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Novel pharmacological effects of poly (ADP-ribose) polymerase inhibitor rucaparib on the lactate dehydrogenase pathway
Biochemical and Biophysical Research Communications 510 (2019) 501e507
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Novel pharmacological effects of poly (ADP-ribose) polymerase inhibitor rucaparib on the lactate dehydrogenase pathway Yuma Nonomiya, Kohji Noguchi*, Kazuhiro Katayama, Yoshikazu Sugimoto Division of Chemotherapy, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo, 105-8512, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 January 2019 Accepted 30 January 2019 Available online 5 February 2019
Poly (ADP-ribose) polymerases (PARPs) are involved in various cellular events, including DNA repair. PARP inhibitors including olaparib and rucaparib, have been specially developed against breast and ovarian cancers deficient in DNA repair systems. In this study, we found that PARP1-defective olaparibresistant A2780 cells (ola-R cells) cells were still sensitive to two PARP inhibitors, rucaparib and veliparib. Metabolomic analysis revealed that rucaparib suppressed the lactate dehydrogenase (LDH)-mediated conversion of pyruvic acid to lactic acid in A2780 cells, although olaparib did not. The inhibition of LDH by siRNA-mediated knockdown or by LDH inhibitors suppressed the growth of ovarian cancer cells. Our results suggested that the suppression of the LDH-associated pathway contributed to the pharmacological effects of rucaparib. © 2019 Elsevier Inc. All rights reserved.
Keywords: Olaparib Rucaparib PARP1 LDHA Ovarian cancer
1. Introduction PARPs hydrolyse nicotine amide adenine dinucleotide (NADþ), catalyse the poly ADP-ribosylation (PARylation) of acceptor proteins on various amino acid residues, and are involved in DNA repair [1]. PARP1 is the most abundant nuclear protein in the PARP family, and PAPR1-mediated PARylation is a critical step for resealing single strand DNA breaks (SSB)s. Inhibition of PARP1 causes an accumulation of unrepaired SSBs, which results in double strand DNA break (DSB). The PARP inhibitors repress PARylation in cells at low concentrations [2], and several PARP inhibitors (olaparib, veliparib, rucaparib, niraparib, and talazoparib) have been developed for clinical use in cancer chemotherapy [3]. The BRCA1/2-inactive cells with low homologous recombination (HR) activity are highly sensitive to DNA damage, and thus the PARP inhibitors show synthetic lethal effect on BRCA-deficient cancer cells [4,5]. However, as a single agent, the cytotoxic activity of these PARP inhibitors is varied. Potent PARP inhibitors trap PARP1 at the base excision repair (BER) intermediate to induce DNA damage, which is known as PARPtrapping, and the PARP-trapping potency of PARP inhibitors is well correlated with their cytotoxic activities [6]. Intriguingly, BRCA mutations are not sufficient for prediction of PARP inhibitor efficacy
* Corresponding author. E-mail address: [email protected] (K. Noguchi). https://doi.org/10.1016/j.bbrc.2019.01.133 0006-291X/© 2019 Elsevier Inc. All rights reserved.
in individual patients. In this study we isolated three olaparib-resistant PARP1deficient cell lines (ola-R-3, -10, and -15) from human ovarian cancer A2780 cells, and found that the ola-R cells were still sensitive to the PARP inhibitor rucaparib, although such cells showed cross-resistance to niraparib and talazoparib. Our metabolomic analysis uncovered rucaparib-induced selective repression of the pyruvate-lactate metabolism. The inhibition of the pyruvate-lactate catalytic enzyme, lactate dehydrogenase (LDH), consistently suppressed the growth of ovarian cancer cell lines. Our experiments provide the first indication that the suppression of LDH-catalysed pyruvate-lactate metabolic pathway may be a novel pharmacological effect of rucaparib. 2. Materials and methods 2.1. Cells, plasmid, transfections, and chemical inhibitors A2780, OVCAR-3, and -8 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 medium (SigmaAldrich, St. Louis, MO, USA) supplemented with 7% fetal bovine serum and 50 mg/mL kanamycin at 37 C in a humidified atmosphere containing 5% CO2. A2780 cells were treated with olaparib (from 1 mmol/L to 40 mmol/L for 4 months) and the surviving colonies were selected to establish olaparib-resistant cell lines (ola-R3, -10, and -15). PARP1 cDNA (GenBank accession: NM_001618.3) was cloned by standard PCR method and PARP1-hemagglutinin
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(HA) stably-expressing A2780 cells were established by the transduction of PARP1-expressing retrovirus (pQCXIP, Clontech Laboratories Inc., Takara Bio Company, Mountain View, CA, USA), followed by selection with 500 ng/mL puromycin (Sigma-Aldrich). Olaparib, veliparib, rucaparib, niraparib, and talazoparib were purchased from Selleck Chemicals (Houston, TX, USA). Cisplatin was purchased from Sigma-Aldrich and SN-38 was provided by Yakult Honsha Co., Ltd. (Tokyo, Japan). 2.2. Cell growth inhibition assay and colony formation assay The cell growth inhibition assay was performed by using cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan) [7,8]. The degree of resistance was calculated by dividing the half-maximal inhibitory concentrations (IC50) of the cells of interest by those of the parent cells. Colony formation assay and the quantification of cell survival were performed as described previously [7]. 2.3. Reverse transcription (RT)ePCR analysis The total RNA was extracted by using an RNeasy kit (QIAGEN Sciences, Germantown, MD, USA). RT-PCR reactions for PARP1/2 were performed by using an RNA LA PCR kit (Takara, Ohtsu, Japan). PCR primer sequences are listed in Table S1. GAPDH cDNA was used as a loading control, as described previously [9]. The PCR conditions for PARP1 were 94 C for 1 min, followed by 25 cycles of 94 C for 30 s, 62 C for 30 s, and 72 C for 1.5 min, and then a final extension step of 72 C for 5 min. The PCR conditions for PARP2 were 94 C for 1 min, followed by 30 cycles of 94 C for 30 s, 62 C for 30 s, and 72 C for 2 min, and a final extension step of 72 C for 5 min. 2.4. Western blotting Western blotting was performed as described previously [7,8]. The antibodies used were directed against PARP1 (F-2), Rad51 (H92), LDH-A (E-9), LDH-B (431.1) (Santa Cruz Biotechnology, Santa Cruz, CA); MDR1þ3 (C219), PARP2 (ab115620), M2III-6 (Abcam, Cambridge, MA); PARG (D8B10), GAPDH (6C5) (Millipore, Billerica, MA); PAR (Trevigen, Helgerman CT, Gaithersburg, MD, USA); BRCA1 and BRCA2 (D9S6V) (Cell Signalling Technology, Danvers, MA, USA); HA (3F10) (Roche Diagnostics, Indianapolis, IN, USA); MRPm6 (Nichirei, Tokyo); M3Ⅱ-9 (Kamiya Biomedical, WA, USA), and antiBCRP polyclonal antibody (3488) (Chemicon, Temecula, CA, USA). GAPDH was used as the loading control.
