Alterations in the mitochondrial responses to PENAO as a mechanism of resistance in ovarian cancer cells

Gynecologic Oncology 138 (2015) 363–371

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Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno

Alterations in the mitochondrial responses to PENAO as a mechanism of resistance in ovarian cancer cells Stéphanie Decollogne a, Swapna Joshi a, Sylvia A. Chung b, Peter P. Luk a, Reichelle X. Yeo a, Sheri Nixdorf b, André Fedier c, Viola Heinzelmann-Schwarz c, Philip J. Hogg a,1, Pierre J. Dilda a,⁎,1 a b c

Tumour Metabolism Group, Adult Cancer Program, Lowy Cancer Research Centre and Prince of Wales Clinical School, University of New South Wales, Sydney, NSW 2052, Australia Neuro Oncology Group, Adult Cancer Program, Lowy Cancer Research Centre and Prince of Wales Clinical School, University of New South Wales, Sydney, NSW 2052, Australia Ovarian Cancer Group, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland

H I G H L I G H T S • PENAO displays anti-proliferative activity on various ovarian cancer cells. • HO-1 induction and metabolism shift provide resistance towards PENAO. • PENAO and mTOR inhibitor synergise to reverse drug resistance.

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Article history: Received 27 February 2015 Received in revised form 10 June 2015 Accepted 12 June 2015 Available online 14 June 2015 Keywords: Ovarian cancer Mitochondria Arsenic-based cancer drugs PENAO Oxidative stress Drug resistance Metabolism mTOR signalling pathway

a b s t r a c t Objective. The purpose of this study was to test PENAO, a promising new organoarsenical that is in phase 1 testing in patients with solid tumours, on a range of ovarian cancer cell lines with different histotypes, and to understand the molecular basis of drug resistance exhibited by the endometrioid ovarian cancer cell line, SKOV-3. Methods. Proliferation arrest and cell death induced by PENAO in serous (OVCAR-3), endometrioid (SKOV-3, TOV112D), clear cell (TOV21G) and mucinous (EFO27) ovarian cancer cells in culture, and anti-tumour efficacy in a murine model of SKOV-3 and OVCAR-3 tumours, were measured. Cells were analysed for cell cycle arrest, cell death mechanisms, reactive oxygen species production, mitochondrial depolarisation, oxygen consumption and acid production. Results. PENAO demonstrated promising anti-proliferative activity on the most common (serous, endometrioid) as well as on rare (clear cell, mucinous) subtypes of ovarian cancer cell lines. No crossresistance with platinum-based drugs was evident. Endometrioid SKOV-3 cells were, however, shown to be resistant to PENAO in vitro and in a xenograft mouse model. This resistance was due to an ability to cope with PENAO-induced oxidative stress, notably through heme oxygenase-1 induction, and a shift in metabolism towards glycolysis. The adaptive glycolytic shift in SKOV-3 was targeted using a mTORC1 inhibitor in combination with PENAO. This strategy was successful with the two drugs acting synergistically to inhibit cell proliferation and to induce cell death via apoptosis and autophagy. Conclusion. Mitochondria/mTOR dual-targeting therapy may constitute a new approach for the treatment of recurrent/resistant forms of epithelial ovarian cancer. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Epithelial ovarian cancer (EOC) is the leading cause of gynaecologic cancer death among developed nations with the majority of women presenting with advanced-stage disease and 5-year survival rates of about 20%. While EOC is generally sensitive to platinum–paclitaxel combination chemotherapy [1], the majority of patients will relapse with a ⁎ Corresponding author. E-mail address: [email protected] (P.J. Dilda). 1 Co-senior authors.

http://dx.doi.org/10.1016/j.ygyno.2015.06.018 0090-8258/© 2015 Elsevier Inc. All rights reserved.

median time-to-recurrence of about one year. A significant number of tumour recurrences become resistant to platinum agents, which significantly hinders successful treatment outcomes. As the prognosis for patients with recurrent/resistant forms of EOC is poor, new therapeutic avenues avoiding known molecular mechanisms of resistance are urgently required. Many cancer cells, including ovarian carcinoma cells, exhibit increased glycolysis in comparison with normal cells. This shift in glucose metabolism in mitochondria competent cells is known as the Warburg effect or aerobic glycolysis [2]. Despite this shift, mitochondria supply anywhere from 40 to 75% of the cellular ATP requirements meaning

