100 ml ethyl acetate on a magnetic stirrer overnight. The next morning, the slurry was filtered through a filter paper and the resulting extract was dried using a rotary evaporator. After freeze drying, the weight of the resulting extract was determined. Southwestern analyses

Genomic DNA was isolated using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The concentration and purity of the DNA were measured photometrically (NanoVueTMPlus; GE Healthcare, Solingen, Germany) and confirmed by agarose gel electrophoresis. 0.5 mg of the DNA was diluted with TE buffer up to 100 ml, denatured by heating (10 min, 951C), and cooled on ice. Afterwards, 100 ml ice cold ammonium acetate (2 mol/l) was added. A nitrocellulose membrane was soaked in 1 mol/l ammonium acetate and placed in the slot-blot apparatus (Roth, Karlsruhe, Germany). The DNA was transferred onto the membrane using a vacuum pump. After washing with 1 mol/l ammonium acetate and water, the membrane was incubated with 5  SSC (10  SSC: 1.5 mol/l NaCl, 150 mmol/l sodium citrate, pH 7.0) for 5 min before it was blocked in 5% nonfat milk in TBS/0.1% Tween 20 (v/v) overnight at 41C. Incubation with the primary antibody (1 : 200), which specifically detects Pt-GG intrastrand cross-links [27], was performed for 1 h at RT, followed by incubation with HRP-coupled anti-rat secondary antibody (1 : 1000) (2 h, RT). Visualization of the Pt-GG intrastrand cross-links was carried out by chemiluminescence. Autoradiographies were analyzed densitometrically and the relative signal intensity in the untreated control was set to 1.0. In addition, the membrane was stained with methylene blue to ensure equal DNA loading. Analysis of DNA damage

For analysis of DNA damage, the level of Ser139phosphorylated H2AX (gH2AX), which is a surrogate marker of DNA damage [32], was analyzed by immunocytochemistry-based analysis of the number of gH2AX foci/cell or fluorophore-linked immunosorbent assaybased method. For immunocytochemical analysis, cells were seeded onto cover slides. After treatment, cells were fixed with 4% paraformaldehyde (v/v) (15 min, RT) and incubated with ice cold methanol (– 201C, 1 h). After treatment with blocking solution [PBS containing 0.3% Triton X-100/5% BSA (w/v), 1 h, RT], antibody specifically detecting phosphorylated (Ser139) histone H2AX (1 : 500) was added. After overnight incubation at 41C and washing, secondary fluorescent-labeled antibody (Alexa Fluor 488; Invitrogen) was added for 2 h (RT in the dark). Afterwards, cells were mounted in Vectashield (Vector Laboratories Inc., Burlingame, California, USA) containing the DNA staining dye DAPI. For microscopical analysis, at least 50 nuclei were evaluated per experimental condition. To detect S139 phosphorylated H2AX

by the fluorophore-linked immunosorbent assay-based method, cells were seeded onto 96-well plates. After fixation and incubation with gH2AX-specific primary and secondary fluorescence-labeled antibody, nuclei were stained with DAPI. gH2AX staining of the cell population is reflected by the intensity of the fluorescence signal (emission wave length 519 nm) and was normalized to the intensity of DAPI staining. The relative level of gH2AX in the untreated control was set to 1.0. Reduction in the mean gH2AX signal intensity by 50% or less in groups that had been cotreated with the NC(s) was considered as not relevant. Determination of cell viability

5–10  103 cells/well (96-well plate; 100 ml culture medium per plate) were seeded for cell viability analyses. In general, viability was analyzed 48 h after treatment with the anticancer drugs using the MTT assay according to the manufacturer’s protocol (Boehringer, Mannheim, Germany). Briefly, 20 ml of the tetrazolium salt [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] stock solution (5 mg/ml in PBS) was added to each well, followed by an incubation period of 30–45 min. During this time, MTT is metabolized to a formazan dye by mitochondrial dehydrogenase, which is active only in living cells. After removal of the medium, 100 ml of DMSO [100% (v/v) (Merck, Darmstadt, Germany)] was added to solubilize the formazan dye. After further incubation for 10 min on a plate shaker (RT), absorption was measured at 560 nm. The relative viability in the corresponding untreated (i.e. nonanticancer drug treated) controls was set to 100%. If not stated otherwise, data shown are the mean±SD from one to three independent experiments, each conducted in quadruplicate. Analysis of synergism/antagonism for combination treatments