foci, and analyzed the g-H2AX-positive cells with the Adobe® Photoshop CS4 Extended software (Adobe Systems Inc., San Jose, CA, USA). At least 100 cells were examined at each experimental point. 2.6. siRNA PARP1-targeting small interfering RNA (siRNA; siGENOME Human PARP1 (142) siRNA-SMARTpool), LDHA-targeting siRNA (siGENOME Human LDHA (3939) siRNA-SMARTpool), and LDHBtargeting siRNA (siGENOME Human LDHB (3945) siRNASMARTpool) were purchased from Dharmacon GE Healthcare (Amersham, UK), and a control scrambled siRNA (AllStars Negative Control siRNA) from Qiagen (Valencia, CA, USA). Lipofectamine 2000 (Invitrogen) was used for the transfection of cells with siRNAs. 2.7. Metabolomic analysis Cells were treated with 20 mmol/L olaparib or rucaparib for 12 h; all samples were prepared in triplicate. Methanol metabolite extraction was conducted in accordance with the Human Metabolome Technologies Inc., (HMT, Inc., Yamagata, Japan), metabolite extraction method for adherent cells. In brief, the cells were washed twice with 5% mannitol and treated with 0.8 mL methanol. The internal standard (0.55 mL of 8 mM internal standard) was added and the methanol extract was collected. After centrifugation, the extracts were subjected to centrifugation filtration through a 5 kDa cut-off filter. The extracts were stored at 80 C until analysis. The metabolomic analysis of 116 metabolites was conducted through a facility service (C-SCOPE) in Human Metabolome Technologies, Inc. (Yamagata) (N ¼ 3 in each group). The concentrations of all charged compounds were measured by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) and capillary electrophoresis tandem mass spectrometry (CE-QqQMS; CEMS/MS). Metabolite concentrations were normalized to viable cell counts. 2.8. Lactate assay The concentration of lactate produced from the cells was evaluated by using a lactate assay kit-WST (Dojindo Laboratories, Kumamoto, Japan). In brief, the cells were treated with PARP inhibitors for 24 h. The sample absorbance at 450 nm was read by using an Infinite M1000 microplate reader (Tecan Japan, Kanagawa, Japan).
2.5. Immunofluorescent microscopy 2.9. Statistical analysis Cells were seeded onto Scientific™ Nuc™ Lab-Tek™ II CC2™ chamber slides (4 104 cells in 1 mL/well; Thermo Fisher Scientific) or Eppendorf Cell Imaging Coverglasses (Eppendorf, Hamburg, Germany). After treatment with PARP inhibitors for 24 h the cells were fixed with ethanol/acetate (50:50) for 5 min at 4 C, and washed with phosphate-buffer saline (PBS) three times. Cells were permeabilized with 0.5% Triton X-100/PBS for 5 min at room temperature, washed three times with PBS, and blocked with 3% bovine serum albumin (BSA) in PBS for 60 min at room temperature. After three times wash with PBS, the cells were incubated with 2 mg/mL of anti-phospho-Histone H2AX Ser 139 (Merck Millipore, Billerica, MA, USA) for 120 min at room temperature. Then cells were probed with 1 mg/mL of Alexa Fluor 488 goat anti-mouse antibody (Invitrogen) and mounted in Prolong™ Gold Antifade reagent with 40 ,6diamidino-2-phenylindole (DAPI) (Invitrogen). The acquisition of images (640 640 pixels) was performed by using FV1000-D IX81 confocal microscopy (Olympus Corp., Tokyo, Japan). We defined the fluorescence signalling diameter as 0.5 mm or more as a g-H2AX
The quantitative results are presented as the mean ± SD (n 3). A scatter plot was generated by using GraphPad Prism 7 software (La Jolla, CA, USA). The Mann-Whitney U test and two-tailed Student's t-test were used to evaluate the statistical significance with IBM Statistics SPSS version 23.0 (IBM Corp., USA). In the metabolome analysis, Welch's t-test was used to compare metabolite concentrations. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Olaparib-resistant clones lacking PARP1 Three olaparib-resistant A2780 cell lines (ola-R-3, -10, and -15), showed strong resistance to olaparib and talazoparib, and moderate resistance to niraparib, compared with the parent cells (Fig. 1A and Supplementary Table S2). However, the ola-R cells showed only
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Fig. 1. Olaparib-resistant clones lacking PARP1 from A2780 cells. (A) Schematic protocol for the isolation of olaparib-resistant cells from A2780 cells. (B) The viabilities of drugtreated cells are shown as the mean ± SD from triplicate experiments. A2780 and ola-R cells were treated with various concentrations of PARP inhibitors olaparib (C), and rucaparib (D) for 24 h. Cells were fixed and g-H2AX was probed with anti-g-H2AX antibody (green signal), and the nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI, blue signal). Representative images are shown (upper images). Scale bar ¼ 10 mm. The number of g-H2AX foci in the g-H2AX foci-positive cells are summarized (counted cell number >100 in each sample) as a dot plot (lower graphs). Statistical significance was determined by using the Mann-Whitney U test, *p < 0.05, **p < 0.01. Horizontal red lines represent the median. (E) Expression of PARP1, PARP2, and other related proteins in the ola-R cells was analyzed by western blotting. (F) The mRNA expression of PARP1 and PARP2 in the ola-R cells was analyzed by using RT-PCR. The mRNA of PARP1 was detected by four different primer sets (Supplementary Table S1). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
marginal levels of cross-resistance to rucaparib and veliparib, and also showed 2- to 5-fold hypersensitivity to cisplatin and the topoisomerase I inhibitor SN-38 (Fig. 1A and Supplementary Table S2), suggesting that DNA damage responses might be compromised in ola-R cells. Histone H2AX phosphorylation (gH2AX) is a marker of DSBs [10], and PARP inhibitors-induced gH2AX foci are associated with levels of cellular DNA damage. Olaparib increased the number of g-H2AX foci in a concentrationdependent manner (upto 100 mmol/L) in parent A2780 cells, whereas the degree of increase was lower in the ola-R cells (Fig. 1C). In A2780 cells, we observed massive cell death after the treatment of rucaparib at 100 mmol/L for 24 h, so that, the maximum concentration of rucaparib was set at 30 mmol/L in Fig. 1D. Compared with the cases in the olaparib-treatment, the induction of g-H2AX foci by rucaparib appeared to be weak in A2780 cells and the ola-R cells. Western blotting showed that PARP1 protein was lost in all the ola-R cells and PARylation in the cell lysates was also lower in ola-R cells than in A2780 cells. (Fig. 1E). The expression of the PARP2, PARG and GAPDH proteins were comparable between A2780 and ola-R cells. RT-PCR analysis showed that mRNA expression of PARP1,
