A novel role of the mitochondrial permeability transition pore in (−)-gossypol-induced mitochondrial dysfunction

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Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev

A novel role of the mitochondrial permeability transition pore in (−)-gossypol-induced mitochondrial dysfunction Verena Warnsmanna, Nina Meyerb, Andrea Hamanna, Donat Kögelb, Heinz D. Osiewacza,

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a Institute of Molecular Biosciences and Cluster of Excellence Frankfurt 'Macromolecular Complexes', Department of Biosciences, J. W. Goethe University, Max-von-LaueStr. 9, 60438 Frankfurt, Germany b Experimental Neurosurgery, Goethe University Hospital, Heinrich-Hoffmann-Str. 7, 60528 Frankfurt, Germany

A R T I C L E I N F O

A B S T R A C T

Keywords: (−)-gossypol Oxidative stress Mitochondrial permeability transition pore Mitochondrial dysfunction Cell death

Gossypol, a natural polyphenolic compound from cotton seeds, is known to trigger different forms of cell death in various types of cancer. Gossypol acts as a Bcl-2 inhibitor that induces apoptosis in apoptosis-competent cells. In apoptosis-resistant cancers such as glioblastoma, it triggers a non-apoptotic type of cell death associated with increased oxidative stress, mitochondrial depolarisation and fragmentation. In order to investigate the impact of gossypol on mitochondrial function, the mitochondrial permeability transition pore and on oxidative stress in more detail, we used the aging model Podospora anserina that lacks endogenous Bcl-2 proteins. We found that treatment with gossypol selectively increases hydrogen peroxide levels and impairs mitochondrial respiration in P. anserina, apoptosis-deficient Bax/Bak double knockout mouse embryonal fibroblasts and glioblastoma cells. Significantly, we provide evidence that CYPD-mediated opening of the mPTP is required for gossypol-induced mitochondrial dysfunction, autophagy and cell death during organismic aging of P. anserina and in glioblastoma cells. Overall, these data provide new insights into the role of the mPTP and autophagy in the antitumor effects of gossypol, a natural compound that is clinically developed for the treatment of cancer.

1. Introduction Gossypol is a polyphenolic compound from the seeds of cotton plants, which has been introduced as a male antifertility agent in animal breeding (Liu, 1957) and later found to have in vitro and in vivo antitumor activity (Dodou et al., 2005). Two enantiomers, (+)-gossypol and (−)-gossypol are known, the latter being more effective as an inhibitor of tumor growth. Various studies on cancer cells with an intact apoptosis machinery demonstrated that (−)-gossypol induces apoptotic cell death (Balakrishnan et al., 2008; Mani et al., 2015; Meng et al., 2008; Wolter et al., 2006). In contrast, in highly apoptosis-resistant cancers, like prostate cancer or glioblastoma, (−)-gossypol has been demonstrated to predominantly trigger an autophagic type of cell death (Lian et al., 2011; Voss et al., 2010). A number of studies provided evidence that mitochondria play a pivotal role in the effect of (−)-gossypol. In glioblastoma it was demonstrated that (−)-gossypol treatment leads to a massive reduction of mitochondrial membrane potential (mtMP) and fragmentation of mitochondria in the absence of prominent Bax and Bak activation and cytochrome c release (Voss et al., 2010). Studies with liver and sperm mitochondria revealed that the drug uncouples respiratory chain-linked

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phosphorylation (Abou-Donia and Dieckert, 1976; Reyes and Benos, 1988). In pancreatic cancer, (−)-gossypol leads to the loss of mitochondrial cristae (Benz et al., 1990). In addition, it has been shown that it is a potent inductor of oxidative stress (Ko et al., 2007; Xu et al., 2014). However, despite this experimental information, the knowledge about the underlying molecular mechanism resulting in (−)-gossypolinduced non-apoptoptic cell death is still limited and remains to be uncovered in detail. In literature a connection between oxidative stress, the mitochondrial permeability transition pore (mPTP) and mtMP is well established. Furthermore, reactive oxygen species (ROS) can cause a loss of mtMP by activation of the mPTP (Jacobson and Duchen, 2002). A well-known regulator of the mPTP opening is the mitochondrial peptidyl-prolylcis,trans-isomerase cyclophilin D (CYPD) (Baines et al., 2005; Basso et al., 2005; Nakagawa et al., 2005; Schinzel et al., 2005). Previous investigations indicated a role of ROS in the regulation of CYPD-induced mPTP (Linard et al., 2009). In the aging model Podospora anserina massive overexpression of PaCypD has recently been shown to lead to autophagic cell death and a reduced organismic lifespan (Brust et al., 2010; Kramer et al., 2016). Based on this existing knowledge, we hypothesized that regulation of mPTP opening by CYPD may play an

Corresponding author. E-mail address: [email protected] (H.D. Osiewacz).

http://dx.doi.org/10.1016/j.mad.2017.06.004 Received 2 March 2017; Received in revised form 7 June 2017; Accepted 30 June 2017 0047-6374/ © 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Warnsmann, V., Mechanisms of Ageing and Development (2017), http://dx.doi.org/10.1016/j.mad.2017.06.004

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essential role in (−)-gossypol-induced mitochondrial dysfunction and cell death. In order to unravel the impact of mPTP and/or CYPD on cell deathinducing mechanisms of (−)-gossypol in more detail, we analyzed (−)-gossypol-induced effects in glioblastoma cells and apoptosis-deficient Bax/Bak double knockout mouse embryonal fibroblasts (Bax/Bak KO MEFs), as well as on mitochondrial function and fitness parameters of a P. anserina CypD deletion strain. Our data uncover that (−)-gossypol-induced mitochondrial dysfunction, autophagy and cell death, as well as lifespan reduction in P. anserina requires the involvement of CYPD. Furthermore, we demonstrate that pharmacological inhibition of mPTP opening is able to rescue mitochondrial function and cell viability in glioblastoma cells treated with (−)-gossypol.

control). Prior to the experiments, the optimal end-concentration of each chemical was assessed: 1 μg/ml oligomycin, 5 μM FCCP for MEF Bax/Bak KO cells and 20 μM FCCP for U343 cells, 0.33 μM rotenone. The time period for the Seahorse protocol steps were set up according to the basal oxygen consumption of the cells. The cell number of at least three wells per condition was assessed and the average was used to normalize the data to 10,000 cells per well. Only the third measurement of basal respiration was used for statistical analysis of the data, as the cultures need to adjust to the conditions. For the other parameters, the three measurements per well were averaged. Each individual well was treated as ‘n’ of one.

2. Material and methods

In this study, the P. anserina wild-type strain ‘s’ (Rizet, 1953) and the previously generated strains PaSod1::Gfp (Zintel et al., 2010), PaSod3H26L::Gfp (Knuppertz et al., 2017), ΔPaCypD (Brust et al., 2010) and ΔPaCypD/PaSod1::Gfp (Kramer et al., 2016) were used. All transgenic strains are in the genetic background of the wild-type strain ‘s’. Strains were grown on standard cornmeal agar (BMM) at 27 °C under constant light (Osiewacz et al., 2013). For spore germination standard cornmeal agar (BMM) with 60 mM ammonium acetate was used and incubated at 27 °C in dark for 2 days. All strains used in this study were derived from monokaryotic ascospores (Osiewacz et al., 2013).

