A novel mechanism of methylglyoxal cytotoxicity in prostate cancer cells

The International Journal of Biochemistry & Cell Biology 45 (2013) 836–844

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A novel mechanism of methylglyoxal cytotoxicity in prostate cancer cells Cinzia Antognelli a , Letizia Mezzasoma a , Katia Fettucciari b , Vincenzo Nicola Talesa a,∗ a b

Department of Experimental Medicine and Biochemical Sciences, University of Perugia, via del Giochetto 06122, Perugia, Italy Department of Clinical and Experimental Medicine, University of Perugia, via Enrico dal Pozzo 06122, Perugia, Italy

a r t i c l e

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Article history: Received 2 July 2012 Received in revised form 2 December 2012 Accepted 8 January 2013 Available online 16 January 2013 Keywords: Methylglyoxal Argpyrimidine AGEs Apoptosis Glyoxalase I Cell proliferation

a b s t r a c t Methylglyoxal is one of the most powerful glycating agents of proteins and other important cellular components and has been shown to be toxic to cultured cells. Methylglyoxal cytotoxicity appears to occur through cell-cycle arrest but, more often, through induction of apoptosis. In this study we examined whether, and through which molecular mechanism, methylglyoxal affects the growth of poorly aggressive LNCaP and invasive PC3 human prostate cancer cells, where its role has not been exhaustively investigated yet. We demonstrated that methylglyoxal is cytotoxic on LNCaP and PC3 and that such cytotoxicity occurs not via cell proliferation but apoptosis control. Moreover, we demonstrated that methylglyoxal cytotoxicity, potentiated by the silencing of its major scavenging enzyme Glyoxalase I, occurred via different apoptotic responses in LNCaP and PC3 cells that also showed a different susceptibility to this metabolite. Finally, we showed that the observed methylglyoxal apoptogenic role involved different molecular pathways, specifically mediated by methylglyoxal or methylglyoxal-derived argpyrimidine intracellular accumulation and NF-kB signaling-pathway. In particular, in LNCaP cells, methylglyoxal, through the accumulation of argpyrimidine, desensitized the key cell survival NF-kB signaling pathway, which was consistent with the modulation of NF-kB-regulated genes, triggering a mitochondrial apoptotic pathway. The results suggest that this physiological compound merits investigation as a potential chemo-preventive/-therapeutic agent, in differently aggressive prostate cancers. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Methylglyoxal (MG) is an extremely reactive ␣-ketoaldehyde endogenously produced by various metabolic pathways, including the dephosphorylation of glycolytic intermediates, metabolites of the polyol pathway, and aminoacetone metabolism (Rabbani and Thornalley, 2012). MG is one of the most powerful glycating agents of proteins and other important cellular components (Lo et al., 1994; Vaca et al., 1994). MG glycation reactions result in the production of advanced glycation end products (AGEs). Among them, argpyrimidine (AP) represents one of the major products

Abbreviations: MG, methylglyoxal; AGEs, advanced glycation end products; AP, argpyrimidine; ROS, reactive oxygen species; p38 MAPK, mitogen-activated protein kinases; JNK, c-Jun N-terminal kinases; GLOI, Glyoxalase I; ATCC, American Type Culture Collection; RIPA buffer, radioimmunoprecipitation assay buffer; HRP, horse radish peroxidase; mAb, monoclonal antibody; ECL, enhanced chemiluminescence; TBST, tris buffer saline TWEEN-20; siGLOI, siRNA oligonucleotides targeting GLOI; siCONTROL, non-targeting siRNA oligos; qRT-PCR, Real Time TaqMan PCR analysis; TRITC, tetramethylrhodamine B isothiocyanate; FITC, fluorescein isothiocyanate; PBS, phosphate buffered saline; DAPI, 4 ,6-diamidino-2-phenylindole. ∗ Corresponding author. Tel.: +39 075 5857483; fax: +39 075 5857483. E-mail addresses: [email protected] (C. Antognelli), [email protected] (L. Mezzasoma), [email protected] (K. Fettucciari), [email protected] (V.N. Talesa). 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2013.01.003

deriving from MG modifications of proteins arginine residues (Kim et al., 2012a,b). It has been shown that MG is toxic to cultured cells (Amicarelli et al., 2001, 1998; Kang et al., 1996; Okado et al., 1996). In this regard, MG shows significant anti-proliferative properties (Gillespie, 1975; Szent-Gyorgyi, 1968) as it can irreversibly modify nucleic acids (Amicarelli et al., 2003; Bair et al., 2010; Kang et al., 1996; Santel et al., 2008). However, it has been recently demonstrated that MG is also able to promote proliferation of vascular smooth muscle cells (Chang et al., 2011). Therefore, a cell-type specific MG proliferative effect exists, and the role of MG in cell proliferation control remains open to question. More often, MG cytotoxicity occurs through the induction of apoptosis (Chan et al., 2007; Ghosh et al., 2011a,b; Huang et al., 2011; Oba et al., 2012). There are multiple mechanisms by which MG can induce apoptosis, e.g., through the generation of reactive oxygen species (ROS) (Amicarelli et al., 2003; Chan et al., 2007; Du et al., 2001; Li et al., 2007), induction of oxidative DNA damage (Kim et al., 2011) or accumulation of a specific MG-derived AGE (Kim et al., 2010). Finally, it has been recently demonstrated that MG induces apoptosis through the inhibition of both glycolysis and mitochondrial respiration and is specific against cancerous cells (Ghosh et al., 2011a). In this regard, further experimental evidences indicated mitochondrial complex I of exclusively malignant cells as the target of MG, strongly suggesting that such a mitochondrial