when targeted to four different parts, was not detected in ola-R cells (Fig. 1F), which indicated that PARP1 was deficient at the mRNA level in the ola-R cells. 3.2. Knockdown of PARP1 in A2780 cells did not confer rucaparibresistance A previous study reported that defect in PARP1 expression causes resistance to PARP1 inhibitors [11]. Transfection of PARP1 siRNA repressed the PARP1 protein selectively in A2780 cells (Fig. 2A), and the number of g-H2AX foci in PARP1 knockdown cells was significantly lower than that in cells transfected with control siRNA after olaparib treatment (Fig. 2BeC). Conversely, the reintroduction of PARP1 in ola-R cells re-sensitized ola-R cells to olaparib-mediated growth inhibition and g-H2AX induction (Supplementary Fig. S1). The colony formation assay showed that PARP1 knockdown conferred resistant to three inhibitors, olaparib, talazoparib and niraparib (Fig. 1D). However, there was no obvious resistance to rucaparib or veliparib in the PARP1 knockdown cells (Fig. 1D). Additionally PARP1 knockdown of another ovarian cancer cell line, OVCAR-8, did not confer resistance to rucaparib
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Fig. 2. Rucaparib sensitivity was unaffected by PARP1 knockdown. (A) Knockdown of PARP1 in A2780 cells. In the left panel, the experiment protocol is shown. In the right panels, the expression of PARP1 was examined by western blotting. Confocal microscopy analysis was performed on cells treated with olaparib (0, 1, 10, and 100 mmol/L) for 24 h. Images of g-H2AX (green signal)- and nuclei by DAPI (blue signal)-staining (B) and the quantification analysis (C) were shown as in Fig. 1. Statistical significance was determined by the Mann-Whitney U test, **p < 0.01. Horizontal red lines represent the medians. Cell survival after the treatment of PARP inhibitors (olaparib, veliparib, rucaparib, talazoparib, and niraparib) in siRNA-transfected A2780 (D) and OVCAR-8 cells (F) were evaluated by using a colony formation assay. Knockdown of PARP1 protein in A2780 cells is shown in A and in OVCAR-8 cells in E. Representative images of surviving stained cells are shown in the upper panels of D and F. The upper triplicate wells were control siRNA-transfected cells, and the lower triplicate wells were PARP1 siRNA-transfected cells. Reproducibility was confirmed twice and representative quantitative results are shown as the mean ± SD from triplicate experiments in the lower graphs of D and F. Statistical significance was determined by using Student's t-test. *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
(Fig. 1EeF). These data suggested that PARP1 was not essential for the induction of cytotoxicity by rucaparib and veliparib, although it was a critical pharmacological target for olaparib, talazoparib, and niraparib. 3.3. Differing effects on metabolic profiles by olaparib and rucaparib We analyzed the metabolic changes after treatment of rucaparib and olaparib for 12 h in A2780 cells (Fig. 3). Quantitative metabolomic analysis was performed on 116 compounds associated with the central carbon metabolic pathways, such as glycolysis, TCA cycle, amino acid synthesis, and the nucleotide synthesis pathways. The change in metabolite concentrations was illustrated by a clustered heat map in Fig. 3A. Principal component analysis revealed a clear separation between the control, olaparib-, and
rucaparib-treated samples (Fig. 3B). Rucaparib-treated samples were distinguishable from control and olaparib-treated samples by the second principal component (PC2)-axis, and the factor loadings (the top nine compounds each for positive and negative values) contributing to the PC2-axis were listed (Fig. 3C). A metabolic pathway map summarized the results (Supplementary Fig. S2A), and the metabolites corresponding to the factor loading contributing to PC2 in the purine metabolic pathway were negatively downregulated, whereas those in the glycolysis pathway were elevated by rucaparib treatment (Fig. 3D). It was reported that rucaparib could inhibit hexose 6-phosphate dehydrogenase (H6PD) as a secondary target, which is mapped in the pentose phosphate pathway upstream of the purine metabolic pathway [12]. Thus, our data would show the first metabolic evidence for the rucaparibselective suppression of some metabolites (PRPP, IMP, and XMP downstream of the pentose phosphate pathway) by rucaparib-
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Fig. 3. Metabolomic profiles after olaparib- and rucaparib-treatment. A2780 cells were treated with olaparib or rucaparib for 12 h and triplicate samples of the methanol extracts were subjected to metabolomic analysis. (A) Standardized relative changes of 96 metabolites from triplicate samples are visualized by clustered heat map, created at the CIMminor site (https://discover.nci.nih.gov/cimminer/home.do). The average linkage was used for the clustered algorithm and the Euclidean algorithm was used for the distance method. (B) The principal component analysis (PCA) separated the metabolic profiles of three groups (n ¼ 3 in each group). (C) The top nine metabolites (positively and negatively) determined from the second principal component (PC2) factor loadings are listed. (D) A summary of the glycolysis pathway and the pentose phosphate pathway is shown. The values shown in the bar graph represent the mean ± S.D. of triplicate samples, and the blue, red, and green bars are the data for the control, olaparib-treated, and rucaparib-treated samples, respectively. The metabolite abbreviations are glucose 6-phosphate (Glucose-6P), fructose 6-phosphate (Fructose-6P), fructose 1,6-bisphosphate (Fructose-1, 6 BP), glyceraldehyde 3-phosphate (Glyceroaldehyde-3P), 1,3-bisphosphoglycerate (1,3-BP Glycerate), 3-phosphoglycerate (3-P Glycerate), 2-phosphoglycerate (2-P Glycerate), phosphoenolpyruvate (P-Enolpyruvate), D-ribose 5-phosphate (D-Ribose-5P), phosphoribosyl diphosphate (PRPP), inosine monophosphate (IMP), and xanthosine monophosphate (XMP). The metabolic enzymes listed are hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD), hexose-6-phosphate dehydrogenase (H6PD), glucose-6-phosphate isomerase (GPI), phosphofructokinase (PFKP), aldolase fructose-bisphosphate A/B/C (ALDOA/B/C), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase 2 (PGAM2), enolase (ENO), pyruvate kinase, muscle/liver and RBC (PKM/LR), lactate dehydrogenase A/B (LDHA/B), and inosine monophosphate dehydrogenase (IMPDH). Statistical significance was determined by Student's t-test. *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