2.4. P. anserina strains and cultivation

2.1. Cell culture Mouse embryonal fibroblasts (MEF) with stable Bax/Bak double knockout (MEF Bax/Bak KO) (Wei et al., 2001) and wild-type cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; +1 g/L glucose, +L-glutamine + pyruvate). Glioma cell lines U343 and MZ-54 were cultivated in DMEM, high glucose, glutamax (+4.5 g/L glucose, +L-glutamine, −pyruvate). Both media were supplemented with 10% fetal calf serum (FCS), 1% L-glutamine and 1% Penicillin-Streptomycin solution. The cells were kept at 37 °C and 5% CO2, medium was changed every 24 h–72 h.

2.5. Growth rate and lifespan determination Determination of the lifespan and the growth rate of P. anserina cultures derived from monokaryotic ascospores was performed on M2 medium at 27 °C and constant light as previously described (Osiewacz et al., 2013). Cultures were treated with 0.02% dimethylsulfoxid (DMSO, Roth, 4720.1) and 20 μM (−)-gossypol (AT 101, Tocris, 3367), respectively, in standard M2 medium. The lifespan of P. anserina is defined as the time period in days (d) of linear hyphal growth while the growth rate is defined as the measured growth (cm) per time period (d).

2.2. Flow cytometry TMRM (ex/em maxima ∼ 553/576, Life Technologies, T668) was used for the detection of depolarized and damaged mitochondria upon treatment. Propidium iodide (PI; ex/em maxima ∼ 535/617 nm) staining was performed for the quantification of cell death. For FACS measurements, 10,000–30,000 cells were plated into 24well plates and treated with different substances: (−)-gossypol (AT 101, Tocris, 3367), A23187 (Sigma-Aldrich, C7522), TRO-19622 (Sigma-Aldrich, T3077). MEF cells were stained with 100 nM TMRM and glioma cells were stained with 50 nM TMRM 20 min before harvesting. Cell pellets were resuspended in 50 μl PBS. For PI measurements, the cell pellets were resuspended in 50 μl FACS buffer with 0.8 μl PI (50 μg/ml, Sigma-Aldrich, P4864). Measurements were performed either with FACS Canto II or FACS Accuri.

2.6. Isolation of mitochondria P. anserina strains were grown on cellophane foil covered solid M2 agar for two days at 27 °C and constant light. Mycelial pieces were transferred to CM-liquid medium and grown at 27 °C and constant light for additional two days. DMSO or (−)-gossypol treatment was carried out by adding 0.02% DMSO or 20 μM (−)-gossypol four hours before isolation. Mitochondria of P. anserina cultures were isolated as previously described by differential centrifugation for measurement of mitochondrial oxygen consumption and by discontinuous sucrose gradient (20-36-50%) ultracentrifugation for BN-PAGE analysis (Osiewacz et al., 2013).

2.3. Determination of the oxygen consumption rate of U343 and MEF Bax/ Bak KO cells The O2 consumption rate of U343 cells and MEF Bax/Bak KO cells was assessed using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience). The assay was performed in a Seahorse 24-well XF cell culture microplate in conjunction with an XF24 sensor cartridge. The day before measurement, 40,000 U343 cells or 20,000 MEF Bax/Bak KO cells were seeded into the wells to obtain a confluent cell layer after 24 h. Treatment of the cells was performed 24 h after seeding. Thereto, the cell culture medium was removed and fresh medium containing (−)-gossypol (15 μM or 30 μM) or DMSO was added. Cells were treated for 2–4 h and measurements were performed directly afterwards. One hour prior measurement the cells were placed in a non-CO2 incubator. The ports of the sensor cartridge were loaded with 10x stock solutions of the particular chemicals (the ATP synthase inhibitor oligomycin (Sigma-Aldrich, 75351), the uncoupler carbonyl-cyanide-(trifluoromethoxy)-phenylhydrazone (FCCP, Abcam, ab120081), the complex I inhibitor rotenone (Sigma-Aldrich, R8875) or DMSO as

2.7. Mitochondrial oxygen consumption Determination of mitochondrial oxygen consumption was performed at 27 °C by high-resolution respirometry (Oxygraph-2k series C and G, Oroboros Instruments, Innsbruck, Austria). 200 μg freshly prepared mitochondria were injected into 2 ml air saturated oxygen buffer (0.3 M sucrose, 10 mM KH2PO4, 5 mM MgCl2, 1 mM EDTA, 10 mM KCl, 0.1% BSA; pH 7.2). To promote the ADP-limited complex I-dependent state 4 respiration (state 4) 10 mM pyruvate (Sigma-Aldrich, P2256) and 2 mM malate (Sigma-Aldrich, M1000) were added. Subsequently, 1.5 mM ADP (Sigma-Aldrich, A5285) was added to determine complex I-dependent state 3 respiration (state 3). To inhibit the F0F1-ATP synthase 2.5 μM oligomycin (Sigma Aldrich, O4876) was added. Data were analyzed using the manufactureŕs software DatLab 6.

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2.13. ‘In-gel’ SOD activity assay

2.8. Blue-native polyacrylamide gels (BN-PAGE) and complex IV ‘in-gel’ activity assay

‘In-gel’ SOD activity assay was performed as described (Osiewacz et al., 2013).

BN-PAGE was performed as previously described (Wittig et al., 2006). For sample preparation, 100 μg of mitochondrial protein extracts were solubilized using a digitonin (Sigma-Aldrich, D141)/protein ratio of 3:1 (w/w). Linear gradient gels (4–13%) overlaid with 3.5% stacking gels were used for separation of the solubilized mitochondrial protein extracts. Respiratory chain components were visualized by Coomassie blue staining and assigned as described previously (Krause et al., 2004). Complex IV ‘in-gel’ activity was performed according to the protocol described in detail by Jung and colleagues (Jung et al., 2000). Therefore, Coomassie blue staining was omitted and the gel was incubated in 50 mM phosphate buffer (pH 7.4) containing 1 mg/ml diaminobenzidine (DAB, Sigma Aldrich, D8001), 24 U/ml catalase (Sigma Aldrich, C1345), 1 mg/ml cytochrome c (Sigma Aldrich, C2506) and 75 mg/ml sucrose (Roth, 4621.2).

2.14. ‘In-gel’ peroxidase and catalase activity assay ‘In-gel’ peroxidase and catalase activity assay was performed as described in a previously published protocol (Wayne and Diaz, 1986). 2.15. Western blot analysis Separation of 100 μg total protein extract by SDS-PAGE and following transfer of proteins to PVDF membranes (Immobilon-FL, Millipore) was performed according to standard protocols (Knuppertz et al., 2014). Blocking and antibody incubation of blotted PVDF membranes were performed in accordance to the Odyssey ‘Western Blot Analysis’ handbook (LI-COR). As primary antibody anti-GFP (mouse, 1:10000 dilution, Sigma-Aldrich, G6795), was used and as secondary antibody a conjugated IRDye CW 800 (1:15000 dilution, goat antimouse 800: 926-32210 LI-COR Biosciences) was used. For detection the Odyssey infrared scanner (LI-COR Biosciences) was used and densitometric quantification was performed with the manufacturer’s software.

2.9. Mitochondrial membrane potential measurement Oxygen consumption measurements were performed with complex I-dependent substrates as described above in the presence of tetramethylrhodamine-methyl-ester-perchlorate (TMRM, Sigma-Aldrich, T5428). Briefly, fresh mitochondria were incubated in air-saturated oxygen buffer to which 1.5 μM TMRM was injected.