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complex might be critically altered in these cells (Ghosh et al., 2011b). The antiproliferative and apoptogenic activity of MG has been investigated for potential pharmacological application in cancer chemotherapy (Milanesa et al., 2000), even though cells are not equally sensitive to its toxicity (Amicarelli et al., 2003; Du et al., 2000; Ghosh et al., 2011b; Talukdar et al., 2009). In addition to MG, AGEs themselves can trigger apoptosis, through increasing oxidative stress or inducing the expression of pro-apoptotic cytokines (Chuang et al., 2011; Denis et al., 2002; Kasper and Funk, 2001; Kim et al., 2012a,b; Lin et al., 2012). Although the action of MG in influencing cellular components has been studied, and amino acid residues affected by MG have been identified, detailed molecular events caused by MG, which activates the intracellular signal transduction pathway and leads the cells to apoptosis, have not yet been completely clarified (Thornalley and Rabbani, 2011). A study reported that MG-induced alterations in growth factor receptor signaling might be implicated in the development of MG cytotoxicity (Cantero et al., 2007). Another study described that MG affects cell viability via desensitization of gp130/STAT3 signaling, which is the key signaling pathway for cell survival in neuroglial cells (Lee et al., 2009). Furthermore, p38 mitogenactivated protein kinases (MAPK) activation was suggested to be a key signaling intermediate of MG-induced apoptosis in kidney cells (Liu et al., 2003) and Schwann cells (Fukunaga et al., 2005), while (Chan et al., 2007) the c-Jun N-terminal kinases (JNK) pathway appears to be important for MG-induced apoptosis in human osteoblasts. Therefore, the apoptogenic role of MG occurs in celltype signaling pathways. In prostate cancer, the role of MG has been scarcely investigated. Two studies on the effect of MG only on PC3 cells showed that it is capable of inducing apoptosis due primarily to a blocking of the cell cycle progression and glycolytic pathway (Milanesa et al., 2000) or to a reduction in specific enzymatic activities (Davidson et al., 2002). However, to our knowledge, signaling pathways involved in MG cytotoxicity have never been investigated in prostate cancer cell models. Glyoxalase system, consisting of Glyoxalase I (GLOI, EC 4.4.1.5) and II (GLOII, EC 3.1.2.6) enzymes, represents the major cellular defence against MG- and AGEs-induced cytotoxicity (Nakadate et al., 2009; Rabbani and Thornalley, 2012). Increased expression of GLOI occurs in some tumors, such as breast and ovarian cancers (Rulli et al., 2001; SmithBeckerman et al., 2005), prostate cancer (Davidson et al., 1999) and melanoma (Bair et al., 2010). In addition, it has been shown that GLOI is involved in resistance of human leukemia cells to antitumor agent-induced apoptosis (Sakamoto et al., 2000) and, more recently, in the survival of aggressive and invasive prostate cancer cells (Antognelli et al., 2012). In the present work we studied whether, and through which mechanism, MG, alone or in combination with GLOI silencing, affects the growth of differently aggressive and invasive LNCaP and PC3 human prostate cancer cell lines, where its cytotoxic role has never or scarcely been investigated (Milanesa et al., 2000; Davidson et al., 2002), respectively. 2. Materials and methods 2.1. Reagents All reagents were purchased from Sigma–Aldrich (Milan, Italy) unless stated otherwise. 2.2. Human prostate cancer cell lines and MG treatment Human prostate adenocarcinoma LNCaP and PC-3 cells were obtained from ATCC (American Type Culture Collection) (Milan, Italy) and routinely maintained at 37 ◦ C in 5% CO2 in RPMI 1640