mediated inhibition of H6PD. We also found that the pyruvate to lactate conversion was suppressed by rucaparib (Fig. 3D), which suggested that this pathway might represent a third target for rucaparib. We reexamined the change in lactate production of PARP inhibitortreated cells by using a conventional assay kit. Consistent with the data from the metabolomic analysis, rucaparib significantly reduced lactate production in A2780, OVCAR-3, and OVCAR-8 cell lines, but olaparib did not (Fig. 4A). The lactate production was not affected by olaparib and talazoparib at doses capable of growth inhibition (Supplementary Fig. S2B). Next, we examined the possible contribution of LDH, which catalyses the reversible conversion of pyruvate to lactate, to the cell growth and survival of ovarian cancer cells. LDH is encoded by
three genes, LDHA, LDHB, and LDHC; LDHA and LDHB are widely expressed [13]. Knockdown of LDHA/B, especially LDHA, significantly suppressed the cell growth of A2780 and OVCAR-3 cells (Fig. 4BeC). We also confirmed the growth inhibitory effects of two LDH inhibitors, FX-11 and NHI-1, on several ovarian cancer cell lines (Supplementary Fig. S3), but we did not see a considerable direct inhibition of LDH enzymatic activity by PARP inhibitors in vitro (Supplementary Fig. S4). These results suggested that the inhibition of LDH-associated metabolic pathway was involved in the pharmacological actions of rucaparib (Supplementary Fig. S5). 4. Discussion Olaparib and rucaparib have similar potencies of PARP-trapping
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Fig. 4. Rucaparib-mediated inhibition of lactate production. (A) A2780, OVCAR-3, and OVCAR-8 cells were treated with various concentrations of rucaparib and olaparib for 24 h. The concentrations of lactate in the medium are shown. (B) The knockdown experiment protocol of LDHA and LDHB in A2780 and OVCAR-3 cells. (C) LDHA and LDHB protein expression after simultaneous transfection of siRNAs were confirmed by western blot analysis (upper panels). The cells were counted at 5 days after transfection (lower graphs). Statistical significance was determined by Student's t-test. **p < 0.01.
mechanisms [11,14], and, consistently, the growth inhibitory effects (IC50 values) of olaparib and rucaparib were similar in A2780 cells (1.1 and 2.4 mmol/L, respectively). However, in this study, we found that PARP1-deficiency conferred olaparib-resistance, but not rucaparib-resistance in A2780 cells. This observation implied that there would be a PARP1-independent cytotoxic action of rucaparib, consistently with previous discussion in other studies [14e16]. Our metabolome analysis additionally suggest that the suppression of the LDH-mediated metabolic pathway is involved in rucaparibmediated cytotoxicity. Principal component analysis of metabolomic experiments distinguished olaparib from rucaparib, and metabolomic analysis
indicated the downregulation of the purine synthetic pathway downstream of H6PD (Fig. 3D). Rucaparib has the ability to inhibit H6PD [12], and the knockdown of H6PD results in the inhibition of cancer cell growth [17,18]. We presumed that H6PD inhibition would be also involved in the rucaparib-induced growth inhibition of PARP1-defective ola-R cells. Moreover, we found that pyruvate was increased and, conversely, lactate was significantly decreased after rucaparib treatment (Fig. 3D), which suggested the selective inhibition of LDH-mediated metabolism by rucaparib. Pyruvate to lactate conversion is a critical step for NADþ production and glycolysis in cancer cells, and LDH has been suggested as a potential therapeutic target in cancer cells [13,19]. We confirmed that LDH
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knockdown and LDH inhibitors suppressed ovarian cancer cell growth (Fig. 4B and C and S3). These data suggested that the LDHmediated metabolic pathway was an additional pharmacological target of rucaparib. As we did not clarify possible off-target effect of a LDH inhibitor FX-11, it would be better to use more specific LDH inhibitor such as GNE-140 to see contribution of LDH enzymatic activity in cell growth inhibition [20]. Moreover, double genetic knockout of LDHA and LDHB which completely inhibits LDH activity, leads to the shifting their metabolism to oxidative phosphorylation with reduction of cell growth, but is not sufficient to abolish tumour cell growth under normoxia condition [20]. Overall, the rucaparib-mediated cytotoxicity would consist of inhibitions of multiple targets, which might distinct pharmacological profile of rucaparib from that of olaparib. Our study has shown a clear difference between the PARP inhibitors rucaparib and olaparib with respect to their PARP1dependent antiproliferative effects and their effect on pyruvatelactate metabolism. A recent study revealed that LDHA-associated lactate production inhibits tumour immunosurveillance by T and NK cells [21]. Thus, the rucaparib-mediated suppression of LDHassociated pathway might contribute to the therapeutic efficacy of rucaparib. Unfortunately, we have not yet clarified the molecular mechanism through which rucaparib represses LDH. Further study would elucidate how the pharmacological variation of each PARP inhibitor might contribute to these anticancer effects. Conflict of interest We have no financial relationships to disclose. Acknowledgement We thank Ayami Iwamoto and Yuri Mae for initial experiments of this study. This work was supported by JSPS KAKENHI Grant numbers 18K06632 to KN and 18K07302 to YS, and a Grant-in-Aid for JSPS Research Fellow 18J11600 to YN. We thank Editage (www. ediateg.jp) for English language editing. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.01.133. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.01.133.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Novel pharmacological effects of poly (ADP-ribose) polymerase inhibitor rucaparib on the lactate dehydrogenase pathway Yuma Nonomiya, Kohji Noguchi*, Kazuhiro Katayama, Yoshikazu Sugimoto Division of Chemotherapy, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo, 105-8512, Japan
a r t i c l e i n f o
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Article history: Received 26 January 2019 Accepted 30 January 2019 Available online 5 February 2019
Poly (ADP-ribose) polymerases (PARPs) are involved in various cellular events, including DNA repair. PARP inhibitors including olaparib and rucaparib, have been specially developed against breast and ovarian cancers deficient in DNA repair systems. In this study, we found that PARP1-defective olaparibresistant A2780 cells (ola-R cells) cells were still sensitive to two PARP inhibitors, rucaparib and veliparib. Metabolomic analysis revealed that rucaparib suppressed the lactate dehydrogenase (LDH)-mediated conversion of pyruvic acid to lactic acid in A2780 cells, although olaparib did not. The inhibition of LDH by siRNA-mediated knockdown or by LDH inhibitors suppressed the growth of ovarian cancer cells. Our results suggested that the suppression of the LDH-associated pathway contributed to the pharmacological effects of rucaparib. © 2019 Elsevier Inc. All rights reserved.