2.16. Determination of free ATP ATP levels were measured by using the luciferin-luciferase assay kit (ATP Bioluminescence Assay Kit CLS II, Roche, Sigma-Aldrich: 11699695001) adapted for use in a microtiter plate format. This assay is based on the reaction: luciferin + ATP + O2 → oxyluciferin + AMP + pyrophosphate + CO2 + light. 100 mg mycelium of each sample was put into 200 μl of ATP-Isolation buffer (100 mM NaCl, 50 mM KH2PO4, pH 6) and then immediately frozen to −80% for 30 min. Samples were submersed in boiling water for 15 min to destroy ATPase activity and to allow diffusion of ATP out of the mycelium. After boiling, the samples were diluted in 300 μl water and smashed with glass beads (Homogenizer ‘Precellys24′, Peqlab) for two minutes and 5000 rpm. Afterwards, probes were additionally submersed in boiling water for 10 min and finally centrifuged (10 min, 14000 rpm). A 1:30 dilution with the supernatant was generated and the assay was performed according to the manufacturer’s instructions.

2.10. Determination of superoxide release Qualitative determination of superoxide release from mycelia was performed by monitoring reduction of nitroblue tetrazolium (NBT, Sigma-Aldrich, N6876) using a modified protocol of Munkres (1990). P. anserina strains were cultivated for 4 days on M2 agar medium in the dark and 27 °C. The plates were floated with 5 ml staining solution for superoxide (contains 5 mM MOPS pH 7.6, 2.5 mM NBT) and incubated for 30 min in the dark and 27 °C. The solution was decanted, the plates were incubated for additional 3 h in the dark and 27 °C to obtain the desired staining intensity.

2.11. Hydrogen peroxide release measurements Qualitative determination of hydrogen peroxide release from mycelia was performed by monitoring the oxidation of diaminobenzidine (DAB, Sigma-Aldrich, D-8001) according to a modified protocol of Munkres (1990). P. anserina strains were cultivated for 4 days on M2 agar medium in the dark and 27 °C. The plates were floated with 5 ml staining solution for hydrogen peroxide (contains 100 mM Tris/HCl pH 6.9, 2.5 mM DAB; dissolved at 60 °C for 10 min) and incubated for 30 min in the dark and 27 °C. The solution was decanted, the plates were incubated for additional 3 h in the dark and 27 °C to obtain the desired staining intensity. Quantitative measurement of hydrogen peroxide was performed as previously described (Kowald et al., 2012).

2.17. Fluorescence microscopy For fluorescence microscopy, the PaSod3H26L::Gfp strain was incubated for 1 d at 27 °C and constant light on a glass slide (Riddell, 1950). Subsequently, grown mycelia were treated with 0.02% DMSO or 20 μM (−)-gossypol immediately before microscopy. Hyphae were visualized and documented using a fluorescence microscope (DM LB/ 11888011, Leica, Wetzlar, Germany) equipped with the appropriate excitation and emission filters. 2.18. Statistical analysis

2.12. Isolation of total protein extract

Significances between different lifespans and growth rates were statistically analyzed using the 2-tailed Mann-Whitney-Wilcoxon U test. Statistical analysis of the FACS measurements and respirometry of MEFs and glioblastoma cells was performed using the Mann-WhitneyWilcoxon U test or the Student’s t-test. All other statistical significances were calculated using the Student's t-test. The minimum level of statistical significance was set at P ≤ 0.05. P ≤ 0.05: *, P ≤ 0.01: **, P ≤ 0.001: ***.

Isolation of total protein extract was performed as previously described (Osiewacz et al., 2013). For ‘in gel’ SOD activity assay DMSO or (−)-gossypol treatment was carried out by adding 0.02% DMSO or 20 μM (−)-gossypol four hours before isolation and for monitoring autophagy by adding 0.2% DMSO or 200 μM (−)-gossypol 24 h before isolation.

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Fig. 1. Effects of (−)-gossypol on mitochondrial depolarization of MEF wt and MEF Bax/Bak KO cells (A), U343 cells (B) and MZ-54 cells (C). Cells were treated with (−)-gossypol (15 (M) for 6 h, 24 h and 48 h. DMSO was used as control (Con). The percentage of TMRM positive cells is equivalent to the amount of mitochondria with an intact membrane potential. The threshold for TMRM positive cells was set according to the control. Measurements were performed by flow cytometry. Data are mean ± SEM from 3 independent experiments with 3–4 samples per experiment (10, 000 cells measured in each sample). *P < 0.05, **P < 0.01, ***P < 0.001 difference from the control (2-tailed Mann-Whitney-Wilcoxon U test).

15 μM (−)-gossypol for 3 h (Fig. 2E). Three hours treatment slightly intensifies the enhancing effect of (−)-gossypol on the OCR during basal respiration and ATPase inhibition. The relative increase of OCR of (−)-gossypol treated cells is slightly but significantly increased after ATPase inhibition compared to control (Fig. 2F). Taken together, mitochondrial respiration is significantly decreased upon 4 h (−)-gossypol treatment with the high concentration of 30 μM, whereas OCR is slightly increased upon treatment with 15 μM (−)-gossypol for 3 h. Collectively, these data suggest that the increase of respiration precedes the drop in OCR. In order to obtain a more detailed insight into the mechanism of (−)-gossypol-induced mitochondrial dysfunction, further analyses were performed in P. anserina, a fungal model organism with a strong mitochondrial etiology of aging and an accessibility of isolated mitochondria for biochemical analysis.

3. Results 3.1. (−)-Gossypol leads to mitochondrial depolarization and reduced mitochondrial oxygen consumption in apoptosis-deficient cells In a set of initial experiments, we investigated the effect of (−)-gossypol on mitochondrial function in apoptosis-deficient and −resistant mammalian cells (Voss et al., 2010). To this end, we used MEF wild-type and MEF Bax/Bak KO cells as well as glioblastoma cell lines U343 and MZ-54. First, we analyzed mtMP by TMRM staining. (−)-Gossypol treatment leads to a significant mitochondrial depolarization in both MEF wild-type and MEF Bax/Bak KO cells, leaving a fraction of only 20–35% of the cells with functional mitochondria after 24 h and 48 h (Fig. 1A). To confirm these results obtained in MEFs, the depolarization of mitochondria was investigated in U343 (Fig. 1B) and MZ-54 (Fig. 1C) cells. Both cell lines react with a continuous and significant decrease of mtMP upon treatment with (−)-gossypol. After 24 h, more than 80% of the cells show a loss in mitochondrial membrane potential. Next, we analyzed the oxygen consumption rate (OCR) of U343 cells and MEF Bax/Bak KO cells via respirometry with the Seahorse XF24 flux analyzer. To monitor the characteristics of mitochondrial respiration in (−)-gossypol treated MEF Bax/Bak KO cells and U343 cells, different parameters of mitochondrial respiratory chain function were measured by injection of the ATPase inhibitor oligomycin, the uncoupling agent FCCP and the complex I inhibitor rotenone to the well at certain time points. Basal respiration is strongly controlled by ATP turnover. Addition of oligomycin shifts the entire ATP production to glycolysis. The respiration rate is then controlled by the proton leak across the inner mitochondrial membrane. The maximum respiration rate can be reached by adding an uncoupling agent such as FCCP (Brand and Nicholls, 2011). The amount of oligomycin, FCCP and rotenone was titrated in preliminary experiments to determine the optimal concentration. In general, oxygen consumption in MEF Bax/Bak KO cells is lower than in U343 cells (Fig. 2A-D). (−)-Gossypol treatment strongly reduces OCR in both cell lines and inhibits the increase of oxygen consumption after addition of FCCP. To assure that the reduced OCR is not a consequence of cell death, the viability of both cell lines was measured by FACS analysis in preliminary experiments. No cell death could be observed upon 30 μM (−)-gossypol treatment for 4 h (data not shown). The next experiments were performed to determine the threshold of (−)-gossypol concentration and treatment time that must be reached for significant reduction of OCRs. Treatment with 30 μM (−)-gossypol for 2 h slightly increases OCR during basal respiration and oligomycin addition, but causes a drop of oxygen consumption during the further measuring process (Fig. S1). FCCP addition is not able to increase oxygen consumption anymore, suggesting that the mitochondria are severely damaged. In contrast, treatment with 15 μM (−)-gossypol for 2 h slightly increases the oxygen consumption rate of U343 cells during the whole measurement (Fig. S1). To find out whether this effect can be intensified by longer treatment periods, the cells were treated with