Dose–response curves for each drug alone and the combinations were monitored using the aforementioned MTT assay. Absorption measurements were normalized to the untreated control. To further analyze the data of the possible antagonistic effects of the spongean alkaloids, the Chou–Talalay method was used [33]. To this end, viability data were entered in CompuSyn software (ComboSyn Inc., Paramus, New Jersey, USA) as fraction affected (Fa) values, ranging between 0 and 1. The CI values obtained were plotted against the Fa values. A CI of more than 1 indicates antagonistic activity. Fluorescence-activated cell sorting-based analysis of cell cycle distribution and cell death

Cell cycle distribution was analyzed by flow cytometry (FACS). To this end, cells were trypsinized, washed twice with PBS, resuspended in 100 ml PBS, and fixed with 2 ml of ice-cold ethanol (– 201C, Z 20 min). After centrifugation (1000g, 5 min, 41C), the supernatant was discarded. The cells were resuspended in PBS containing RNase (1 mg/ml)

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920 Anti-Cancer Drugs 2014, Vol 25 No 8

and incubated for 1 h at RT. After adding propidium iodide (50 mg/ml), the cells were subjected to FACS analysis for quantification of the percentage of cells present in the G1, S, and G2-phases of the cell cycle. The subG1-fraction is considered a measure of dead (apoptotic) cells. Western blot analysis

After treatment, total cell extracts were prepared by lysing the cells in 100–200 ml of Roti-Load1 buffer (Carl Roth GmbH, Karlsruhe, Germany). After heating to 951C for 5 min, 20–30 ml of the protein extract (containing B20–30 mg protein) was separated by SDS-PAGE [12% gel (v/v)] using the Mini-Protean system from Biorad (Munich, Germany). After electrophoresis (30 mA/gel), proteins were transferred onto a nitrocellulose membrane by wet blotting using the aforementioned system from Biorad [transfer buffer: 25 mmol/l Tris, 192 mmol/l glycin, 20% (v/v) MeOH]. After blocking in 5% nonfat milk in TBS (w/v)/0.1% Tween

(v/v) for 1 h at RT, incubation with the primary antibodies (1 : 200 to 1 : 1000) was performed overnight at 41C. After washing with TBS/0.1% Tween (v/v) (3  5 min, RT), incubation with peroxidase-conjugated secondary antibody (1 : 2000) was performed for 2 h at RT. After washing, the bound antibodies were visualized using the Fusion FX7 imaging system (Peqlab, Erlangen, Germany). Statistical analysis

Student’s t-test was used for statistical analysis. A P value of 0.05 or less was considered a statistically significant difference between cells that were treated only with cisplatin and cells that had been cotreated with the NCs.

Results and discussion In the present study, we addressed the question of whether pretreatment of rat kidney tubular epithelial cells (NRK-52E) with various NC can confer cytoprotection

Fig. 1

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Influence of polyphenolic plant substances on anticancer drug sensitivity of rat tubular cells. (a, c) Logarithmically growing NRK-52E cells were pretreated with a substance pool (PP) (10 mmol/l), which contains five different purified plant polyphenols as described in the Materials and methods section. Twentyfour hours later, cisplatin (a) or doxorubicin (c) was added at the indicated concentrations. After a further incubation period of 48 h, cell viability was monitored. Data shown are the mean±SD from n = 1–3 independent experiments, each conducted in quadruplicate. – PP, without plant polyphenols; + PP, in the presence of plant polyphenols. The effects of PP on cell viability are shown in Supplementary Figure S2A (http://links.lww.com/ACD/A58). (b, d) Twenty-four hours after pretreatment with polyphenolic substance pool (PP) (10 mmol/l) cells were pulse treated for 4 and 8 h with doxorubicin and cisplatin, respectively. After a postincubation period of 16–20 h, DNA damage was monitored by measuring the level of gH2AX as described in the Materials and methods section. Shown are the mean±SD from one to two independent experiments, each conducted in quadruplicate.