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supplemented with 10% heat inactivated (1 h at 56 ◦ C) FBS, 1× lglutamine, 1 mM sodium pyruvate, 1× non-essential amino acids, 100 units/ml of penicillin and 0.1 mg/ml of streptomycin (Invitrogen, Milan, Italy). The cells were incubated with MG (purified by distillation before use) in appropriate cell culture conditions for 24, 48 and 72 h, in preliminary experiments, and for 72 h in the subsequent experiments. 2.3. Cell proliferation Cell proliferation was determined by [3H]thymidine incorporation assay (Tso et al., 2000). 2.4. Cell cycle analysis and apoptosis evaluation by flow cytometry Cell cycle distribution and apoptosis were evaluated as previously described (Antognelli et al., 2012). 2.5. TUNEL assay (ApoAlert® DNA fragmentation assay) The nuclear DNA fragmentation was evaluated by a commercial kit (ApoAlert® DNA Fragmentation Assay, Clontech Laboratories, Inc.) in accordance with the manufacturer’s instructions. The ApoAlert® DNA fragmentation assay kit detects apoptosis-induced nuclear DNA fragmentation via a fluorescence assay. The assay is based on terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL). TdT catalyzes incorporation of fluorescein-dUTP at the free 3 -hydroxyl ends of fragmented DNA. Fragments that incorporate the fluorescent probe can be quantified with a flow cytometer using  = 520 nm (FITC channel). Each sample is also treated with propidium iodide (PI) fluorescent at  > 620 nm, which gets incorporated in apoptotic and nonapoptotic cells. The percentage of green fluorescent cells and red fluorescent cells is considered as an apoptotic hallmark (Russo et al., 2010). 2.6. Whole-cell protein extraction and Western blot For extraction of total proteins, cells were lysed in pre-cooled radioimmunoprecipitation assay (RIPA) lysis buffer (Fettucciari et al., 2006). For subcellular fractionation, cells were resuspended in Mitobuffer (Fettucciari et al., 2006). For Western blot, samples of equal protein concentration (40 ␮g) were treated with Laemmli buffer (Invitrogen, Milan, Italy), boiled for 5 min, resolved on 10, 12 or 15% SDS-PAGE and then blotted onto a nitrocellulose membrane, using iBlot Dry Blotting System (Invitrogen Life Technologies, Milan, Italy). Non-specific binding sites were blocked in Roti-Block (Roth, Germany) for 1 h at room temperature, incubated overnight at 4 ◦ C with an appropriate dilution of the primary specific Abs (mouse anti GLOI mAb, BioMac, GmbH, Leipzig, Germany; mouse anti-AP mAb, Antibodies-online, GmbH, Aachen, Germany; mouse anti-␤-actin mAb, rabbit anti-Bcl-XL polyclonal Ab, rabbit anti-Bax (N20)polyclonal Ab, Santa Cruz Biotechnology, Heidelberg, Germany; rabbit anti caspase-3 polyclonal Ab, rabbit anti phospho-I-kappa-B-alpha (Ser32) (14D4) and anti I-kappa-B-alpha (44D4) mAbs, Cell Signaling Technology, Milan, Italy; mouse antiBcl-2 mAb, DAKO, Milan, Italy; mouse anti-Cytochrome c (Cyt c) mAb, BD Pharmingen, Milan, Italy; mouse anti-Cyt c oxidase subunit IV (Cox IV) mAb, Molecular Probes, Monza, Italy). After washing with TBST, antigen–antibody complexes were detected by incubation of the membranes for 1 h at room temperature with the appropriated HRP-conjugated secondary Ab and revealed using ECL system (Amersham Pharmacia, Milan, Italy). Densitometry analyses were performed in ImageJ software. As internal loading controls and for protein expression normalizing purpose, all

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membranes were subsequently stripped of the first Ab in a stripping buffer (100 mM 2-ME, 2% SDS, and 62.5 mM Tris–HCl, pH 6.8) and re-probed with anti-␤-actin mAb. To confirm results, membranes were also re-probed with mouse anti-pan-cytokeratin mAb (clone PCK-26) (Sigma–Aldrich, Milan, Italy) that yielded comparable results (data not shown). 2.7. Methylglyoxal (MG) assay Intracellular MG was measured as previously described (Antognelli et al., 2012; Barua et al., 2011). 2.8. siRNA-GLOI transfection LNCaP and PC3 cells were transiently transfected with a 50 nmol or 100 nmol pool of four siRNA oligonucleotides (oligos) targeting GLOI (siGLOI) or with non-targeting siRNA oligos (siCONTROL), using the Dharmafect 3 or 2 transfection reagent (M-Medical, Milan, Italy) and following a standard procedure (Cabello et al., 2009). Cells were incubated with the siRNAs in appropriate cell culture conditions for 24, 48 and 72 h, in preliminary experiments, and for 72 h in the subsequent experiments. The effect of silencing was evaluated by measuring the relative expression of GLOI vs ␤-actin by quantitative RT-PCR (qRT-PCR) and analyzing the specific protein knockdown by Western blot using the specific Ab, and ␤-actin as loading control. In mock-transfection, all vehicles were used except for the siRNA. Cells were vital throughout the course of all experiments as determined by Trypan Blue exclusion assay. In all transfection experiments, mock transfection and transfection with control non-targeting siRNA (to exclude off-target effects of GLOI silencing) did not affect all the studied parameters in both cell lines, providing results comparable with those obtained from untreated LNCaP and PC3 cells (data not shown).