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that disrupting oxidative phosphorylation remains a valid strategy to significantly interrupt the cancer cell's energy supply [3]. With the discovery of the remarkable efficacy of arsenic trioxide for the treatment of acute promyelocytic leukaemia (APL) this metalloid has been further investigated for its efficacy in other cancers [4]. As a result, and because of their better toxicity profile compared to inorganic derivatives, new organic arsenicals are currently being investigated for the treatment of various cancers. PENAO, (4-(N-(Spenicillaminylacetyl)amino) phenylarsonous acid), a cysteine mimetic analogue of the cysteine-S-conjugate metabolite of GSAO [5–7], has been investigated. PENAO inactivates adenine nucleotide translocase (ANT) of the inner-mitochondrial membrane by cross-linking the sulphur atoms of cysteine residues 57 and 257 on the matrix face of the protein [8,9]. ANT is the most abundant protein found on the inner mitochondrial membrane and it is responsible for the exchange of matrix ATP for cytosolic ADP across the inner mitochondrial membrane. Disruption of its function has been shown to have major impacts on mitochondrial integrity and cell survival [5,10]. PENAO selectively induces proliferation arrest in a variety of proliferative endothelial and cancer cell lines [8,11]. Interestingly, PENAO has a strong anti-proliferative activity against glioblastoma cell lines [12,13] and primary isolates of diffuse intrinsic pontine glioma [14]. In vivo, PENAO demonstrated preclinical activity without signs of toxicity in murine tumour models of glioblastoma [15] and pancreatic carcinoma [8]. Large scale animal toxicity studies demonstrated that PENAO was well tolerated. It is currently being tested in a phase 1 clinical trial in patients with solid tumours refractory to standard therapy. This study addressed the question of how PENAO interferes with the proliferation of various types of ovarian carcinoma cells. We show here that PENAO induces an oxidative stress and interferes at low micromolar range with the proliferation of ovarian carcinoma cell lines established from four histological subtypes. One endometrioid ovarian carcinoma cell line (SKOV-3) was found to be resistant towards PENAO in an in vitro proliferation assay and in a tumour mouse model. When compared to a sensitive cell line (OVCAR-3), we show that the observed resistance is not due to a higher expression of multidrug resistance associated proteins. Rather, we found that heme oxygenase-1 is strongly induced in SKOV-3 cells which may contribute to resistance against PENAO-induced oxidative stress. SKOV-3 mitochondria produced less superoxide ions and were subsequently less sensitive to depolarisation than mitochondria from OVCAR-3 cells. Importantly, and in contrast to PENAO sensitive cells, we found that SKOV-3 cells shift their glucose metabolism from oxidative phosphorylation to glycolysis. Moreover, inhibition of the mTOR pathway, known to decrease glycolytic metabolism, reverses SKOV-3 resistance and synergizes with PENAO to block cell proliferation. These findings indicate that the resistance towards PENAO activity results from their ability to switch their metabolism to glycolysis and warrant further studies involving combinations with therapeutics that modulate glycolytic metabolism.

and TOV112D (endometrioid ovarian cancer (16)) cells were cultured in DMEM containing 15% v/v foetal bovine serum and 2 mM glutamine. EFO-27 (Mucinous ovarian cancer [17], DSMZ, Germany) cells were cultured in DMEM containing 20% v/v foetal bovine serum and 2 mM glutamine. CH-1 and CH-1 CisR cells (ovarian adenocarcinoma) were cultured in DMEM containing 10% v/v foetal bovine serum and 2 mM glutamine. MRC5 cells were cultured in MEM medium supplemented with 1 mM sodium pyruvate, 1% nonessential amino acids (NEAA), and 1.5 g/L sodium bicarbonate. HOSE 17.1 cells (normal ovarian surface epithelium) were grown in 1:1 Medium 199:MCDB 105. MCF 10A cells (non-cancerous breast cells) were grown in 1:1 DMEM:F12 supplemented with epidermal growth factor (20 μg/mL), hydrocortisone (1 mg/mL), insulin (4 mg/mL) and horse serum (10%). Normal human astrocytes (Lonza, Mount Waverley, Australia) were cultured in astrocyte growth medium (AGM™, Lonza, Mount Waverley, Australia). HCT1161ch3 and HCT1161ch2 were obtained from Dr. M. Koi [18]. Both cell lines as well as the parental HCT116 cells were maintained in Iscove's modified Dulbecco's medium (Life Technologies, Basel, Switzerland) supplemented with 2 mM L-glutamine and 10% foetal bovine serum. 2.3. Proliferation assay SKOV-3, OVCAR-3, CH-1, CH-1CisR and EFO27 cells were seeded at a density of 4 × 103 cells/well in 96-well plates. TOV112D and TOV-21G cells were seeded at a density of 2 × 103 cells/well in 96-well plates. HOSE17.1, MCF10A, MRC5 cells as well as normal astrocytes were seeded at a density of 2 × 104 cells/well in 96-well plates. Cells were allowed to adhere for 24 h at 37 °C in a 5% CO2, 95% air atmosphere and then treated with drugs (see figure legends for details) for 72 h. Viable cells were determined using the vital dye, MTT, according to the manufacturer's instructions. 2.4. In vivo studies Female BALB/c nude mice, 6–8 weeks old were injected subcutaneously in the proximal midline with 5 × 106 SKOV-3 or OVCAR-3 tumour cells in Matrigel™ (BD Biosciences). Mice bearing established ~ 100 mm3 tumours were randomized into groups of 7–9 and microosmotic pumps (Alzet model 2002, Cupertino, CA) were subcutaneously implanted. Mice were treated with vehicle, 1 or 3 mg/kg/day PENAO for 27 days. Tumour size (callipers) and animal weight were recorded 2–3 times a week. Tumour volume was expressed as relative tumour volumes (RTV). The mean of these values was used to calculate the ratio between treatment and control tumours (T/C × 100%) as an indicator of drug efficacy. Tumour volume quadrupling time (TVQT) and growth delay (GD) were calculated as previously described [19]. The tumour growth curves were compared using repeated measures two-way analysis of variance (ANOVA) using GraphPad Prism 6. The median times to quadrupling was estimated for each group of mice from the Kaplan–Meier survival distribution and compared by a log-rank test [20].