3.2. Lifespan reduction of P. anserina by (−)-gossypol is dose-dependent Since (−)-gossypol-induced cell death in glioblastoma is linked to mitochondrial depolarization, we analyzed the effect of the drug on P. anserina lifespan and growth, which are strongly affected by mitochondrial function. To exclude the possibility that DMSO, in which (−)-gossypol is dissolved, influences the (−)-gossypol impact, we first examined the effect of DMSO and found that DMSO does not influence lifespan and growth rate at the tested concentration (Fig. S2A-C). Next we investigated whether (−)-gossypol treatment affects these fitness parameters. In concordance with the induction of cell death in cancer cells, we found a lifespan reduction of P. anserina cultures on agar containing 20 μM (−)-gossypol, a concentration that does not affect the growth rate of the fungus (Fig. 3A-C). Compared to the DMSO control, (−)-gossypol treatment results in a 10% reduction of mean lifespan (Fig. 3B). Treatment with a higher dose leads to a greater reduction in lifespan of the P. anserina wild type (Fig. 3D). For instance, 200 μM (−)-gossypol reduced the mean lifespan by 25% (Fig. 3E) and the growth rate by 45% (Fig. 3F). Next, we investigated the question of whether the reduced lifespan depends on apoptotic cell death. We analyzed a P. anserina strain in which PaMCA1, one of two metacaspases involved in execution of apoptosis (Hamann et al., 2007), is ablated. In this mutant (ΔPaMca1), 20 μM (−)-gossypol had no effect on lifespan (Fig. 3G, H) and induced only a slight decrease in growth rate (Fig. 3I). These data suggest that despite the lack of Bcl-2 proteins in P. anserina and in contrast to some cancer models (Lian et al., 2011; Voss et al., 2010), the dose-dependent, (−)-gossypol-triggered cell death associated with lifespan reduction in the wild type results from increased apoptosis. 3.3. Long-term incubation of mitochondria with (−)-gossypol leads to mitochondrial dysfunction Since a link between lifespan, mitochondrial function and quality control in P. anserina and other fungi is well documented (Adam et al., 2012; Fischer et al., 2015; Francis et al., 2007; Kurashima et al., 2013; Scheckhuber et al., 2011) and since we found that (−)-gossypol impairs 4

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Fig. 2. Effects of (−)-gossypol on the oxygen consumption rate of MEF Bax/Bak KO cells and U343 cells. (A-D) Cells were treated with (−)-gossypol (30 μM) (Gossy) or DMSO (Con) for 4 h. Following parameters of mitochondrial respiratory chain function were measured: basal oxygen consumption, ATP-linked respiration, maximal respiratory capacity and non-mitochondrial respiration. Data A and C are mean ± SEM from n = 4 samples. Data B and D are 3 independent experiments with 3–4 wells per condition (n = 11-12) ((−)-gossypol treated or control) and 3 measurements per respiration parameter. (E + F) Cells were treated with (−)-gossypol (15 μM) (Gossy) or DMSO (Con) for 3 h. Data E are mean ± SEM from n = 10 samples. Data F are 3 independent experiments with 10 wells per condition ((−)-gossypol treated or control) and three measurements per respiration parameter. *P < 0.05, **P < 0.01, ***P < 0.001 difference of cells treated with (−)-gossypol and control (2-tailed Mann-Whitney-Wilcoxon U test for B and D, 1-tailed Student’s t-test for F).

respiration, a situation in which electron transport is not linked (coupled) to ATP synthesis due to proton leakage across the inner mitochondrial membrane which causes a reduced mtMP. Hence, we determined the mtMP using the fluorescent dye tetramethylrhodaminemethyl-ester-perchlorate (TMRM), a fluorescent dye that accumulates in mitochondria depending on the mtMP (Fig. 4B). In concordance with a role as an uncoupler (Abou-Donia and Dieckert, 1976; Reyes and Benos, 1988), (−)-gossypol leads to a strong reduction of the mtMP in state 4 respiration. The uncoupling of respiration upon short-term (−)-gossypol treatment is accompanied with a fragmentation of mitochondria compared to the DMSO control (Fig. S4A-B). This is consistent with earlier published data of mitochondria fragmentation preceding cell death after (−)-gossypol treatment of glioma cells (Voss et al., 2010). Uncoupling of the respiratory chain over a longer period of time is

mitochondrial function also in glioblastoma cells (Fig. 2), we next investigated the effect of (−)-gossypol on mitochondrial function in P. anserina. First, we analyzed the OCR of isolated mitochondria by highresolution respirometry. The complex I-dependent OCR was determined using mitochondria isolated from wild-type cultures grown in standard growth medium and subsequently treated with DMSO or (−)-gossypol during the measurement. Compared to the DMSO control, (−)-gossypol significantly increased state 4 (no addition of ADP) OCR but not state 3 OCR (addition of ADP) (Fig. 4A). To exclude the possibility that this effect results from the incubation of mitochondria in DMSO, OCR of mitochondria was determined after DMSO treatment and compared to the OCR of untreated mitochondria. In concordance with the fitness parameters of P. anserina cultures, DMSO has no effect on the OCR of isolated mitochondria (Fig. S3). An increase in respiration can be attributed to uncoupling of 5

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Fig. 3. Effect of (−)-gossypol on P. anserina wild-type lifespan. (A) Survival curves of P. anserina wild-type cultures grown on M2 containing 0.02% of the solvent DMSO (Con; n = 24; median lifespan=∼18 d) or 20 μM (−)-gossypol dissolved in DMSO (Gossy; n = 27; median lifespan=∼16 d; P = 0.0067). (B) Relative mean lifespans, (C) relative mean growth rates of P. anserina wild type treated with 20 μM (−)-gossypol (Gossy; n = 27) compared to 0.02% DMSO (Con; n = 24). (D) Survival curves of P. anserina wild-type cultures grown on M2 containing 0.2% of the solvent DMSO (Con; n = 10; median lifespan=∼19 d) or 200 μM (−)-gossypol dissolved in DMSO (Gossy; n = 10; median lifespan =∼14 d; P = 0.00001). (E) Relative mean lifespans, (F) relative mean growth rates of P. anserina wild type treated with 200 μM (−)-gossypol (Gossy; n = 10) compared to 0.2% DMSO (Con; n = 10). (G) Survival curves of P. anserina ΔMca1 cultures grown on M2 containing 0.02% of the solvent DMSO (Con; n = 10) or 20 μM (−)-gossypol dissolved in DMSO (Gossy; n = 10; P = 0.203). (H) Relative mean lifespans, (I) relative mean growth rates of P. anserina ΔMca1 treated with 20 μM (−)-gossypol (Gossy; n = 10) compared to 0.02% DMSO (Con; n = 10). Error bars correspond to the standard deviation and P-values were determined by 2tailed Mann-Whitney-Wilcoxon U test. *P < 0.05, **P < 0.01, ***P < 0.001.