et al. (2003). GLOI activity was assayed according to Mannervik et al. (1981). 2.12. DNA fragmentation assay DNA was extracted from both floating and attached cells after a 72 h treatment with 1 mM MG or siGLOI and DNA fragmentation assay was carried out by agarose gel electrophoresis (Hoque et al., 2008). 2.13. Immunofluorescence microscopy LNCaP and PC3 cells were grown in 4-well culture dishes to approximately 60% confluence before treatment with 1 mM MG in combination with siRNA targeting GLOI, for 72 h. For immunofluorescence staining of F-actin cells were washed with PBS and fixed in 3.7% paraformaldehyde. The slides were, then, washed with PBS three times and permeabilized with 0.1% Triton X-100 and, then, after washing, incubated with tetramethylrhodamine B isothiocyanate (TRITC)-phalloidin mAb. The cells were briefly rinsed in PBS + TWEEN-20 0.1%, followed again by three washes in PBS + TWEEN-20 0.1% and one wash in PBS. After washing with PBS, nuclear counter staining (DAPI) was performed for 5 min. For immunofluorescence staining of ␣-tubulin the slides were washed with PBS and then fixed in cold methanol (−20 ◦ C), After washing with PBS, cells were permeabilized with 0.1% Triton X-100 and blocked with 3% BSA. Slides were then incubated with primary ␣-tubulin mAb. After washing with PBS, the slides were incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse Ab followed by washes in phosphate buffered saline (PBS) + TWEEN-20 0.1%. Cells were nuclear stained with 4 ,6diamidino-2-phenylindole (DAPI). Coverslips were then mounted on slides with PBS/glycerol (1:1) and images were obtained using a fluorescent light microscopy (Zeiss, Germany).

2.9. RNA isolation and cDNA synthesis Total cellular RNA was isolated using TRIzol Reagent (Invitrogen, Milan, Italy). cDNA was then synthesized from 1 ␮g of RNA using the RevertAidTM H Minus First Strand cDNA Synthesis Kit (M-Medical, Milan, Italy).

2.14. Affinity purification of the 40 kDa MG-modified protein AP and amino acid sequence analysis Purification and identification of MG-modified protein Argpyrimidine (AP) was performed according to Sakamoto et al. (2002).

2.10. Real Time TaqMan PCR analysis (qRT-PCR)

2.15. Statistical analysis

GLOI expression vs ␤-actin was evaluated by quantitative Real Time TaqMan PCR analysis (qRT-PCR) on a MX3005P Real-Time PCR System (Agilent Technology, Milan, Italy). The sequences of oligonucleotide primers and TaqMan probes were as follows: GLOI: 5 -ctctccagaaaagctacactttgag-3 (sense, 400 nM), 5 -cgagggtctgaattgccattg-3 (antisense, 400 nM), (TaqMan Probe, 5 -FAM-tgggtcgcatcatcttcagtgccc-TAMRA-3 200 nM); ␤-actin: 5 -cactcttccagccttccttcc-3 (sense, 600 nM), 5 -acagcactgtgttggcgtac-3 (antisense, 600 nM), 5 -TEXASREDtgcggatgtccacgtcacacttca-BHQ-3 (TaqMan Probe, 200 nM). PCR reactions were performed in a total volume of 25 ␮l, containing 250 ng of cDNA, 1× Brilliant QPCR master mix (Agilent Technology, Milan, Italy), 0.5 ␮l of ROX Reference Dye (Agilent Technology, Milan, Italy) and a concentration of specific primers and probes. The thermal cycling conditions were as follows: 1 cycle at 95 ◦ C for 10 min, followed by 45 cycles at 95 ◦ C for 20 s and 55 ◦ C for 1 min. Data for comparative analysis of gene expression were obtained using the Ct method (Livak and Schmittgen, 2001).

Unless differently indicated, the results are given as means ± SD of three independent experiments performed in quadruplicate. The data were analyzed by Student’s t test (*P < 0.01; **P < 0.001). In figures, results have been reported as fold-change, to allow a comparative analysis of parameters expressed with different units.

2.11. GLOI enzymatic activity Cell extract preparation for GLOI enzymatic activity and protein content measurement were performed according to Amicarelli

3. Results 3.1. MG affects LNCaP and PC3 cell growth not via cell proliferation but apoptosis control The effect of 1 mM MG on LNCaP and PC3 cell growth was studied by evaluating proliferation and apoptosis. LNCaP exhibited a lower rate of proliferation compared to PC3 cells (Fig. 1A). Analysis of cell cycle distribution revealed a significantly higher amount of PC3 cells in S-phase [mean (%) ± SD, 16.4 ± 2.0] and G2/M-phase [mean (%) ± SD, 12.8 ± 6.1] compared to LNCaP cells in S-phase [mean (%) ± SD, 5.6 ± 0.6] and G2/M-phase [mean (%) ± SD, 6.7 ± 0.9] (Fig. 1B). Following MG administration, no significant changes were observed in comparison to the typical proliferative trends (Fig. 1A) and cell cycle phases (Fig. 1B) of both cell lines. Comparable results (data not shown) were obtained after GLOI genetic antagonism (Fig. 2). Mean values of GLOI activity, expressed as

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Fig. 1. Methylglyoxal (MG) effect on LNCaP and PC3 cell proliferation. (A) proliferation curves. Number of cells at seeding: 1 × 105 in both cell lines. *P < 0.01 or **P < 0.001 compared to cell number at seeding (0 h); (B) cell-cycle analysis. Results are presented as the mean ± SD of three independent experiments tested in quadruplicate.