2. Methods 2.5. Western blotting 2.1. Reagents Except otherwise mentioned, all the reagents and chemicals were from Sigma (St Louis, MO). 2.2. Cell culture Except otherwise mentioned, cells were from ATCC (Bethesda, VA) and all culture media, serum, antibiotics and supplements were from Invitrogen (Mulgrave, VIC, Australia). All cultures contained 20 units/ mL penicillin and 20 units/mL streptomycin. OVCAR-3 cells (high-grade serous ovarian cancer (16)) and SKOV-3 (endometrioid ovarian cancer (16)) were cultured in RPMI 1640 medium containing 10% v/v foetal bovine serum and 2 mM glutamine. TOV21G (clear cell ovarian cancer (16))

Proteins from SKOV-3 and OVCAR-3 cell lysates were resolved by SDSPAGE. Primary antibodies: anti-MRP-1 and anti-MRP-2 (Enzo Life Science); anti-c-PARP-1 (Cell Signaling); anti-LC3B (Cell Signaling), antiHO (Enzo Life Science) and anti-β actin (Abcam). Lysates from MCF-7/ VP [21] and MDCKII-MRP2 [22] cells were employed as positive controls for MRP-1 and -2 expressions, respectively. Images were acquired by using an ImageQuant LAS 4000 system (GE Healthcare Life Sciences). 2.6. Drug accumulation PENAO accumulation in SKOV-3 and OVCAR-3 cells was determined as previously described [8]. Briefly, ovarian cells were incubated for 4 h with PENAO in the presence or absence of reversan, washed twice with

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ice-cold phosphate-buffered saline, and lysed with 70% w/w nitric acid. Lysates were analysed for arsenic atoms by Inductively Coupled Plasma Spectrometry. 2.7. Cell cycle

Table 1 Anti-proliferative activity of PENAO, GSAO and two platinum-based therapeutics for ovarian cancer cell lines from various histotypes. Cell growth was revealed after 72 h of contact with the compounds by MTT assay. CisPt: cisplatin; Carbo: carboplatin; IC50: concentration of compound that resulted in half-maximal proliferation arrest; SD: standard deviation. IC50 values presented are means ± SD from at least 3 experiments performed in triplicates.