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Nephroprotection by the spongean alkaloids Funk et al. 921

against a subsequent treatment with cisplatin. To handicap the identification of substances that unspecifically confer resistance to anticancer drugs, the anthracycline derivative doxorubicin was included in the study as a control. The cytotoxicity caused by the various compounds on their own was determined in preliminary experiments by dose–response analyses using the MTT assay. For subsequent combination experiments with the aforementioned anticancer drugs, a subtoxic or only a weakly cytotoxic dose of a given plant extract, substance pool, or purified substance was used. Analysis of the influence of various plant extracts on cytotoxicity and genotoxicity caused by cisplatin

Plant polyphenols (PP) are reported to exert pleiotropic biological effects. Therefore, we examined their potency to modulate the cytotoxicity of cisplatin and doxorubicin in NRK-52E cells. For the analyses, a substance pool containing

a selected subset of five purified polyphenols (see the Materials and methods section and Supplementary Table 3, Supplemental Digital Content 2, http://links.lww.com/ACD/ A57) was used. For combination treatment with cisplatin, a concentration of 10 mmol/l of the PP pool was used, which was found to be subtoxic in NRK-52E cells (Supplementary Fig. S2A, Supplemental digital content 4, http://links.lww.com/ ACD/A58). The data show that the polyphenolic substance pool neither affected cisplatin nor doxorubicin-induced loss of cell viability (Fig. 1a and c). The level of cisplatin-induced DNA damage, as monitored by serine-139 phosphorylation of histone H2AX (gH2AX), also remained unaltered by the plant extract (Fig. 1b and d). Extracts from medicinal plants are used frequently in phytotherapy (e.g. arnica or calendula extracts), and hence might be principally useful for therapeutic purposes. Therefore, we also included a subset of them in our analyses (see Supplementary Table 2, Supplemental digital content 1, http://links.lww.com/ACD/A59).

Fig. 2

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Harpagoside, the major ingredient of Harpagophytum procumbens, does not confer cisplatin resistance. (a, c) Logarithmically growing NRK-52E cells were pretreated with a nontoxic concentration of harpagoside (100 mmol/l) (see Supplementary Figure S2B, http://links.lww.com/ACD/A58) for 24 h before cisplatin (a) or doxorubicin (c) was added. After a postincubation period of 48 h, cell viability was monitored as described in the Materials and methods section. Data are the mean±SD from a single experiment conducted in quadruplicate. (b, d) Twenty-four hours after pretreatment with harpagoside (100 mmol/l) cells were pulse treated for 4 and 8 h with doxorubicin and cisplatin, respectively. After a postincubation period of 16–20 h, DNA damage was monitored by measuring the level of gH2AX as described in the Materials and methods section. Shown are the mean±SD from one to two independent experiments, each conducted in quadruplicate.

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922 Anti-Cancer Drugs 2014, Vol 25 No 8

Fig. 3

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Ingredients of marine sponges confer cisplatin resistance to rat tubular cells. (a, c) Logarithmically growing NRK-52E cells were pretreated with a substance pool containing five different purified substances isolated from marine sponges (SP) (5 mmol/l) as described in the Materials and methods section. At this concentration, SP induces a moderate decrease in cell viability (see Supplementary Figure S2C, http://links.lww.com/ACD/A58). Twenty-four hours later, cisplatin (a) or doxorubicin (c) was added at the indicated concentrations. After a further incubation period of 48 h, cell viability was monitored as described in the Materials and methods section. Shown is the relative viability following anticancer drug treatment as related to the corresponding control (i.e. cells not treated with anticancer drugs), which was set to 100% (absolute viability data are presented in Supplementary Figures S3, Supplemental digital content 7, http://links.lww.com/ACD/A63). Data shown are the mean±SD from n = 2–3 independent experiments, each conducted in quadruplicate. SP, marine sponge pool. *P r 0.05 as compared with the corresponding non-SP cotreated cells. (b, d) Twenty-four hours after pretreatment with the marine SP (5 mmol/l) cells were pulse treated for 4 and 8 h with doxorubicin and cisplatin, respectively. After a postincubation period of 16–20 h, DNA damage was monitored as described in the Materials and methods section. Shown are the mean±SD obtained from one to two independent experiments, each conducted in quadruplicate. #Reduction by Z 50% as compared with the non-SP cotreated control.