␮mol/min/mg protein, in non transfected (NT) LNCaP and PC3 cells were (mean ± SD) 1.45 ± 0.027 and 0.85 ± 0.01, respectively, while in silenced LNCaP and PC3 cells, 0.26 ± 0.058 and 0.18 ± 0.014, respectively. On the contrary, an increase in LNCaP (about 10-fold) and PC3 cells (about 7-folds) apoptosis was observed, compared to the spontaneous one, confirmed by DNA fragmentation visualization (Fig. 3), and paralleled by MG or AP intracellular levels modifications in both cell lines (Fig. 4). In particular, a significant increase in both MG and AP or only MG intracellular levels was observed in LNCaP and PC3 cells, respectively, compared to controls (Fig. 4). Mean values of intracellular MG content, expressed as pmol/107 cells, were in LNCaP (mean ± SD) 78.90 ± 2.6 vs 10.52 ± 0.4 (controls) and in PC3 63.12 ± 2.5 vs 13.15 ± 0.9 (controls). All these modifications were further enhanced in the presence of GLOI silencing. In fact, the genetic antagonism of GLOI dramatically sensitized LNCaP cells to apoptosis (Fig. 5A), also inducing an additional increase in both MG (315.6 ± 0.8 vs 10.52 ± 0.4) and AP intracellular levels (Fig. 5B), while moderately sensitized PC3 cells to apoptosis (Fig. 5A), inducing an increase only in MG levels (134.13 ± 0.8 vs 13.15 ± 0.9) (Fig. 5C). Again, DNA fragmentation visualization confirmed the apoptotic responses (Fig. 5A). In order to investigate whether the observed increase in apoptosis, paralleled by no changes in cell proliferation, after MG administration, influenced cell growth, the percentage of apoptotic cells was measured by the TUNEL method, which detects apoptosisinduced nuclear DNA fragmentation via a fluorescence and which is thought to be paralleled by the degree of apoptosis. The percentage of apoptotic cells measured after MG administration, calculated by the ratio between the values of green fluorescence (apoptotic cells) and red fluorescence (total amount of cells) by flow cytometry (FITC or PI channel), was about 56% for LNCaP and 31% for PC3 cells.

Fig. 2. Glyoxalase I (GLOI) silencing by small interfering RNA (siRNA) in LNCaP and PC3 cells. Cells were transfected for 72 h with vehicle alone (Mock), control nontargeting siRNA (siCONTROL) and GLOI-siRNA (siGLOI) or non-transfected (NT). (A) GLOI mRNA expression by Real Time TaqMan PCR analysis (qRT-PCR) performed with 250 ng of cDNA and a concentration of specific primers and probes; (B) GLOI protein level (40 ␮g proteins) by using anti-GLOI Ab and anti-␤-actin as loading control and densitometric analysis from Western blots; (C) GLOI enzymatic specific activity. Data are expressed as percentage of NT. All the data are means ± SD of four independent experiments done in triplicate; **P < 0.001.

3.2. MG, in the presence of GLOI silencing, induces LNCaP and PC3 cytoskeleton disorganization In order to investigate whether the observed MG-induced apoptosis was also paralleled by modification in cytoskeleton organization, LNCaP and PC3 cells were immunostained for F-actin and ␣-tubulin, after MG and siGLOI co-treatment that induced the maximum apoptotic effect. A dramatic change in cell morphology and a complete disorganization of the actin cytoskeleton was observed in both cell lines, compared to controls (Fig. 6A). Similarly, a disruption of the ␣-tubulin cytoskeleton organization was

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Fig. 3. Methylglyoxal (MG) effect on LNCaP and PC3 cells apoptosis. Apoptosis was evaluated by FACS and confirmed by DNA fragmentation (inserts). Histograms indicate means ± SD of three different cultures tested in quadruplicate. **P < 0.001 vs untreated cells.

observed, as indicated by a decrease in intracellular ␣-tubulin staining (Fig. 6B). 3.3. MG triggers a mitochondrial apoptotic pathway in LNCaP cells via AP and nuclear factor-kB We have, recently, demonstrated that the AP intracellular accumulation triggers a mitochondrial apoptotic pathway, by modifying the expression of molecules typically involved in such apoptotic mechanism, through NF-kB signaling pathway (Antognelli et al., 2012). Therefore, in order to investigate the molecular mechanism of the observed apoptotic response after MG administration, alone or in combination with siGLOI, paralleled in some cases by AP accumulation, we evaluated the anti-apoptotic Bcl-2 or Bcl-XL and the pro-apoptotic Bax proteins levels, Cytochrome c (Cyt c) release and the activation of procaspase-3, typically involved in the mitochondrial apoptotic pathway, after the treatment with MG alone or in

Fig. 5. Methylglyoxal (MG) and Glyoxalase I (GLOI) silencing effect on the apoptosis, MG or MG-derived argpyrimidine (AP) intracellular levels in LNCaP and PC3 cells. (A) Apoptosis was evaluated by FACS and confirmed by DNA fragmentation (inserts); (B and C) MG and AP intracellular levels were measured by HPLC and densitometric analysis from Western blot detection, respectively. Western blots (40 ␮g proteins) were obtained by using a mouse anti-AP mAb. The blots were stripped of the bound Ab and reprobed with mouse anti-␤-actin, to confirm equal loading. Western blots are representative of three separate experiments. Histograms indicate means ± SD of three different cultures tested in quadruplicate. siGLOI: GLOI-siRNA; **P < 0.001 vs untreated cells (−).