long-term (−)-gossypol treatment. First we determined the release of superoxide anions and hydrogen peroxide by cultures treated with DMSO and (−)-gossypol, respectively (Fig. 5A-D). Superoxide anion and hydrogen peroxide release by P. anserina cultures was measured histochemically (Munkres, 1990). We observed no differences between the superoxide anion release from cultures treated with (−)-gossypol compared to DMSO controls. In contrast, we found a concentrationdependent increase of hydrogen peroxide release from (−)-gossypol treated cultures (Fig. 5B, D). To confirm the results obtained by this qualitative assay we performed a quantitative photometric analysis of hydrogen peroxide release. Consistently, in cultures treated with 20 μM (−)-gossypol a strong significant increase of hydrogen peroxide release was measured which further increased in cultures grown on media with increased (−)-gossypol concentration (Fig. 5E). Nearly five to ten times more hydrogen peroxide is released by (−)-gossypol treated cultures than DMSO treated controls. This increased release is an indirect measure of increased ROS levels in different cellular compartments including the cytoplasm and mitochondria. In the latter hydrogen peroxide can be generated by PaSOD3 in the matrix and by PaSOD1 in the intermembrane space. Subsequently, it can be translocated to the cytoplasm and across the plasmalemma out of the cell. Together with the observed reduction of the growth rate on medium containing 200 μM (−)-gossypol, this strong increase of hydrogen peroxide release is an indication for oxidative stress induction. This increased hydrogen peroxide release may be caused by an increased production or a decreased reduction of hydrogen peroxide. To

known to lead to negative consequences for mitochondrial function (Kwon et al., 2011; Nishio and Ma, 2016). We therefore analyzed the complex I-dependent OCR from mitochondria isolated from cultures grown on medium containing DMSO and (−)-gossypol, respectively. We found that this long-term treatment with (−)-gossypol indeed impairs respiration resulting in a reduction of OCR in both respiratory states (Fig. 4C). Next, we analyzed the composition of mitochondrial respiratory chain by BN-PAGE after long-term (4 h) (−)-gossypol treatment. We found that this treatment leads to an increase in mitochondrial respiratory supercomplexes (mtRSCs) S1 and S2 which consist of complexes CI1CIII2CIV1 and CI1CIII2CIV2, respectively (Fig. 4D). Previous studies revealed that mtRSC are characterized by improved respiratory electron flow (Bianchi et al., 2003; Bianchi et al., 2004; Schägger and Pfeiffer, 2000). The observed increase in mtRSCs in (−)-gossypoltreated cultures thus may be a compensation mechanism counteracting the negative effect of (−)-gossypol on respiration. 3.4. Hydrogen peroxide abundance is increased by (−)-gossypol Since it is known that mitochondrial impairments lead to increased ROS production and previously studies showed (−)-gossypol-induced oxidative stress (Ko et al., 2007; Xu et al., 2014), we next analyzed the production and the depletion of ROS, respectively. Therefore, we investigated ROS levels and the activity or the amount of selected ROS scavenging enzymes under mitochondrial damaging conditions via 6

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Fig. 4. Effect of (−)-gossypol on P. anserina wild-type mitochondria. (A) Complex I-dependent oxygen consumption rate (OCR) of wild-type mitochondria which were directly treated with 0.02% DMSO (Con) or 20 μM (−)-gossypol (Gossy) during measurement (5 different mitochondrial preparations = biological replicates with a total number of 15–20 technical replicates). State 4 OCR of DMSO treated mitochondria (Con) was set to 100%. (B) Mitochondrial membrane potential (mtMP) determined by the mtMP-dependent accumulation of TMRM in the mitochondria (2 biological replicates with a total number of 6 technical replicates). mtMP during state 4 OCR of mitochondria from DMSO treated cultures was set to 100%. (C) Complex I-dependent oxygen consumption rate (OCR) of wild-type mitochondria from cultures treated 4 h with 0.02% DMSO (Con) or 20 μM (−)-gossypol (4 different mitochondrial preparations = biological replicates with a total number of 11–17 technical replicates). State 4 OCR of mitochondria from DMSO treated cultures was set to 100%. (D) Representative BN-PAGE analysis of isolated mitochondria from 4 h treated with 20 μM (−)-gossypol compared to untreated wild-type cultures (each tree biological replicates). The CI1CIII2CIV0–2 (S0–2) supercomplexes, dimeric complexes III and V (III2 and V2) as well as monomeric complexes I1, IV1 and V1 were visualized by Coomassie staining. (E) Complex IV staining. Error bars correspond to the standard deviation and P-values were determined by 2-tailed Student’s t-test test. *P < 0.05, **P < 0.01, ***P < 0.001.

3.5. Autophagy induction by (−)-gossypol is cyclophilin D-dependent

discriminate between these possibilities we next investigated the activity of the superoxide dismutase isoforms of P anserina, using an ‘ingel’ staining method. In comparison to the DMSO treated cultures, we found an increase in cytosolic PaSOD1 activity in cultures treated with 20 μM (−)-gossypol (Fig. 6A). Consistent to previous finding that PaSOD1 is regulated post-translational (Borghouts et al., 2002; Wiemer and Osiewacz, 2014), we found no significantly differences in the protein amount of PaSOD1 upon (−)-gossypol treatment compared to the control (Fig. 6B, C). In contrast, PaSOD2 activity, a secreted SOD of P. anserina (Zintel et al., 2010; Fig. 6A) and of PaSOD3 located in the mitochondrial matrix (not shown) did not change. Another possible route of reducing superoxide anion abundance is scavenging by the mitochondrial protein cytochrome c (PaCYTc). Analysis of the amount of this mitochondrial protein revealed no significant differences in (−)-gossypol treated and control strains (Fig. 6D, E). Next, we analyzed the reduction of hydrogen peroxide levels by different scavenging enzymes. Analysis of peroxidase activity by ‘in-gel’ staining indicated no significantly difference between (−)-gossypol treated and control strains (Fig. 7A). Also, no significant differences were found for catalase activity (Fig. 7B) and peroxiredoxin protein abundance (Fig. 7C-D) in (−)-gossypol treated and in control strains, respectively. Overall, these data demonstrate that PaSOD1 and not peroxidases, catalases or peroxiredoxin leads to increased hydrogen peroxide abundance in (−)-gossypol treated P. anserina strains.