The data, which are summarized in Supplementary Table 1 (Supplemental digital content 5, http://links.lww.com/ACD/ A60), show that extracts from Curcuma xanthorrhiza, Salix spp., Arnica montana, and Glycyrrhiza glabra did not increase the viability of the rat tubular cells following cisplatin treatment, while sensitizing the cells to doxorubicin. Calendula officinalis extract was found to increase the sensitivity to cisplatin and doxorubicin (Supplementary Table 1, Supplemental digital content 5, http://links.lww.com/ ACD/A60). Out of the different medicinal plant extracts tested, only the extract from Harpagophytum procumbens provided protection against cisplatin-induced loss of cell viability (Supplementary Table 1, Supplemental digital content 5, http://links.lww.com/ACD/A60 and supplementary Fig. S1A, Supplemental digital content 6, http://links.lww. com/ACD/A61), which was paralleled by genoprotection

(Supplementary Table 1, Supplemental digital content 5, http://links.lww.com/ACD/A60 and Supplementary Fig. S1D, Supplemental digital content 6, http://links.lww.com/ACD/A61). Cytoprotection and genoprotection by H. procumbens extract seems to be specific because at the same time it sensitized the cells toward doxorubicin (Supplementary Table 1, Supplemental digital content 5, http://links.lww.com/ACD/A60 and Supplementary Figs S1E, Supplemental digital content 6, http://links.lww.com/ACD/A61). FACS-based cell cycle analyses and microscopical examination yielded identical results (Supplementary Figs S1B and S1C, Supplemental digital content 6, http://links.lww.com/ACD/A61). As harpagoside is known to be the major bioactive ingredient of H. procumbens extract, we investigated its effect on cisplatin-induced cytotoxicity and genotoxicity. To this end, harpagoside was used at a concentration of 100 mmol/l, which was found to be

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Nephroprotection by the spongean alkaloids Funk et al. 923

Fig. 4

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Fraction affected (Fa)

Influence of aaptamine (Aa) and aeroplysinin-1 (Ap) on the sensitivity of rat kidney cells to cisplatin (CisPt). (a) Rat tubular kidney cells (NRK-52E) were pretreated for 24 h with either Aa (2 mmol/l) or Ap (2 mmol/l) before CisPt was added at the concentrations indicated. After a further incubation period of 48 h, cell viability was monitored. Data shown are the mean±SD from n = 3 independent experiments, each conducted in quadruplicate. *P r 0.05 as compared with non-Aa cotreated cells. #P r 0.05 as compared with non-Ap-treated cells. Dose response of NRK-52E cells following Aa treatment is shown in Supplementary Figure S2D (http://links.lww.com/ACD/A58). Ap was also subtoxic at the concentration used (see Supplementary Figure S2E, http://links.lww.com/ACD/A58). (b) Rat glomerular endothelial cells (RGE) were pretreated for 24 h with either Aa (1 mmol/l) or Ap (1 mmol/l) before CisPt was added at the concentrations indicated. After a further incubation period of 48 h, cell viability was monitored as described in the Materials and methods section. Data shown are the mean±SD from n = 3 independent experiments, each conducted in quadruplicate. *P r 0.05 as compared with non-Aa cotreated cells. #P r 0.05 as compared with non-Ap cotreated cells. (c, d) Chou–Talalay analysis of the combined effects of CisPt and Aa or Ap on the viability of NRK-52E (c) and RGE (d) cells was carried out as described in the Materials and methods section. A combination index (CI) of >1 is indicative of antagonistic activity.

subtoxic in NRK-52E cells (Supplementary Fig. S2B, Supplemental digital content 4, http://links.lww.com/ACD/ A58). Yet, as shown in Fig. 2, harpagoside did not protect against cisplatin-induced loss of viability (Fig. 2a) and DNA damage induction (Fig. 2b). Harpagoside caused a slight sensitization of NRK-52E cells to doxorubicin (Fig. 2c). The data show that the beneficial effects of H. procumbens extract against cytotoxicity and genotoxicity following cisplatin treatment are independent of harpagoside, but rather rest on other yet unknown ingredient(s) of H. procumbens.

Cytoprotective effects of natural products from marine sponges on cisplatin-induced toxicity

In a next step, the cytoprotective potency of a pool of five selected purified substances (see the Materials and methods section) isolated from marine sponges (Porifera) was investigated. For combined treatment, a concentration of 5 mmol/l was used, which showed moderate cytotoxicity in NRK-52E cells (see Supplementary Fig. S2C, Supplemental digital content 4, http://links.lww.com/ACD/A58). The ingredients of the marine sponge pool (SP) significantly increased

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924 Anti-Cancer Drugs 2014, Vol 25 No 8