Fig. 4. Methylglyoxal (MG) and MG-derived argpyrimidine (AP) intracellular levels after MG administration in LNCaP and PC3 cells. MG and AP intracellular levels were measured by HPLC and densitometric analysis from Western blot detection, respectively. Western blots (40 ␮g proteins) were obtained by using a mouse anti-AP mAb. The blots were stripped of the bound Ab and reprobed with mouse anti-␤actin, to confirm equal loading. Western blots are representative of three separate experiments. Histograms indicate means ± SD of three different cultures tested in quadruplicate. C: controls. **P < 0.001 vs C.

combination with siGLO. Results showed a significant decrease in Bcl-2 and Bcl-XL and a marked increase in Bax proteins (Fig. 7A), paralleled by Cyt c release into the cytosol (Fig. 7B) and procaspase3 activation (Fig. 7C), in LNCaP cells, where a marked increase in AP intracellular levels was achieved. Conversely, in PC3 cells, where no accumulation in AP levels was obtained, only a slight decrease in Bcl-2 and Bcl-XL was observed (Fig. 7A). The subsequent investigation of NF-kB signaling pathway involvement in the regulation of its Bcl-2, Bcl-XL and Bax target genes, showed that, in LNCaP cells,

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Fig. 6. Immunofluorescence staining of F-actin and ␣-tubulin after methylglyoxal (MG) administration in the presence of Glyoxalase I (GLOI) silencing in LNCaP and PC3 cells. Immunofluorescence staining of F-actin (A) and ␣-tubulin (B) showed a dramatic change in the architecture and morphology of LNCaP and PC3 cells with a complete dismantling of the actin and tubulin cytoskeleton in exposed compared to non-treated (NT) cells. Representative pictures from three experiments done independently, which gave the same results. Distance scale is 20 ␮m.

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Fig. 7. Methylglyoxal (MG) and Glyoxalase I (GLOI) silencing trigger a mitochondrial apoptotic pathway via Nuclear Factor kB in LNCaP cells. (A) Anti-apoptotic Bcl-2 and Bcl-XL or pro-apoptotic Bax proteins, (B) Cytochrome c (Cyt c), (C) caspase-3 (intact protein, solid arrow; active fragment, open arrow) and (D) phospho-IkB␣ or total IkB␣ proteins expression (40 ␮g proteins) in untreated (−) and treated (+) cells. Western blot analysis of ␤-actin or Cox IV expression is provided to show equal loading of the samples. Serine 32-phosphorylated IkB␣ mAb was used as a marker of NF-kB activation. Blots are representative of three different experiments done independently which gave the same results. siGLOI: Glyoxalase I-siRNA.

MG administration, alone or in combination with siGLOI, markedly reduced NF-kB activation, as indicated by the decrease in serine 32phosphorylated IkB␣ and the increase in total IkB␣ levels (Fig. 7D). Conversely, in PC3 cells, only a slight decrease in NF-kB activation was observed (Fig. 7D). The use of the monoclonal antibody that detects endogenous levels of serine 32-phosphorylated IkB␣, is an excellent marker of NF-kB activation (Baeuerle and Baltimore, 1988), being phosphorylation of IkB␣ at Ser32 essential for the release of active NF-kB. 3.4. Identification of the 40 kDa MG-modified AP protein To purify the detected 40 kDa MG-modified AP protein, LNCaP cells lysate was applied to immunoaffinity chromatography using the anti-AP mAb. Eluted proteins were subjected to SDS-PAGE in a gel followed by Coomassie Blue staining. As shown in Fig. 8A, the protein fraction mainly contained a 40-kDa polypeptide. To determine the identity of the 40-kDa protein it was digested and resolved as individual peptides by HPLC. As a result, the internal peptides

Fig. 8. Identification of MG-modified protein Argpyrimidine (AP). (A) proteins eluted from an argpyrimidine-affinity column were subjected to SDS-PAGE in a gel, followed by Coomassie Blue staining. The molecular weight of the protein standards (M) and molecular size markers (in kDa) are indicated. (B) Sequence analysis of the 40 kDa protein is compared with that of the human Hsp 40 protein.

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identified with human Hsp 40 upon comparison with standard sequencing databases in the public domain (BLAST) (Fig. 8B).