Since hydrogen peroxide is known to be a potent inducer of autophagy in mammalian cells (Lee et al., 2012) and since (−)-gossypol induces autophagic cell death in glioma cells (Voss et al., 2010) we next analyzed the role of autophagy in (−)-gossypol-induced cell death of the fungus. First, we investigated the effect of (−)-gossypol in an autophagy deficient mutant (ΔPaAtg1). Similar to what we have found in the wild type, 20 μM (−)-gossypol reduced the lifespan of ΔPaAtg1 (Fig. 8A, B), demonstrating that the lifespan reduction of P. anserina by (−)-gossypol is not autophagy dependent. Next, we analyzed whether (−)-gossypol can induce autophagy in P. anserina. For visualisation of autophagy, we used a recently established biochemical assay (Knuppertz et al., 2014). In this assay, the vacuolar degradation of the cytoplasmic reporter protein PaSOD1::GFP is examined. If general autophagy is induced, the SOD1 part of the PaSOD1::GFP fusion reporter protein is degraded, while the GFP part (‘free GFP’) remains stable in the vacuole and can be quantified by western blot analysis with a GFP antibody. First, we investigated autophagy in (−)-gossypol treated P. anserina wild type expressing PaSod1::Gfp. We found no significantly differences in the ‘free GFP’ in strains treated with 20 μM (−)-gossypol compared to control strains (Fig. 8C, E), demonstrating no induction of autophagy. In order to

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Fig. 5. Effect of (−)-gossypol on P. anserina ROS level. (A + C) Qualitative determination of superoxide in P. anserina wild-type cultures (n = 4) by NBT staining. (B + D) Qualitative determination of hydrogen peroxide in P. anserina wild-type cultures (n = 4) by DAB staining. (E) Quantitative measurement of H2O2 release from P. anserina wild-type cultures treated with 0.02% DMSO (Con) or 20 μM or 200 μM (−)-gossypol (Gossy) (5 different cultures = biological replicates with 3 technical replicates). The DMSO treated wild type was set to 1. Error bars correspond to the standard deviation and P-values were determined by two-tailed Student's t test (C, E, H). **P < 0.01.

Recently it has been shown that PaCYPD plays a role in autophagy induction in P. anserina (Kramer et al., 2016). Therefore, we next analyzed whether PaCYPD plays a role in (−)-gossypol-dependent autophagy induction. In striking contrast to the wild type, the amount of

check whether autophagy induction in P. anserina is possible by higher concentrations of (−)gossypol, we increased (−)-gossypol to 200 μM and found a slight, 2-fold increase of ‘free GFP’ compared to the control (Fig. 8C, E). 8

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Fig. 6. Effect of (−)-gossypol on P. anserina superoxide anion scavenging enzymes. (A) Representative ‘in-gel’ superoxid dismutases (PaSOD1 and PaSOD2) activity staining from total potein extracts of P. anserina wild-type cultures treated 4 h with 0.02% DMSO (Con) or 20 μM (−)-gossypol (Gossy). (B) Western blot analysis of total protein extract of P. anserina wild type cultures treated 4 h with 0.02% DMSO (Con) or 20 μM (−)-gossypol (Gossy) (each three biological replicates). (C) Quantification of PaSOD1 protein level normalized to the Coomassie stained gel. Protein level in DMSO treated cultures was set to 1. (D) Western blot analysis of mitochondrial protein extract of P. anserina wild-type cultures treated 4 h with 0.02% DMSO (Con) or 20 μM (−)-gossypol (Gossy) (each three biological replicates). (E) Quantification of PaCYTc protein level normalized to the Coomassie stained gel. Protein level in DMSO treated cultures was set to 1.

‘free GFP’ was only slightly and not significantly increased in the in a PaCypD deletion mutant after (−)-gossypol treatment (Fig. 8D, F) demonstrating a crucial role of PaCYPD in the induction of autophagy by (−)-gossypol.

revealed a diminished inhibitory effect of oligomycin on F0F1-ATP synthase after (−)-gossypol treatment of isolated mitochondria (Fig. 9A). This effect may be related to a role of the F0F1-ATP synthase as a component of the mPTP (Bernardi, 2013; Giorgio et al., 2009; Giorgio et al., 2013) which is regulated by CYPD. To address this possibility experimentally, we investigated the effect of (−)-gossypol in a PaCypD deletion mutant (ΔPaCypD). First, we determined the lifespan of the PaCypD deletion mutant treated with DMSO and (−)-gossypol, respectively. Strikingly, in comparison to the wild type, the lifespan of ΔPaCypD is not affected by (−)-gossypol treatment (Fig. 9B). In contrast to the observed (−)-gossypol-dependent reduction of mean lifespan of the wild type, it does not significantly differ in the mutant treated with (−)-gossypol in comparison

3.6. The mitochondrial permeability transition pore is required for (−)-gossypol-induced cell death and mitochondrial dysfunction Together with this role of PaCYPD and the fact that hydrogen peroxide induces the CYPD-mediated opening of the mPTP (Baines et al., 2005; Nguyen et al., 2011), a role of PaCYPD in (−)-gossypol-induced cell death and mitochondrial dysfunction is likely. Indeed, OCR measurements

Fig. 7. Effect of (−)-gossypol on P. anserina hydrogen peroxide scavenging enzymes. (A) Representative ‘in-gel’ peroxidase activity staining from total potein extracts of P. anserina wild type cultures treated 4 h with 0.02% DMSO (Con) or 20 μM (−)-gossypol (Gossy). (B) Representative ‘ingel’ catalases activity staining from total potein extracts of P. anserina wild-type cultures treated 4 h with 0.02% DMSO (Con) or 20 μM (−)-gossypol (Gossy). (C) Western blot analysis of mitochondrial protein extract of P. anserina wild-type cultures treated 4 h with 0.02% DMSO (Con) or 20 μM (−)-gossypol (Gossy) (each three biological replicates). (D) Quantification of PaPRX protein level normalized to the Coomassie stained gel. Protein level in DMSO treated cultures was set to 1.Error bars correspond to the standard deviation and P-values were determined by two-tailed Student's t test (C, E, H).

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Fig. 8. Effect of (−)-gossypol on autophagy in P. anserina. (A) Survival curves of P. anserina ΔAtg1 cultures grown on M2 containing 0.02% of the solvent DMSO (Con; n = 12) or 20 μM (−)-gossypol dissolved in DMSO (Gossy 20 μM; n = 12; P = 0.0089). (B) Relative mean lifespan of P. anserina ΔAtg1 treated with 20 μM (−)-gossypol (Gossy 20 μM; n = 12) compared to 0.02% DMSO (Con; n = 12). (C) Monitoring autophagy by western blot analysis of total protein extract from PaSod1::Gfp cultures treated with 0.02% DMSO, 0.2% DMSO, 20 μM or 200 μM (−)-gossypol with a GFP-antibody (3 biological replicates). (D) Monitoring autophagy by western blot analysis of total protein extract from ΔPaCypD/PaSod1::Gfp cultures treated with 0.2% DMSO or 200 μM (−)-gossypol compared with a GFP-antibody (3 biological replicates). (E) Quantification of ‘free GFP’ protein level in total protein extract from PaSod1::Gfp cultures normalized to the Coomassie stained gel. Protein level in DMSO treated cultures were set to 1. (F) Quantification of ‘free GFP’ protein level in total protein extract from ΔPaCypD/PaSod1::Gfp cultures normalized to the Coomassie stained gel. Protein level in DMSO treated cultures was set to 1. Error bars correspond to the standard deviation and P-values were determined by two-tailed Mann-Whitney-Wilcoxon U test (B) and by two-tailed Student's t test (E + F). **P < 0.01, ***P < 0.001.