Keywords: Olaparib Rucaparib PARP1 LDHA Ovarian cancer
1. Introduction PARPs hydrolyse nicotine amide adenine dinucleotide (NADþ), catalyse the poly ADP-ribosylation (PARylation) of acceptor proteins on various amino acid residues, and are involved in DNA repair [1]. PARP1 is the most abundant nuclear protein in the PARP family, and PAPR1-mediated PARylation is a critical step for resealing single strand DNA breaks (SSB)s. Inhibition of PARP1 causes an accumulation of unrepaired SSBs, which results in double strand DNA break (DSB). The PARP inhibitors repress PARylation in cells at low concentrations [2], and several PARP inhibitors (olaparib, veliparib, rucaparib, niraparib, and talazoparib) have been developed for clinical use in cancer chemotherapy [3]. The BRCA1/2-inactive cells with low homologous recombination (HR) activity are highly sensitive to DNA damage, and thus the PARP inhibitors show synthetic lethal effect on BRCA-deficient cancer cells [4,5]. However, as a single agent, the cytotoxic activity of these PARP inhibitors is varied. Potent PARP inhibitors trap PARP1 at the base excision repair (BER) intermediate to induce DNA damage, which is known as PARPtrapping, and the PARP-trapping potency of PARP inhibitors is well correlated with their cytotoxic activities [6]. Intriguingly, BRCA mutations are not sufficient for prediction of PARP inhibitor efficacy
* Corresponding author. E-mail address: [email protected] (K. Noguchi). https://doi.org/10.1016/j.bbrc.2019.01.133 0006-291X/© 2019 Elsevier Inc. All rights reserved.
in individual patients. In this study we isolated three olaparib-resistant PARP1deficient cell lines (ola-R-3, -10, and -15) from human ovarian cancer A2780 cells, and found that the ola-R cells were still sensitive to the PARP inhibitor rucaparib, although such cells showed cross-resistance to niraparib and talazoparib. Our metabolomic analysis uncovered rucaparib-induced selective repression of the pyruvate-lactate metabolism. The inhibition of the pyruvate-lactate catalytic enzyme, lactate dehydrogenase (LDH), consistently suppressed the growth of ovarian cancer cell lines. Our experiments provide the first indication that the suppression of LDH-catalysed pyruvate-lactate metabolic pathway may be a novel pharmacological effect of rucaparib. 2. Materials and methods 2.1. Cells, plasmid, transfections, and chemical inhibitors A2780, OVCAR-3, and -8 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 medium (SigmaAldrich, St. Louis, MO, USA) supplemented with 7% fetal bovine serum and 50 mg/mL kanamycin at 37 C in a humidified atmosphere containing 5% CO2. A2780 cells were treated with olaparib (from 1 mmol/L to 40 mmol/L for 4 months) and the surviving colonies were selected to establish olaparib-resistant cell lines (ola-R3, -10, and -15). PARP1 cDNA (GenBank accession: NM_001618.3) was cloned by standard PCR method and PARP1-hemagglutinin
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(HA) stably-expressing A2780 cells were established by the transduction of PARP1-expressing retrovirus (pQCXIP, Clontech Laboratories Inc., Takara Bio Company, Mountain View, CA, USA), followed by selection with 500 ng/mL puromycin (Sigma-Aldrich). Olaparib, veliparib, rucaparib, niraparib, and talazoparib were purchased from Selleck Chemicals (Houston, TX, USA). Cisplatin was purchased from Sigma-Aldrich and SN-38 was provided by Yakult Honsha Co., Ltd. (Tokyo, Japan). 2.2. Cell growth inhibition assay and colony formation assay The cell growth inhibition assay was performed by using cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan) [7,8]. The degree of resistance was calculated by dividing the half-maximal inhibitory concentrations (IC50) of the cells of interest by those of the parent cells. Colony formation assay and the quantification of cell survival were performed as described previously [7]. 2.3. Reverse transcription (RT)ePCR analysis The total RNA was extracted by using an RNeasy kit (QIAGEN Sciences, Germantown, MD, USA). RT-PCR reactions for PARP1/2 were performed by using an RNA LA PCR kit (Takara, Ohtsu, Japan). PCR primer sequences are listed in Table S1. GAPDH cDNA was used as a loading control, as described previously [9]. The PCR conditions for PARP1 were 94 C for 1 min, followed by 25 cycles of 94 C for 30 s, 62 C for 30 s, and 72 C for 1.5 min, and then a final extension step of 72 C for 5 min. The PCR conditions for PARP2 were 94 C for 1 min, followed by 30 cycles of 94 C for 30 s, 62 C for 30 s, and 72 C for 2 min, and a final extension step of 72 C for 5 min. 2.4. Western blotting Western blotting was performed as described previously [7,8]. The antibodies used were directed against PARP1 (F-2), Rad51 (H92), LDH-A (E-9), LDH-B (431.1) (Santa Cruz Biotechnology, Santa Cruz, CA); MDR1þ3 (C219), PARP2 (ab115620), M2III-6 (Abcam, Cambridge, MA); PARG (D8B10), GAPDH (6C5) (Millipore, Billerica, MA); PAR (Trevigen, Helgerman CT, Gaithersburg, MD, USA); BRCA1 and BRCA2 (D9S6V) (Cell Signalling Technology, Danvers, MA, USA); HA (3F10) (Roche Diagnostics, Indianapolis, IN, USA); MRPm6 (Nichirei, Tokyo); M3Ⅱ-9 (Kamiya Biomedical, WA, USA), and antiBCRP polyclonal antibody (3488) (Chemicon, Temecula, CA, USA). GAPDH was used as the loading control.