SKOV-3 and OVCAR-3 cells were seeded at 1.2 × 105 cells per well, in 6-well plates. Cells were allowed to adhere overnight and were then treated with PENAO for 48 h. Cell cycle progression was analysed using flow cytometry with propidium iodide (10 μg/mL; PI) staining [23]. 2.8. Apoptosis assays Apoptosis was detected using Annexin-V-FLUOS Staining Kit (Roche Applied Science) according to the manufacturer's instructions. SKOV-3 and OVCAR-3 cells were seeded at a density of 1.2 × 105 cells per well were allowed to adhere in 6-well plates for 24 h and then treated with PENAO for 24 h. Cells were detached then stained with AnnexinV and PI for 15 min before being analyzed [19]. 2.9. Acridine orange staining of acidic vesicles Detection of autolysosomes was performed 24 h after drug(s) exposure with Acridine Orange (0.25 μg/mL, Life Technologies) staining for 15 min under normal culture conditions. Photographs were imaged through the green (BP530-585nm) and red (BP450-490nm) fluorescence channels and processed on the Zen2012 (Carl-ZeissAxioVert.A.1 Fluorescence microscope) [24]. 2.10. Superoxide production assays SKOV-3 and OVCAR-3 cells were seeded at 1.2 × 105 cells per well in 6-well plates. Cells were allowed to adhere for 24 h and then treated with PENAO for 16 h. Mitochondrial superoxide production was measured by flow cytometry using the MitoSox Red dye (5 μM; Invitrogen) as previously described [23]. Sytox Blue (1 μM; Invitrogen) was added to counter-stain for dead cells. 2.11. Mitochondrial membrane potential assays SKOV-3 and OVCAR-3 cells were treated with PENAO for 16 h then stained with JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylimidacarbocyanine iodide; 2 μM; Sigma) for 15 min in the dark at 37 °C and analyzed by flow cytometry according to the manufacturer's instructions. 2.12. Acid production and oxygen consumption assays SKOV-3 and OVCAR-3 cells were seeded in XF 24-well cell culture microplates at 1 × 104 cells/well for 24 h followed by PENAO treatment for another 24 h. After 48 h cell medium was changed to unbuffered medium containing the same treatment as the previous 24 h. After calibration of the XF24 sensor cartridge, the cell plate was loaded in the analyser and oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were recorded over 1 h. At the end of the assay, cells were harvested and the numbers of viable cells were determined by flow cytometry. The measurements of OCR and ECAR were normalized using the viable cell count. 2.13. Statistical analyses In vitro results are presented as mean ± SD. All analyses were performed using GraphPad Prism (GraphPad, San Diego, CA). All tests of statistical significance were two-sided and p values b 0.05 were considered statistically significant.

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1.3 10.6 1.1 10.9

15.7 114.0 6.6 95.0

1.9 8.8 0.2 4.2

3.9 24.8 7.9 83.3

1.1 4.5 1.6 28.9

2.6 13.5 7.7 102.7

0.4 2.1 0.9 19.4

3.5 21.5 5.9 81.3

0.5 2.1 2.1 10.3

3. Results 3.1. SKOV-3 cells are resistant towards PENAO both in vitro and in vivo PENAO as well as two standard chemotherapeutics (cisplatin, carboplatin) and the first generation organoarsenical-based drug developed in our laboratory (GSAO) were tested and their IC50 for proliferation arrest determined (Table 1). PENAO displays anti-proliferative activities in the low micromolar range. PENAO IC50 values were between 2.6 and 15.7 μM, SKOV-3 being the most resistant cell line tested. Consistent with a recent study [11], PENAO demonstrated only moderate anti-proliferative activity on a variety of non-cancerous cells including ovarian (HOSE17.1), breast (MCF10A), and lung (MRC5) cells and normal human astrocytes. For these cells, the IC50 values for proliferation inhibition were between 6.1 ± 0.5 and 8.7 ± 0.8 μM allowing an appreciable selectivity when compared with the most sensitive ovarian cancer cells tested. As previously observed [8], PENAO demonstrated stronger anti-proliferative activity than GSAO on all cancer cell lines tested. Across all ovarian cell lines employed here, SKOV-3 was consistently the most resistant one towards the arsenic- and platinum-based drugs tested. Notably, PENAO anti-proliferative activity was not impaired when tested on ovarian cells (CH1-CisR) which developed platinum drug resistance due to proficiency in DNA repair [25] (Supplementary Table S1). This observation was confirmed using HCT116 ch3 (MLH1+/+) and HCT116 ch2 (MLH1−/−) cells [26,27]: no significant difference in PENAO anti-proliferative activity could be detected between proficient and deficient cells for the DNA mismatched repair protein MLH1 (Supplementary Table S1). Human ovarian tumours from PENAO-sensitive OVCAR-3 and PENAOresistant SKOV-3 cells (Fig. 1A) were established subcutaneously in the proximal midline of immunodeficient mice. Established tumours in the mice were treated by continuous systemic administration of 1 and 3 mg/kg/day PENAO. PENAO treatment had no significant effect on SKOV-3 tumours, but inhibited the growth of OVCAR-3 tumours (Fig. 1B, C). The rate of OVCAR-3 tumour growth was inhibited by 51% (T/C: 49%) at 1 mg/kg/day and 48% (T/C: 52%) at 3 mg/kg/day of PENAO at day 27. The calculated growth delays (GD) over 2 tumour doubling times were 11.5 and 9.2 days for OVCAR-3 tumours treated with PENAO 1 and 3 mg/kg/day. No appreciable growth delay was observed for SKOV-3 tumours with GD values of 1.5 and −0.5 (Supplementary Table S2). 3.2. SKOV-3 cells express higher levels of MRP-1 than OVCAR-3 cells Organic and mineral arsenical compounds are known substrates for multidrug resistance associated proteins (MRP). PENAO, as well as GSAO, anti-proliferative activities are blunted by the expression of MRP1 and MRP-2 at the surface of cancer and endothelial cells [8,28]. The levels of expression of MRP-1 and MRP-2 in SKOV-3 and OVCAR-3 were determined by Western blot (Fig. 2A). MRP-2 was not detected in either of the lines. SKOV-3 cells express much higher levels of MRP-1 than