positive control for mPTP opening. Furthermore, quantification of cell death revealed that olesoxime significantly rescues cell viability of U343 cells after (−)-gossypol treatment (Fig. 10B), indicating that mPTP opening is contributing to (−)-gossypol-induced cell death. In addition, inhibition of autophagy with the phosphoinositide (PI) 3-kinase inhibitor 3-methyladenine significantly decreases (−)-gossypol induced cell death, suggesting an autophagy dependent form of cell death (Fig. 10C). Of note, 3-MA mimicked the cell death-inhibiting effects of the mPTP inhibitor olesoxime, suggesting a functional link between mPTP, autophagy and cell death in glioma cells. Overall, our data demonstrate that the (−)-gossypol-induced cell death in both P. anserina and mammalian cells is linked to mitochondrial dysfunction and the mPTP. The mPTP and CYPD as a key regulator of this protein complex emerge as new players in cancer development (Bigi et al., 2016) and antitumor therapy, consistent with the concept that the mtPTP may represent a selective target carrying the potential to bypass the apoptosis resistance mechanisms of cancer (Suh et al., 2013).

to DMSO controls (Fig. 9C), demonstrating that the reduced lifespan of (−)-gossypol treated P. anserina wild type requires PaCYPD. Next, we analyzed the effect of short-term and long-term (−)-gossypol treatment on mitochondria from ΔPaCypD. Similar to the wild type, we observed that short-term treatment of the mutant with (−)-gossypol leads to uncoupling of mitochondria (Fig. 9D). In contrast, long-term treatment with (−)-gossypol has no effect on state 4 and state 3 OCR of mitochondria from the deletion mutant (Fig. 9E). This suggests a mechanistic role of PaCYPD and the mPTP in (−)-gossypol-induced lifespan reduction and mitochondria impairments, which may subsequently lead to either apoptotic (P. anserina) or non-apoptotic cell death (glioblastoma, Bax/Bak-deficient MEFs). To analyze the hypothesis that this functional connection is also observed in mammalian cells, we investigated the role of the mPTP in (−)-gossypol treated glioblastoma cells (Fig. 10). Indeed, we found that the mPTP inhibitor olesoxime (TRO-19622) (Bordet et al., 2007) is able to significantly reduce mitochondrial depolarization upon treatment with (−)-gossypol (Fig. 10A). Olesoxime also decreases mitochondrial depolarization induced by the calcium ionophor A23187, which was used as a 10

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Fig. 9. Effect of (−)-gossypol on lifespan and mitochondrial function of a P. anserina CypD deletion strain. (A) Complex I-dependent oxygen consumption rate (OCR) of wild-type mitochondria, which were directly treated with 0.02% DMSO (Con) or 20 μM (−)-gossypol (Gossy) during measurement. State 4 OCR of DMSO treated mitochondria (Con) was set to 100%. (B) Survival curves of P. anserina ΔCypD cultures grown on M2 containing 0.02% of the solvent DMSO (Con; n = 40; median lifespan= ∼ 19 d), or 20 μM (−)-gossypol dissolved in DMSO (Gossy; n = 40; median lifespan= ∼ 20 d). (C) Relative mean lifespans of P. anserina ΔCypD treated with 0.02% DMSO (Con; n = 40) or 20 μM (−)-gossypol (Gossy; n = 40) compared to untreated wild type (n = 40, set to 100%). (D) Complex I-dependent oxygen consumption rate (OCR) of ΔPaCypD mitochondria treated with 0.02% DMSO (Con) or 20 μM (−)-gossypol (Gossy) compared to untreated mitochondria (3 different mitochondrial preparations = biological replicates with a total number of 11 technical replicates). State 4 OCR of untreated mitochondria was set to 100%. (E) Complex I-dependent oxygen consumption rate (OCR) of ΔPaCypD mitochondria from cultures treated 4 h with 0.02% DMSO (Con) or 20 μM (−)-gossypol compared to mitochondria from untreated cultures (3 different mitochondrial preparations = biological replicates with a total number of 11 technical replicates). State 4 OCR of mitochondria from untreated cultures was set to 100%. Error bars correspond to the standard deviation and P-values were determined by 2-tailed Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Fig. 10. Effect of olesoxime (TRO-19622) on mitochondrial depolarization and cell death of (−)-gossypoltreated U343 cells. (A) For TMRM measurements the cells were pre-treated with TRO-19622 (5 μM) and after 15 min, (−)-gossypol (30 μM) or A23187 (20 μM) was added for 3 h and 2 h respectively. The percentage of TMRM positive cells is equivalent to the amount of mitochondria with an intact membrane potential. The threshold for TMRM positive cells was set according to the control. (B) Cell death was measured by PI staining. Cells were pre-treated with TRO-19622 (2 μM) and (−)-gossypol (15 μM) was added after 24 h for additional 48 h. Measurements were performed by flow cytometry. (C) Cells were pre-treated with 3-methyladenine (3-MA, 2 mM) for 2 h, then (−)-gossypol was added for additional 48 h. Cell death was measured by PI staining. Data are mean ± SEM from 3 independent experiments with 3–4 samples per experiment (10,000 cells measured in each sample). *P < 0.05, **P < 0.01, ***P < 0.001 difference of cells treated with (−)-gossypol+/− TRO19622 or A23187+/− TRO-19622 (1-tailed MannWhitney-Wilcoxon U test).

4. Discussion

Here we report that CYPD-mediated opening of the mPTP is required for (−)-gossypol-induced mitochondrial dysfunction, the induction of autophagy, cell death and organismic aging. We started our work with investigating the (−)-gossypol effect on mitochondrial function in apoptosis-deficient cells. In line with previously published findings (Voss et al., 2010), our data demonstrated a reduced mtMP in MEF Bax/Bak KO cells and glioblastoma cells after (−)-gossypol treatment. This (−)-gossypol-induced reduction of the mtMP is well consistent with what has been previously reported for other polyphenols in different cancers, like quercetin, resveratrol and green tea

Mitochondria are eukaryotic organelles with fundamental pro-survival and pro-death functions. They are essential for cellular energy transduction and homeostasis, but also for sensing danger signals and the regulation of different types of cell death. Therefore, mitochondria are key targets for cancer therapy. Gossypol is a natural polyphenolic compound and BH3 mimetic that is capable to induce either apoptosis or autophagic cell death and is currently investigated as an anticancer drug. 11

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Most importantly, in the current analysis we found that the induction of autophagy by (−)-gossypol is dependent on the mPTP regulator CYPD. This is consistent with previous results in P. anserina, which suggest that PaCYPD plays a functional role in activating autophagy in response to oxidative stress (Kramer et al., 2016). Concordantly, we found in P. anserina a concentration-dependent increased release of hydrogen peroxide from mycelia after (−)-gossypol treatment accompanied with a strong reduction of growth rate. This indicates an increased cellular level of this ROS and assumes an induction of oxidative stress, which is known to induce the CYPD-mediated opening of the mPTP (Baines et al., 2005; Nguyen et al., 2011). The assumed induction of oxidative stress by (−)-gossypol correlates with findings observed in mammalian cell cultures (Ko et al., 2007; Xu et al., 2014). In addition to autophagy, we found that the (−)-gossypol-induced mitochondrial dysfunction and lifespan reduction are also CYPD-dependent. Moreover, in glioblastoma cells we could demonstrate that the mPTP inhibitor TRO-19622 rescues mitochondrial function and cell viability upon (−)-gossypol treatment confirming a role of mPTP opening in glioma cell death. Overall, the results of the current study identified an evolutionary conserved cellular effect of gossypol as an uncoupler of mitochondrial respiration that induces cellular responses to compensate impairments in mitochondrial function. These attempts are however not efficient enough to prevent mitochondrial induced cell death leading to a reduced lifespan of P. anserina. Most importantly, the described effects of (−)-gossypol depend on the induction of mPTP opening in P. anserina and glioblastoma cells. This new impact of CYPD and the mPTP identifies them as suitable targets of components like (−)-gossypol and warrants further exploration of strategies aimed at exploiting mPTPdependent cell death for the development of novel cancer therapies.