foci, and analyzed the g-H2AX-positive cells with the Adobe® Photoshop CS4 Extended software (Adobe Systems Inc., San Jose, CA, USA). At least 100 cells were examined at each experimental point. 2.6. siRNA PARP1-targeting small interfering RNA (siRNA; siGENOME Human PARP1 (142) siRNA-SMARTpool), LDHA-targeting siRNA (siGENOME Human LDHA (3939) siRNA-SMARTpool), and LDHBtargeting siRNA (siGENOME Human LDHB (3945) siRNASMARTpool) were purchased from Dharmacon GE Healthcare (Amersham, UK), and a control scrambled siRNA (AllStars Negative Control siRNA) from Qiagen (Valencia, CA, USA). Lipofectamine 2000 (Invitrogen) was used for the transfection of cells with siRNAs. 2.7. Metabolomic analysis Cells were treated with 20 mmol/L olaparib or rucaparib for 12 h; all samples were prepared in triplicate. Methanol metabolite extraction was conducted in accordance with the Human Metabolome Technologies Inc., (HMT, Inc., Yamagata, Japan), metabolite extraction method for adherent cells. In brief, the cells were washed twice with 5% mannitol and treated with 0.8 mL methanol. The internal standard (0.55 mL of 8 mM internal standard) was added and the methanol extract was collected. After centrifugation, the extracts were subjected to centrifugation filtration through a 5 kDa cut-off filter. The extracts were stored at 80 C until analysis. The metabolomic analysis of 116 metabolites was conducted through a facility service (C-SCOPE) in Human Metabolome Technologies, Inc. (Yamagata) (N ¼ 3 in each group). The concentrations of all charged compounds were measured by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) and capillary electrophoresis tandem mass spectrometry (CE-QqQMS; CEMS/MS). Metabolite concentrations were normalized to viable cell counts. 2.8. Lactate assay The concentration of lactate produced from the cells was evaluated by using a lactate assay kit-WST (Dojindo Laboratories, Kumamoto, Japan). In brief, the cells were treated with PARP inhibitors for 24 h. The sample absorbance at 450 nm was read by using an Infinite M1000 microplate reader (Tecan Japan, Kanagawa, Japan).
2.5. Immunofluorescent microscopy 2.9. Statistical analysis Cells were seeded onto Scientific™ Nuc™ Lab-Tek™ II CC2™ chamber slides (4 104 cells in 1 mL/well; Thermo Fisher Scientific) or Eppendorf Cell Imaging Coverglasses (Eppendorf, Hamburg, Germany). After treatment with PARP inhibitors for 24 h the cells were fixed with ethanol/acetate (50:50) for 5 min at 4 C, and washed with phosphate-buffer saline (PBS) three times. Cells were permeabilized with 0.5% Triton X-100/PBS for 5 min at room temperature, washed three times with PBS, and blocked with 3% bovine serum albumin (BSA) in PBS for 60 min at room temperature. After three times wash with PBS, the cells were incubated with 2 mg/mL of anti-phospho-Histone H2AX Ser 139 (Merck Millipore, Billerica, MA, USA) for 120 min at room temperature. Then cells were probed with 1 mg/mL of Alexa Fluor 488 goat anti-mouse antibody (Invitrogen) and mounted in Prolong™ Gold Antifade reagent with 40 ,6diamidino-2-phenylindole (DAPI) (Invitrogen). The acquisition of images (640 640 pixels) was performed by using FV1000-D IX81 confocal microscopy (Olympus Corp., Tokyo, Japan). We defined the fluorescence signalling diameter as 0.5 mm or more as a g-H2AX
The quantitative results are presented as the mean ± SD (n 3). A scatter plot was generated by using GraphPad Prism 7 software (La Jolla, CA, USA). The Mann-Whitney U test and two-tailed Student's t-test were used to evaluate the statistical significance with IBM Statistics SPSS version 23.0 (IBM Corp., USA). In the metabolome analysis, Welch's t-test was used to compare metabolite concentrations. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Olaparib-resistant clones lacking PARP1 Three olaparib-resistant A2780 cell lines (ola-R-3, -10, and -15), showed strong resistance to olaparib and talazoparib, and moderate resistance to niraparib, compared with the parent cells (Fig. 1A and Supplementary Table S2). However, the ola-R cells showed only
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Fig. 1. Olaparib-resistant clones lacking PARP1 from A2780 cells. (A) Schematic protocol for the isolation of olaparib-resistant cells from A2780 cells. (B) The viabilities of drugtreated cells are shown as the mean ± SD from triplicate experiments. A2780 and ola-R cells were treated with various concentrations of PARP inhibitors olaparib (C), and rucaparib (D) for 24 h. Cells were fixed and g-H2AX was probed with anti-g-H2AX antibody (green signal), and the nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI, blue signal). Representative images are shown (upper images). Scale bar ¼ 10 mm. The number of g-H2AX foci in the g-H2AX foci-positive cells are summarized (counted cell number >100 in each sample) as a dot plot (lower graphs). Statistical significance was determined by using the Mann-Whitney U test, *p < 0.05, **p < 0.01. Horizontal red lines represent the median. (E) Expression of PARP1, PARP2, and other related proteins in the ola-R cells was analyzed by western blotting. (F) The mRNA expression of PARP1 and PARP2 in the ola-R cells was analyzed by using RT-PCR. The mRNA of PARP1 was detected by four different primer sets (Supplementary Table S1). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
marginal levels of cross-resistance to rucaparib and veliparib, and also showed 2- to 5-fold hypersensitivity to cisplatin and the topoisomerase I inhibitor SN-38 (Fig. 1A and Supplementary Table S2), suggesting that DNA damage responses might be compromised in ola-R cells. Histone H2AX phosphorylation (gH2AX) is a marker of DSBs [10], and PARP inhibitors-induced gH2AX foci are associated with levels of cellular DNA damage. Olaparib increased the number of g-H2AX foci in a concentrationdependent manner (upto 100 mmol/L) in parent A2780 cells, whereas the degree of increase was lower in the ola-R cells (Fig. 1C). In A2780 cells, we observed massive cell death after the treatment of rucaparib at 100 mmol/L for 24 h, so that, the maximum concentration of rucaparib was set at 30 mmol/L in Fig. 1D. Compared with the cases in the olaparib-treatment, the induction of g-H2AX foci by rucaparib appeared to be weak in A2780 cells and the ola-R cells. Western blotting showed that PARP1 protein was lost in all the ola-R cells and PARylation in the cell lysates was also lower in ola-R cells than in A2780 cells. (Fig. 1E). The expression of the PARP2, PARG and GAPDH proteins were comparable between A2780 and ola-R cells. RT-PCR analysis showed that mRNA expression of PARP1,