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Fig. 1. In vitro and in vivo effects of PENAO on OVCAR-3 and SKOV-3 tumour. A, Representative anti-proliferative activity of PENAO on OVCAR-3 and SKOV-3 ovarian cancer cells after 72 h of contact with the drug (MTT assay). Human ovarian tumours were established subcutaneously from B, OVCAR-3 and C, SKOV-3 cells in the proximal midline of female Balb/C nude mice. Mice bearing ~100 mm3 tumours were randomized into three groups (n = 7–9 per group) and implanted with subcutaneous Alzet micro-osmotic 2002 pumps in the flank that delivered vehicle, 1 or 3 mg/kg/day PENAO. Tumour volume was calculated using the relationship length × height × width × 0.523 and are expressed as relative tumour volumes (RTV), where the tumour volume at any given time is divided by the starting tumour volume. The data points are mean ± SE of the tumour volumes. *: p b 0.05, **: p b 0.01, ***: p b 0.001.

OVCAR-3 cells and this difference correlates with PENAO accumulation levels (Fig. 2B). SKOV-3 cells accumulate significantly less (2.6-fold; p b 0.001) PENAO compared to OVCAR-3 cells (Fig. 2B). The inhibition of MRP-1 by Reversan resulted in a significant increase in accumulation in both cell lines (p b 0.001). Interestingly, the level of PENAO accumulation in the presence of the inhibitor appeared to be similar (p = 0.685) in both cell lines (Fig. 2B). However, when MRP-1 was inhibited, SKOV-3 cells remained more resistant compared to OVCAR-3 (Fig. 2C), indicating that other mechanisms were responsible for SKOV-3 resistance towards PENAO.

early apoptotic cells, was clearly identified but no necrotic (PI+) cells were observed (Fig. 3B). Interestingly, for SKOV-3 cells, low percentages of Annexin-V+ and PI+ cells were detected (Fig. 3B) suggesting mixed cell death mechanisms: apoptosis and necrosis. The mechanisms of cell death were further investigated by Western blot for the detection of cleaved PARP and an autophagy marker (Fig. 3C). The cleavage of PARP-1 and the autophagy marker LC3B were detected in both cell lines. The induction of autophagy in SKOV-3 and OVACR-3 cells treated by PENAO was further demonstrated by the detection of acidic vesicular organelles (AVO) (Fig. 3D).

3.3. Effect of PENAO on cell cycle and cell death mechanisms

3.4. Effects on mitochondria

PENAO demonstrated anti-proliferative activity on a variety of ovarian cancer cell lines (Table 1). We further examined how PENAO perturbed the cell cycle in SKOV-3 and OVCAR-3 cell lines (Fig. 3A). After 48 h of exposure to PENAO, both cell lines displayed a moderate but significant cell cycle arrest in G2/M phase without modification of S phase. The mechanism of cell death induction by PENAO was examined by flow cytometry analysis (Supplementary Fig. S1). PENAO induced a dose-dependent increase in double Annexin-V/PI staining in both cell types, representing late apoptotic and dead cells (Fig. 3B). For OVCAR-3 cells, a population of Annexin-V + cells, representing

3.4.1. Oxidative stress The induction of heme oxygenase-1 (HO-1), a well-known cellular marker for oxidative stress was detected by Western blot in SKOV-3 and OVCAR-3 cell lysates in response to PENAO treatment (Fig. 4A). PENAO treatment increased superoxide production in a concentrationdependent manner in both cell lines. However, at comparable PENAO concentrations we observed that OVCAR-3 cells were producing more mitochondrial superoxide ions than SKOV-3 (Fig. 4B). Modulations of intracellular glutathione level, a main actor in cellular redox control, demonstrated further the oxidative stress-driven effect of PENAO. The