4. Discussion MG, directly or through the formation of MG-derived products (AGEs), is a cytotoxic glycolysis-derived by-product (Ghosh et al., 2011a,b; Kim et al., 2012a,b; Oba et al., 2012; Rabbani and Thornalley, 2012). Among AGEs, argpyrimidine (AP) represents one of the major products deriving from MG modifications of proteins arginine residues (Kim et al., 2012a,b). In the present work we studied the role of MG in poorly aggressive LNCaP and invasive PC3 human prostate cancer cells, where it has never or scarcely been investigated (Milanesa et al., 2000; Davidson et al., 2002), respectively. We provided the first systematic demonstration that MG cytotoxic role occurs not via cell proliferation but apoptosis control in these cell lines. In fact, MG, alone or in combination with GLOI silencing, did not induce any significant change in comparison to the typical proliferation curves and cell cycle phases of both cell lines, providing novel evidence in the controversial role of this molecule in the proliferative ambit and suggesting that its involvement in this context is an intrinsic characteristic of some cell types. Besides, up to today, some studies have suggested an antiproliferative activity (Bair et al., 2010; Kang et al., 1996; Santel et al., 2008) of MG, while, more recently, MG has been demonstrated to promote proliferation of vascular smooth muscle cells (Chang et al., 2011), thus leaving its role in proliferation control still open to question. On the contrary, MG induced a significant increase in apoptosis, paralleled by MG or AP intracellular levels modifications, in both cell lines. In particular, in LNCaP, where a major MG-induced apoptotic effect was observed, both MG and AP intracellular levels were increased, while in PC3, showing a lower apoptotic response, only an increase in MG intracellular levels was observed. These data showed the involvement of these metabolites in the observed specific apoptotic responses and a different susceptibility of the two cell lines to MG. As known, cell growth results from a ratio between cell proliferation and cell death. A change of such a ratio in favor of cell death is thought to negatively influence cell growth. We found that MG, without affecting cell proliferation, induced a percentage of apoptotic cells (TUNEL positive) by far major than 20%, thus reasonably to let us judging that both cell lines growth is significantly influenced by this metabolite. Since GLOI represents the major cellular MG scavenging enzyme and defence against MG- and AGEs-induced cytotoxicity (Nakadate et al., 2009; Rabbani and Thornalley, 2012), we hypothesized that GLOI silencing could potentiate MG-induced apoptotic effect, through the endogenous accumulation of these metabolites. In fact, we found a dramatic enhancement in apoptosis as well as MG and AP intracellular levels in LNCaP, and a minor but still significant apoptotic increment, paralleled by an increase only in MG intracellular levels, in PC3. These results confirmed our hypothesis as well as the involvement of MG or AP in the observed specific apoptotic responses and the different susceptibility of LNCaP and PC3 cells to MG. Besides, MG apoptogenic role, direct or through AGEs (Chan et al., 2007; Denis et al., 2002; Ghosh et al., 2011a,b; Kasper and Funk, 2001; Kim et al., 2012a,b; Rabbani and Thornalley, 2012), and a cell typeor concentration-dependent sensitivity to it has been previously described in other cellular models (Amicarelli et al., 2003; Du et al., 2000; Thornalley et al., 2010). We have, recently, demonstrated that the AP intracellular accumulation triggers a mitochondrial apoptotic pathway, by modifying the expression of molecules typically involved in such apoptotic mechanism, through NF-kB signaling pathway (Antognelli et al., 2012). Moreover, it has been reported that MG is involved in the negative regulation of anti-apoptotic proteins (Chen et al., 2010; Thornalley et al., 2010) and both MG

(Chen et al., 2010) and AGEs (Alikhani et al., 2005) in the positive regulation of pro-apoptotic proteins, with a predominant effect by AGEs (Alikhani et al., 2005; Wang et al., 2011; Yamagishi et al., 2002). Therefore, we then investigated the molecular mechanism of the observed apoptotic responses, evaluating the anti-apoptotic Bcl-2 or Bcl-XL and the pro-apoptotic Bax proteins levels, Cyt c release and the activation of procaspase-3, typically involved in ˜ the mitochondrial apoptotic pathway (Munoz-Pinedo, 2012). In LNCaP, the increase in MG and AP intracellular levels was paralleled by the enhanced expression of the pro-apoptotic Bax, and the decreased expression of the anti-apoptotic Bcl-2 and Bcl-XL proteins, triggering the mitochondrial apoptotic program. Conversely, in PC3, a mitochondrium-independent apoptotic program, via intracellular accumulation of MG, was induced, likely through the involvement of specific MG-adducts different from the one we detected. Besides, previous studies have established that MG adduction of the heat shock protein 27 (Hsp27) is essential for repressing mitochondrial-mediated caspase activation (Sakamoto et al., 2002). Alternatively, MG could modify other proteins with important pro-apoptotic functions in caspase-independent apoptosis (Bidère and Senik, 2001; Sukumari-Ramesh et al., 2011). Since, as above mentioned, we previously demonstrated that the AP intracellular accumulation triggers a mitochondrial apoptotic mechanism, through NF-kB signaling pathway (Antognelli et al., 2012), we also investigated the involvement of such transcription factor, constitutively activated in LNCaP and PC3 cells (Gupta et al., 2002; Lindholm et al., 2000), where it up-regulates Bcl-2 and BclXL antiapoptotic target genes (Gasparian et al., 2002; Shukla and Gupta, 2004; Suh et al., 2002), playing a pivotal role in the protection of these cells from apoptosis (Suh et al., 2002). We found that MG administration, alone or in combination with siGLOI, markedly reduced NF-kB activation only in LNCaP, where was paralleled by a significant down-regulation of Bcl-2 or Bcl-XL and up-regulation of Bax proteins, demonstrating NF-kB involvement in the mitochondrial apoptotic pathway occurring in this cell line. Therefore, our data indicated that MG affects LNCaP cells viability via desensitization of NF-kB signaling, which is the key signaling pathway for cell survival, thereby promoting cytotoxicity. In line with our recently published study (Antognelli et al., 2012), we again demonstrated and corroborated that the modulation of NF-kB activation occurred through AP intracellular accumulation, being achieved only in LNCaP cells, where AP intracellular levels were enhanced. In the attempt to understand how, in LNCaP cells, MG-induced AP accumulation triggered an apoptotic mechanism, we identified AP and found to be a heat-shock protein 40 (Hsp 40). It is known that HSPs family are agents mainly protective against programmed cell death. However, in particular conditions, some of these pro´ ´ teins may promote apoptosis (Kazmierczuk and Kilianska, 2010), as observed for the identified MG-adduct AP (Hsp 40) in this study. It has been shown that the anti-apoptotic functions of another member of the HSPs family, Hsp27, can be effectively compromised after MG adduction in glomerular mesangial cells (Padival et al., 2003) and that MG promotes the formation of large aggregates containing Hsp 40, Hsc70-interacting protein (CHIP) and ubiquitin, in a human retinal pigment epithelium cell line (Bento et al., 2010). Consistently with this latter observation, increased levels of MG led to accumulation of modified proteins and decreased cell viability (Bento et al., 2010). We speculate that similar MG-induced modifications might also occur for the Hsp 40 function in our study. Furthermore, it has been described that Hsp27 MG-adduction may compromise also other functions of Hsp27. For example, Hsp27 serves as a chaperone to stabilize cytoskeletal proteins (Lavoie et al., 1995). Cytoskeletal breakdown is a key event in the initiation of apoptosis (Castoldi et al., 2000). Therefore, it is reasonable also to assume that, similarly, the MG-adducted Hsp 40 in our study might serve to trigger the observed cytoskeleton disorganization. In order