when targeted to four different parts, was not detected in ola-R cells (Fig. 1F), which indicated that PARP1 was deficient at the mRNA level in the ola-R cells. 3.2. Knockdown of PARP1 in A2780 cells did not confer rucaparibresistance A previous study reported that defect in PARP1 expression causes resistance to PARP1 inhibitors [11]. Transfection of PARP1 siRNA repressed the PARP1 protein selectively in A2780 cells (Fig. 2A), and the number of g-H2AX foci in PARP1 knockdown cells was significantly lower than that in cells transfected with control siRNA after olaparib treatment (Fig. 2BeC). Conversely, the reintroduction of PARP1 in ola-R cells re-sensitized ola-R cells to olaparib-mediated growth inhibition and g-H2AX induction (Supplementary Fig. S1). The colony formation assay showed that PARP1 knockdown conferred resistant to three inhibitors, olaparib, talazoparib and niraparib (Fig. 1D). However, there was no obvious resistance to rucaparib or veliparib in the PARP1 knockdown cells (Fig. 1D). Additionally PARP1 knockdown of another ovarian cancer cell line, OVCAR-8, did not confer resistance to rucaparib
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Fig. 2. Rucaparib sensitivity was unaffected by PARP1 knockdown. (A) Knockdown of PARP1 in A2780 cells. In the left panel, the experiment protocol is shown. In the right panels, the expression of PARP1 was examined by western blotting. Confocal microscopy analysis was performed on cells treated with olaparib (0, 1, 10, and 100 mmol/L) for 24 h. Images of g-H2AX (green signal)- and nuclei by DAPI (blue signal)-staining (B) and the quantification analysis (C) were shown as in Fig. 1. Statistical significance was determined by the Mann-Whitney U test, **p < 0.01. Horizontal red lines represent the medians. Cell survival after the treatment of PARP inhibitors (olaparib, veliparib, rucaparib, talazoparib, and niraparib) in siRNA-transfected A2780 (D) and OVCAR-8 cells (F) were evaluated by using a colony formation assay. Knockdown of PARP1 protein in A2780 cells is shown in A and in OVCAR-8 cells in E. Representative images of surviving stained cells are shown in the upper panels of D and F. The upper triplicate wells were control siRNA-transfected cells, and the lower triplicate wells were PARP1 siRNA-transfected cells. Reproducibility was confirmed twice and representative quantitative results are shown as the mean ± SD from triplicate experiments in the lower graphs of D and F. Statistical significance was determined by using Student's t-test. *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
(Fig. 1EeF). These data suggested that PARP1 was not essential for the induction of cytotoxicity by rucaparib and veliparib, although it was a critical pharmacological target for olaparib, talazoparib, and niraparib. 3.3. Differing effects on metabolic profiles by olaparib and rucaparib We analyzed the metabolic changes after treatment of rucaparib and olaparib for 12 h in A2780 cells (Fig. 3). Quantitative metabolomic analysis was performed on 116 compounds associated with the central carbon metabolic pathways, such as glycolysis, TCA cycle, amino acid synthesis, and the nucleotide synthesis pathways. The change in metabolite concentrations was illustrated by a clustered heat map in Fig. 3A. Principal component analysis revealed a clear separation between the control, olaparib-, and
rucaparib-treated samples (Fig. 3B). Rucaparib-treated samples were distinguishable from control and olaparib-treated samples by the second principal component (PC2)-axis, and the factor loadings (the top nine compounds each for positive and negative values) contributing to the PC2-axis were listed (Fig. 3C). A metabolic pathway map summarized the results (Supplementary Fig. S2A), and the metabolites corresponding to the factor loading contributing to PC2 in the purine metabolic pathway were negatively downregulated, whereas those in the glycolysis pathway were elevated by rucaparib treatment (Fig. 3D). It was reported that rucaparib could inhibit hexose 6-phosphate dehydrogenase (H6PD) as a secondary target, which is mapped in the pentose phosphate pathway upstream of the purine metabolic pathway [12]. Thus, our data would show the first metabolic evidence for the rucaparibselective suppression of some metabolites (PRPP, IMP, and XMP downstream of the pentose phosphate pathway) by rucaparib-
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Fig. 3. Metabolomic profiles after olaparib- and rucaparib-treatment. A2780 cells were treated with olaparib or rucaparib for 12 h and triplicate samples of the methanol extracts were subjected to metabolomic analysis. (A) Standardized relative changes of 96 metabolites from triplicate samples are visualized by clustered heat map, created at the CIMminor site (https://discover.nci.nih.gov/cimminer/home.do). The average linkage was used for the clustered algorithm and the Euclidean algorithm was used for the distance method. (B) The principal component analysis (PCA) separated the metabolic profiles of three groups (n ¼ 3 in each group). (C) The top nine metabolites (positively and negatively) determined from the second principal component (PC2) factor loadings are listed. (D) A summary of the glycolysis pathway and the pentose phosphate pathway is shown. The values shown in the bar graph represent the mean ± S.D. of triplicate samples, and the blue, red, and green bars are the data for the control, olaparib-treated, and rucaparib-treated samples, respectively. The metabolite abbreviations are glucose 6-phosphate (Glucose-6P), fructose 6-phosphate (Fructose-6P), fructose 1,6-bisphosphate (Fructose-1, 6 BP), glyceraldehyde 3-phosphate (Glyceroaldehyde-3P), 1,3-bisphosphoglycerate (1,3-BP Glycerate), 3-phosphoglycerate (3-P Glycerate), 2-phosphoglycerate (2-P Glycerate), phosphoenolpyruvate (P-Enolpyruvate), D-ribose 5-phosphate (D-Ribose-5P), phosphoribosyl diphosphate (PRPP), inosine monophosphate (IMP), and xanthosine monophosphate (XMP). The metabolic enzymes listed are hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD), hexose-6-phosphate dehydrogenase (H6PD), glucose-6-phosphate isomerase (GPI), phosphofructokinase (PFKP), aldolase fructose-bisphosphate A/B/C (ALDOA/B/C), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase 2 (PGAM2), enolase (ENO), pyruvate kinase, muscle/liver and RBC (PKM/LR), lactate dehydrogenase A/B (LDHA/B), and inosine monophosphate dehydrogenase (IMPDH). Statistical significance was determined by Student's t-test. *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