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Fig. 2. SKOV-3 cells express higher levels of MRP-1 compared to OVCAR-3 and are more resistant to PENAO exposure. A, MRP-1 and -2 expressions in SKOV-3 and OVCAR-3 cells (20 μg of protein) determined by Western blot. MRP-1 and -2 positive controls are cell lysates from MCF-7 cells (1 μg) and MDCKI cells (20 μg), respectively. The blots presented are representative of 2 experiments. B, PENAO accumulation in SKOV-3 and OVCAR-3 cells. The cells were incubated with PENAO (50 μM) for 4 h in the presence or absence of the MRP-1 inhibitor, reversan (5 μM). Cellular accumulation of arsenic was determined by ICP. Values are mean ± SD of triplicate determinations. ***, p b 0.001. #, p b 0.001 vs no reversan. Results are representative of 2 experiments performed in triplicates. C, Inhibition of MRP-1 enhanced PENAO activity in both SKOV-3 and OVCAR-3 cells. IC50 values for PENAO anti-proliferative activity were determined by MTT assay after 72 h in the presence or absence of increasing concentrations of reversan. Results shown are representative of 2 experiments performed in triplicate.

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anti-oxidant and glutathione precursors N-acetyl cysteine (NAC) as well as membrane permeable glutathione ethyl-ester (GSH-EE) protected ovarian cells from PENAO anti-proliferative activity (Fig. 4C). On the contrary, glutathione depletion using L-buthionine sulfoximine (BSO, Fig. 4D) enhanced PENAO activity.

3.4.2. Mitochondrial perturbations The mitochondrial trans-membrane potential, an indicator of mitochondrial integrity, was measured using the JC-1 dye. PENAO treatment disrupted the mitochondrial trans-membrane potential (Fig. 5A). A dose-dependent effect on mitochondrial trans-membrane potential is

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Fig. 3. Cell cycle arrest and cell death mechanisms induced by PENAO in SKOV-3 and OVCAR-3 cells. SKOV-3 (left panels) and OVCAR-3 (right panels) cells were exposed to PENAO. PENAO concentrations used are expressed in micromolar or as fractions of IC50 values for proliferation inhibition at 72 h (see Table 1). A, Cell cycle: relative percentages of PI+ cell populations as a function of PENAO concentration are presented for SKOV-3 and OVCAR-3 cells. Interference in cell cycle was revealed after 48 h of contact with PENAO. *, p b 0.05; **, p b 0.01; ***, p b 0.001 vs G2/M population of untreated control. Data presented are from at least two experiments performed in triplicate. B, Annexin V/PI staining: relative percentages of stained cell populations as a function of PENAO concentration are presented for SKOV-3 and OVCAR-3 cells. The cells were treated for 24 h with PENAO. Data presented are from at least two experiments performed in duplicate. C, Cell death marker detection: the detection of apoptotic and autophagy markers by Western blot in response to increasing concentrations of PENAO was performed after 24 h exposure. Loading control is β-actin. The blots presented are representative of 2 experiments. D, Autophagy detection. An accumulation of acidic vesicular organelles (AVO) (red fluorescence) indicative of autophagy is observed in both SKOV-3 and OVCAR-3 cells in response to 17 μM and 5 μM PENAO treatment for 24 h, respectively. Pictures are representative fields from two distinct experiments. Magnification: 400×.

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Fig. 4. Oxidative stress induction by PENAO. A, Heme-oxygenase-1 detection by Western blot in response to increasing concentrations of PENAO was performed after 24 h of exposure. Loading control was β-actin. The blots presented are representative of 2 experiments. B, PENAO induces mitochondrial superoxide ion production. Dose-dependent mitochondrial superoxide ion production in response to PENAO (16 h after exposure) was measured using MitoSox Red. **, p b 0.01; ***, p b 0.001 vs untreated control. Data presented are from at least two experiments performed in duplicate. C, Modulation of glutathione levels influences oxidative stress-driven PENAO anti-proliferative activity in ovarian cells (SK, SKOV-3; OV, OVCAR-3). Nacetyl cysteine (NAC) and glutathione ethyl-ester (GSH-EE) protected ovarian cells from PENAO anti-proliferative activity. D, Glutathione depletion using BSO enhanced PENAO activity. Cell proliferation was measured by MTT assay after 72 h of exposure. Results are representative of two experiments performed in triplicate.

presented. Notably, OVCAR-3 cells had a basal percentage of depolarised mitochondria significantly higher (p b 0.001) than what was observed for SKOV-3 cells: 13.11 ± 4.25% and 2.59 ± 0.37%, respectively. In response to PENAO treatment, mitochondrial depolarisation appeared to be more pronounced in OVCAR-3 than in SKOV-3 cells (Fig. 5A).