Fig. 5

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Influence of aaptamine (Aa) and aeroplysinin-1 (Ap) on the sensitivity of rat kidney cells to oxaliplatin (OxaliPt). (a) Rat kidney tubular cells (NRK-52E) (b) or rat glomerular endothelial cells (RGE) were pretreated for 24 h with either Aa (2 mmol/l) or Ap (2 mmol/l) before OxaliPt was added at the concentrations indicated. After a further incubation period of 48 h, cell viability was monitored. Data shown are the mean±SD from n = 3 independent experiments, each conducted in quadruplicate. *P r 0.05 as compared with non-Aa cotreated cells. #P r 0.05 as compared with non-Ap cotreated cells. (b) Rat glomerular endothelial cells (RGE) were pretreated for 24 h with either Aa (1 mmol/l) or Ap (1 mmol/l) before OxaliPt was added at the concentrations indicated. After a further incubation period of 48 h, cell viability was monitored. Data shown are the mean±SD from n = 3 independent experiments, each conducted in quadruplicate. *P r 0.05 as compared with non-Aa cotreated cells. #P r 0.05 as compared with non-Ap cotreated cells.

Fig. 6

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Aaptamine (Aa)-mediated protection of NRK-52E cells from cisplatin (CisPt)-induced cytotoxicity. Rat kidney cells (NRK-52E) were pretreated for 24 h with different concentrations of Aa before CisPt (30 mmol/l) was added. After a further incubation period of 48 h, cell viability was monitored. Data shown are the mean±SD from n = 2–5 independent experiments, each conducted in quadruplicate. *P r 0.05 as compared with non-Aa cotreated cells.

the resistance of NRK-52E cells against cisplatin (Fig. 3a), while not influencing the sensitivity of the tubular cells toward doxorubicin (Fig. 3c). Protection toward cisplatin by SP ingredients was paralleled by at least a 50% decrease in the level of DNA damage (Fig. 3b) as analyzed 24 h after drug treatment. Taken together, the marine SP could confer specific and substantial resistance to cisplatin treatment and, hence, was subjected to further analyses.

Next, we aimed to identify the bioactive compound(s) of the SP that conferred cisplatin resistance. The SP contained equimolar concentrations of the following substances: ( – )agelasin D, (+)-agelasidin C, aeroplysinin-1, aaptamine, and aerothionin. Aeroplysinin-1 is reported to be an inhibitor of the EGF receptor [34]. Moreover, aeroplysinin-1 was found to be antiproliferative in human endothelial cells and inhibits the expression of inflammatory cytokines [35]. In addition, it is known to have antiangiogenic potency by inducing apoptosis in bovine endothelial cells [36]. Aeroplysinin-1 causes growth arrest and apoptosis also in Ehrlich ascites and HeLa tumor cells, which likely involves the formation of free radicals [37]. The spongean alkaloid aaptamine causes a p53independent induction of p21 in chronic myeloid leukemia cells [38]. A p53-independent activation of the p21 promotor by aaptamine (20–50 mmol/l) was also reported in osteosarcoma cells and is accompanied by G2/M arrest [39]. At lower concentrations, including the IC50 concentration, aaptamine induced G2/M arrest in human embryonal carcinoma NT2 cells, whereas higher concentrations resulted in the induction of apoptosis [40]. Proteome-based pathway analyses suggested that aaptamine interferes with the myc and p53 network [41] and, moreover, mediates hypusine modification of the eukaryotic initiation factor 5A [40]. Furthermore, the cytotoxic and anti-viral activity of aaptamine was related to its ability to intercalate into the DNA [42]. Agelasin D and agelasidin C are described as inhibitors of Na + /K + ATPase [43] and aerothionin as an antibacterial [37]. Bearing in mind that EGF signaling and p53/p21 are well known to influence drug resistance, we speculated that aaptamine and/or aeroplysinin-1 might be of particular relevance for mediating cisplatin resistance. To assay the

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Nephroprotection by the spongean alkaloids Funk et al. 925

Fig. 7

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Aaptamine (Aa) does not protect RT-112 bladder tumor cells against cisplatin (CisPt)-induced cytotoxicity. Human bladder carcinoma cells (RT-112) were pretreated for 24 h with either Aa (1 mmol/l) or aeroplysinin-1 (Ap) (1 mmol/l) before CisPt (a) or oxaliplatin (OxaliPt) (b) was added at the concentrations indicated. Aa and Ap treatment showed only weak cytotoxicity (decrease in viability by

Spongean alkaloids protect rat kidney cells against cisplatin-induced cytotoxicity.

Nephrotoxicity is the major dose-limiting adverse effect of cisplatin (CisPt) and results from CisPt-induced damage of tubular cells. Nephroprotective...
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