mediated inhibition of H6PD. We also found that the pyruvate to lactate conversion was suppressed by rucaparib (Fig. 3D), which suggested that this pathway might represent a third target for rucaparib. We reexamined the change in lactate production of PARP inhibitortreated cells by using a conventional assay kit. Consistent with the data from the metabolomic analysis, rucaparib significantly reduced lactate production in A2780, OVCAR-3, and OVCAR-8 cell lines, but olaparib did not (Fig. 4A). The lactate production was not affected by olaparib and talazoparib at doses capable of growth inhibition (Supplementary Fig. S2B). Next, we examined the possible contribution of LDH, which catalyses the reversible conversion of pyruvate to lactate, to the cell growth and survival of ovarian cancer cells. LDH is encoded by
three genes, LDHA, LDHB, and LDHC; LDHA and LDHB are widely expressed [13]. Knockdown of LDHA/B, especially LDHA, significantly suppressed the cell growth of A2780 and OVCAR-3 cells (Fig. 4BeC). We also confirmed the growth inhibitory effects of two LDH inhibitors, FX-11 and NHI-1, on several ovarian cancer cell lines (Supplementary Fig. S3), but we did not see a considerable direct inhibition of LDH enzymatic activity by PARP inhibitors in vitro (Supplementary Fig. S4). These results suggested that the inhibition of LDH-associated metabolic pathway was involved in the pharmacological actions of rucaparib (Supplementary Fig. S5). 4. Discussion Olaparib and rucaparib have similar potencies of PARP-trapping
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Fig. 4. Rucaparib-mediated inhibition of lactate production. (A) A2780, OVCAR-3, and OVCAR-8 cells were treated with various concentrations of rucaparib and olaparib for 24 h. The concentrations of lactate in the medium are shown. (B) The knockdown experiment protocol of LDHA and LDHB in A2780 and OVCAR-3 cells. (C) LDHA and LDHB protein expression after simultaneous transfection of siRNAs were confirmed by western blot analysis (upper panels). The cells were counted at 5 days after transfection (lower graphs). Statistical significance was determined by Student's t-test. **p < 0.01.
mechanisms [11,14], and, consistently, the growth inhibitory effects (IC50 values) of olaparib and rucaparib were similar in A2780 cells (1.1 and 2.4 mmol/L, respectively). However, in this study, we found that PARP1-deficiency conferred olaparib-resistance, but not rucaparib-resistance in A2780 cells. This observation implied that there would be a PARP1-independent cytotoxic action of rucaparib, consistently with previous discussion in other studies [14e16]. Our metabolome analysis additionally suggest that the suppression of the LDH-mediated metabolic pathway is involved in rucaparibmediated cytotoxicity. Principal component analysis of metabolomic experiments distinguished olaparib from rucaparib, and metabolomic analysis
indicated the downregulation of the purine synthetic pathway downstream of H6PD (Fig. 3D). Rucaparib has the ability to inhibit H6PD [12], and the knockdown of H6PD results in the inhibition of cancer cell growth [17,18]. We presumed that H6PD inhibition would be also involved in the rucaparib-induced growth inhibition of PARP1-defective ola-R cells. Moreover, we found that pyruvate was increased and, conversely, lactate was significantly decreased after rucaparib treatment (Fig. 3D), which suggested the selective inhibition of LDH-mediated metabolism by rucaparib. Pyruvate to lactate conversion is a critical step for NADþ production and glycolysis in cancer cells, and LDH has been suggested as a potential therapeutic target in cancer cells [13,19]. We confirmed that LDH
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knockdown and LDH inhibitors suppressed ovarian cancer cell growth (Fig. 4B and C and S3). These data suggested that the LDHmediated metabolic pathway was an additional pharmacological target of rucaparib. As we did not clarify possible off-target effect of a LDH inhibitor FX-11, it would be better to use more specific LDH inhibitor such as GNE-140 to see contribution of LDH enzymatic activity in cell growth inhibition [20]. Moreover, double genetic knockout of LDHA and LDHB which completely inhibits LDH activity, leads to the shifting their metabolism to oxidative phosphorylation with reduction of cell growth, but is not sufficient to abolish tumour cell growth under normoxia condition [20]. Overall, the rucaparib-mediated cytotoxicity would consist of inhibitions of multiple targets, which might distinct pharmacological profile of rucaparib from that of olaparib. Our study has shown a clear difference between the PARP inhibitors rucaparib and olaparib with respect to their PARP1dependent antiproliferative effects and their effect on pyruvatelactate metabolism. A recent study revealed that LDHA-associated lactate production inhibits tumour immunosurveillance by T and NK cells [21]. Thus, the rucaparib-mediated suppression of LDHassociated pathway might contribute to the therapeutic efficacy of rucaparib. Unfortunately, we have not yet clarified the molecular mechanism through which rucaparib represses LDH. Further study would elucidate how the pharmacological variation of each PARP inhibitor might contribute to these anticancer effects. Conflict of interest We have no financial relationships to disclose. Acknowledgement We thank Ayami Iwamoto and Yuri Mae for initial experiments of this study. This work was supported by JSPS KAKENHI Grant numbers 18K06632 to KN and 18K07302 to YS, and a Grant-in-Aid for JSPS Research Fellow 18J11600 to YN. We thank Editage (www. ediateg.jp) for English language editing. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.01.133. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.01.133.
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