3.4.3. Metabolic perturbations The results presented above indicate that PENAO is interfering to some extent with normal mitochondrial function. Its effect on oxygen consumption and acid production in SKOV-3 and OVCAR-3 cells was tested. Treatment of SKOV-3 cells with PENAO resulted in a significant drop in oxygen consumption (Fig. 5B) and a concomitant and significant increase in acid production (Fig. 5C). These observations were consistent with an inhibition of mitochondrial respiration followed by an adaptive glycolytic metabolism. Interestingly, OVCAR-3 cells were shown to be more sensitive than SKOV-3 in terms of oxygen consumption (Fig. 5B), but were unable to adapt their metabolism towards glycolysis (Fig. 5C). In an attempt to interfere with SKOV-3 adaptive metabolism shift, we combined PENAO with a mTOR inhibitor (temsirolimus) known to decrease glycolytic metabolism. When combined in a fixed ratio, PENAO and temsirolimus synergised to inhibit SKOV-3 proliferation (Fig. 5D). The calculated combination index (CI) was 0.74 ± 0.07. In variable ratio combination experiments, temsirolimus (15 μM) increased PENAO activity on SKOV-3 cell proliferation by 6.9 ± 1.2 fold, according to IC50 values (not shown). The combination of the two drugs resulted in increased cell death induction by apoptosis and autophagy as demonstrated by cPARP-1 and LC3B detection (Fig. 5E and Supplementary Fig. S2).

4. Discussion Overcoming the acquired or innate chemotherapy resistance of malignant tumours has been an ongoing challenge ever since the development of the first chemotherapeutic agents. Despite considerable efforts to improve early detection and advances in chemotherapy, patients suffering from undifferentiated high stage ovarian cancer have estimated five-year survival rates of less than 20%. The two most common histological types of EOC are serous (75%) and endometrioid carcinoma (10%). Clear cell and mucinous carcinomas are rare and together account for only ~11% of cases [29]. Despite the relatively small number of cases, these subtypes of ovarian cancers are of significant interest to many investigators because they are characterized by poor chemosensitivity and possibly a worse prognosis than the more common serous cell type [30]. PENAO has strong anti-proliferative activity against a range of cancer cell lines with limited activity on normal cells [8,11]. In preclinical models of pancreatic carcinoma [8] and glioblastoma multiforme [13], PENAO demonstrated efficacy without signs of toxicity. This compound is currently in clinical Phase 1 testing in patients with solid tumours refractory to standard therapy. We have tested PENAO on the most common (serous, endometrioid) as well as on rare (clear cell, mucinous) subtypes of ovarian cancer cell lines. PENAO induced proliferation arrest was compared with two standard platinum-based chemotherapeutics (cisplatin and carboplatin). Except for SKOV-3 cells (endometrioid) [16], which are known for being resistant to cisplatin and taxoids [31, 32], PENAO displayed anti-proliferative activities in the low micromolar range similar to what was observed with cisplatin. Confirming our previous observations [8], PENAO is consistently more potent than the first

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Concentration (fractions of IC 50) Fig. 5. Mitochondrial and metabolism perturbations. A, PENAO disrupts mitochondrial transmembrane potential. The percentage of cells with depolarised mitochondria (measured by JC-1 dye) as a function of PENAO concentration is presented. The data points and error bars are the mean and SD of at least 2 determinations performed in duplicate. B, C, PENAO perturbs the metabolism of ovarian cancer cells. Oxygen consumption rate (OCR, B) and extracellular acid production (ECAR, C) were measured in real time using a cell metabolism analyser 24 h after exposure to various concentrations of PENAO. Data presented are representative of two separate experiments performed in duplicate. D, PENAO and temsirolimus (TEM) synergistically inhibit SKOV-3 cell proliferation. Comb, fixed ratio combination of PENAO and temsirolimus. Cell proliferation was measured by MTT assay after 72 h of exposure. Results are representative of two experiments performed in duplicate. E, Cell death marker detection: the detection of apoptotic (cPARP-1) marker by Western blot in response to PENAO (0.5 × IC50) and TEM (0.5 × IC50) as single agent or in combination (Comb) was performed after 24 h exposure. The detection of autophagy (LC3B) marker in response to PENAO (5 μM) and TEM (10 μM) as single agent or in combination (Comb) was performed after 24 h exposure. Loading control was β-actin. Blots presented are representative of two experiments.