extract (Mahyar-Roemer et al., 2001; Mouria et al., 2002; Qanungo et al., 2005). It also demonstrates that this drug can bypass Bax/Bakdependent activation of the intrinsic apoptosis pathway to induce mitochondrial dysfunction and ensuing cell death. The mtMP is the result of electron transport processes during respiration and the transport of protons across the inner mitochondrial membrane. Consistently, we found that the reduction of the mtMP by (−)-gossypol treatment of MEF Bax/Bak KO and glioblastoma cells is linked to a reduced OCR. Such an effect of (−)-gossypol treatment on the OCR of isolated mitochondria was also observed in P. anserina. Furthermore, the effect of (−)-gossypol on OCR in all three biological systems of our study is dose- and time-dependent and corresponds to the early recognition that gossypol acts as an uncoupler of respiration (Abou-Donia and Dieckert, 1976; Reyes and Benos, 1988). These effects are also linked to reduction of the P. anserina wild-type lifespan and growth rate which corresponds to increasing (−)-gossypol concentrations. Unexpectedly, despite the observed increase in mtRSCs, mitochondria of (−)-gossypol treated cultures from the P. anserina wild type, in which an improved electron flow (Bianchi et al., 2003; Bianchi et al., 2004; Schägger and Pfeiffer, 2000) and reduced ROS generation (Ghelli et al., 2013; Indo et al., 2007; Lenaz et al., 2010; Maranzana et al., 2013) should occur, we found a decline in OCR and increased ROS. It appears, that the induction of mtRSCs, as a cellular stress response, is not efficient enough to counteract the strong (−)-gossypol induced impairments of the respiratory chain. Another line of compensation may be a comparatively moderate induction of autophagy, which is known to act in a pro-survival manner in P. anserina (Knuppertz et al., 2014). This hypothesis is supported by evidence demonstrating that (−)-gossypol simultaneously triggers apoptosis and a cytoprotective form of autophagy in several cancer models including breast and bladder cancer (Antonietti et al., 2016; Gao et al., 2010; Mani et al., 2015). Furthermore, an induction of pro-survival autophagy has also been reported for other polyphenols. For instance, in P. anserina it was demonstrated that the lifespan extension by curcumin depends on autophagy induction (Warnsmann and Osiewacz, 2016). In C. elegans the resveratrol-induced lifespan extension is mediated by autophagy (Morselli et al., 2010). Despite these observations, it should be emphasized that the effects of autophagy on cell death are highly dependent on the cellular context, as both pro-survival and pro-death functions of autophagy have been repeatedly reported in mammalian cells (Fulda and Kögel, 2015; Marino et al., 2014) and lower organisms such as C. elegans, Drosophila melanogaster and Dictyostelium discoideum (Berry and Baehrecke, 2007; Calvo-Garrido et al., 2010; Samara et al., 2008). It was proposed early that the extent and duration of autophagy is a major determinant in the switch between pro-survival and pro-death autophagy (Levine, 2007). This dual function of autophagy in cell death is also observed in P. anserina (Knuppertz et al., 2014; Kramer et al., 2016). In fact, unphysiological overexpression of PaCypD was associated with induction of autophagic cell death in the latter study, consistent with the hypothesis that dysregulation of the mPTP may trigger a pro-death form of autophagy in certain settings. Similar to the apoptosis-deficient ΔPaMca1 mutant, the ΔPaCypD deletion mutant did not show significant changes in lifespan after (−)-gossypol treatment. Since deletion of PaCYPD prevents both mPTP opening and drug-induced autophagy in parallel, it is not possible to distinguish whether the reduction of (−)-gossypol-induced cell death during organismic aging (in comparison to the wild type) can be attributed exclusively to the lack of mPTP opening or in part also to the lack of autophagy induction. Still, our data clearly demonstrate that in P. anserina, (−)-gossypol-induced opening of the mPTP is associated with downstream activation of apoptotic cell death. In contrast, the lack of (−)-gossypol-induced apoptosis induction in glioblastoma cells may be attributed to alternative resistance mechanisms such as overexpression of the anti-apoptotic co-chaperone BAG3 (Antonietti et al., 2017).

Acknowledgements This work was supported by grants of the Deutsche Forschungsgemeinschaft through SFB 1177 to DK and HDO and by the LOEWE excellence initiative (project: Integrated Fungal Research) of the state of Hesse (Germany) to HDO. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mad.2017.06.004. References Abou-Donia, M.B., Dieckert, J.W., 1976. Gossypol: uncoupling of respiratory chain and oxidative phosphorylation. Life Sci. 14, 1955–1963. Adam, C., Picard, M., Dequard-Chablat, M., Sellem, C.H., Hermann-Le Denmat, S., Contamine, V., 2012. Biological roles of the Podospora anserina mitochondrial Lon protease and the importance of its N-domain. PLoS One 7, e38138. Antonietti, P., Gessler, F., Düssmann, H., Reimertz, C., Mittelbronn, M., Prehn, J.H., Kögel, D., 2016. AT-101 simultaneously triggers apoptosis and a cytoprotective type of autophagy irrespective of expression levels and the subcellular localization of BclxL and Bcl-2 in MCF7 cells. Biochim. Biophys. Acta 1863, 499–509. Antonietti, P., Linder, B., Hehlgans, S., Mildenberger, I.C., Burger, M.C., Fulda, S., Steinbach, J.P., Gessler, F., Rödel, F., Mittelbronn, M., Kögel, D., 2017. Interference with the HSF1/HSP70/BAG3 pathway primes glioma cells to matrix detachment and BH3 mimetic-induced apoptosis. Mol. Cancer Ther. 16, 156–168. Baines, C.P., Kaiser, R.A., Purcell, N.H., Blair, N.S., Osinska, H., Hambleton, M.A., Brunskill, E.W., Sayen, M.R., Gottlieb, R.A., Dorn, G.W., Robbins, J., Molkentin, J.D., 2005. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434, 658–662. Balakrishnan, K., Wierda, W.G., Keating, M.J., Gandhi, V., 2008. Gossypol, a BH3 mimetic, induces apoptosis in chronic lymphocytic leukemia cells. Blood 112, 1971–1980. Basso, E., Fante, L., Fowlkes, J., Petronilli, V., Forte, M.A., Bernardi, P., 2005. Properties of the permeability transition pore in mitochondria devoid of Cyclophilin D. J. Biol. Chem. 280, 18558–18561. Benz, C.C., Keniry, M.A., Ford, J.M., Townsend, A.J., Cox, F.W., Palayoor, S., Matlin, S.A., Hait, W.N., Cowan, K.H., 1990. Biochemical correlates of the antitumor and antimitochondrial properties of gossypol enantiomers. Mol. Pharmacol. 37, 840–847. Bernardi, P., 2013. The mitochondrial permeability transition pore: a mystery solved?

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