generation organoarsenical compound we developed, GSAO. In contrast to PENAO, GSAO is a prodrug that requires activation at the cell surface [19,33]. The clear cell and mucinous ovarian carcinoma cell lines employed were shown to be sensitive to PENAO. To test the possibility of cross resistance between platinum drugs and PENAO, cells lines with adaptive DNA repair strategies known to compensate the impact of cisplatin DNA crosslinking were employed. None of them demonstrated resistance towards PENAO when compared to their parental counterparts, indicating that proficiency in DNA repair mechanisms has no deleterious effect on overall PENAO anti-proliferative activity. The well documented chemo-resistant profile of the endometrioid SKOV-3 cells was confirmed in the present study with subcutaneously implanted SKOV-3 tumours not responding to PENAO treatment. It was reported that SKOV-3 cells induce the expression of multidrug resistance proteins such as P-glycoprotein (P-gp) [34] when exposed to plant alkaloids. Knowing that PENAO is a substrate for both MRP-1 and MRP-2 [8] and that overlaps exist in drug selectivity between Pgp and MRP family members, a comparison of expression levels and drug accumulation was performed for SKOV-3 cells and its sensitive counterpart, OVCAR-3. Despite a much higher MRP-1 expression, this multidrug resistance-associated protein had no or limited role in SKOV-3 resistance towards PENAO. Indeed, when MRP-1 activity is abolished, the SKOV-3 cells remain 5.7 ± 0.9 fold more resistant than OVCAR-3 cells. Cell cycle studies were performed to further investigate the anti-proliferative effect of PENAO on ovarian cancer cells. A moderate but significant increase of cell proportions in the G2/M phase in

response to PENAO exposure observed in both cell lines is consistent with a recent study performed on commercial and primary glioblastoma (GBM) cells [23]. As cancer cells are most sensitive to radiation therapy in the G2/M phase of the cell cycle [35], these findings suggest that PENAO treatment may sensitize cancer cells to ionizing irradiation. In terms of cell death mechanisms, a clear induction of apoptosis was observed in the two ovarian cancer cell lines, confirming what has been recently observed with breast and glioblastoma tumour cells [11,23]. Notably, PENAO, like other arsenic-based cancer drugs [36,37] also induces autophagy. This observation along with the known PENAO mitochondrial tropism [9] and its ability to induce ROS production [23] led us to investigate the potential role of oxidative stress in ovarian cell death mechanism. We found that PENAO activity correlates with enhanced mitochondrial production of ROS and to the subsequent effects of oxidative stress. The inducible form of HO-1 is known to play important roles in protection against oxidative and chemical stress including from arsenic-based drugs [38]. A recent study [39] describes that HO-1 translocates from endoplasmic reticulum to mitochondria where it promotes mitochondrial dysfunction and autophagy. Interestingly, the link between HO-1 induction and autophagy was also observed in our study: HO-1 and LC3B inductions in response to PENAO treatment were consistently stronger in SKOV-3 cells compared to OVCAR-3 cells. With HO-1 providing defence against oxidative stress by accelerating the degradation of pro-oxidant heme and hemoproteins to the radical scavenging bile pigments, SKOV-3 cells can adapt better than OVCAR-3 cells to the

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mitochondrial stress induced by PENAO. The important role of HO-1 in SKOV-3 resistance towards PENAO was confirmed using a HO-1 inhibitor (Supplementary Fig. S3). Taken together, experiments using antioxidant and pro-oxidant compounds demonstrate the central role of oxidative stress in PENAO activity. Through its direct interaction with the mitochondria, PENAO interferes with oxidative phosphorylation. Notably and in contrast to OVCAR-3 cells, the drug-resistant SKOV-3 cells can shift their metabolism towards glycolysis in response to PENAO treatment. The mTOR signalling pathway, and more specifically mTORC1, is known to be critical for maintaining the glycolytic metabolism state of cancer cells [40]. We targeted the adaptive glycolytic shift in SKOV-3 by using a mTORC1 inhibitor in combination with PENAO. This strategy was successful with the two drugs acting synergistically to inhibit cell proliferation and induce cell death via apoptosis and autophagy. A similar approach using dichloroacetate (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor that reverses the Warburg effect, on breast cancer cells and GBM cells also produced interesting combinatorial profiles with PENAO [11, 23]. In summary, PENAO inhibits the proliferation of ovarian cancer cells derived from various histological subtypes, including rare clear cell and mucinous ovarian carcinoma cell lines. In vitro and in vivo PENAO resistance displayed by endometrioid cells has been attributed to, (i) a strong heme oxygenase-1 induction providing defence against oxidative stress and limiting subsequent mitochondrial injuries and, (ii) to an ability to shift metabolism towards glycolysis. Targeting this adaptive glycolytic shift by combining a mTOR signalling pathway inhibitor with PENAO synergistically enhanced proliferation arrest and cell death induction. Taken together, the findings of this study warrant further evaluation of this dual-targeting therapy in vivo, which may constitute a new approach for the treatment of recurrent/resistant forms of EOC. Conflict of interest statement The authors have no conflicts of interest to declare. Dr. Dilda and Dr. Hogg have a patent entitled “Organo-arsenoxide compounds and use thereof” issued.

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