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to exclude the action of intracellular inhibitors that may prevent cells from undergoing apoptosis also in the presence of an activated apoptotic molecular pathway (Kelly et al., 2011), we finally studied DNA fragmentation and cytoskeleton disorganization, after MG treatment, alone or along with GLOI silencing. We again confirmed MG pro-apoptotic effect and the specific susceptibility of LNCaP and PC3 to this molecule. 5. Conclusions We demonstrated that MG is cytotoxic on LNCaP and PC3 by inducing apoptosis and that such cytotoxicity, potentiated by the silencing of its major scavenging enzyme GLOI, occurs via different apoptotic responses in LNCaP and PC3 cells that also showed a different susceptibility to this metabolite. Finally, we showed that in LNCaP cells, MG, through the accumulation of AP, inhibited constitutive NF-kB activity, which was consistent with the modulation of NF-kB-regulated genes, triggering a mitochondrial apoptotic pathway. The observed MG apoptogenic action, enhanced in the presence of GLOI silencing, suggests that, this physiologic compound merits investigation as a potential chemopreventive/-therapeutic agent, in differently aggressive prostate cancers. Disclosure statement The authors have no conflict of interest. Acknowledgement The authors thank Mrs. Roberta Frosini for the excellent technical assistance. References Alikhani Z, Alikhani M, Boyd CM, Nagao K, Trackman PC, Graves DT. Advanced glycation end products enhance expression of pro-apoptotic genes and stimulate fibroblast apoptosis through cytoplasmic and mitochondrial pathways. Journal of Biological Chemistry 2005;280:12087–95. Amicarelli F, Bucciarelli T, Poma A, Aimola P, Di Ilio C, Ragnelli AM, et al. Adaptive response of human melanoma cells to methylglyoxal injury. Carcinogenesis 1998;19:519–23. Amicarelli F, Colafarina S, Cattani F, Cimini A, Di Ilio C, Ceru MP, et al. Scavenging system efficiency is crucial for cell resistance to ROS-mediated methylglyoxal injury. Free Radical Biology and Medicine 2003;35:856–71. Amicarelli F, Colafarina S, Cesare P, Aimola P, Di Ilio C, Miranda M, et al. Morphofunctional mitochondrial response to methylglyoxal toxicity in Bufo bufo embryos. International Journal of Biochemistry and Cell Biology 2001;33: 1129–39. Antognelli C, Mezzasoma L, Fettucciari K, Mearini E, Talesa VN. Role of glyoxalase I in the proliferation and apoptosis control of human LNCaP and PC3 prostate cancer cells. Prostate 2012., http://dx.doi.org/10.1002/pros.22547. Baeuerle PA, Baltimore D. I kappa B: a specific inhibitor of the NF-kappa B transcription factor. Science 1988;242:540–6. Bair 3rd WB, Cabello CM, Uchida K, Bause AS, Wondrak GT. GLO1 overexpression in human malignant melanoma. Melanoma Research 2010;20:85–96. Barua M, Jenkins EC, Chen W, Kuizon S, Pullarkat RK, Junaid MA. Glyoxalase I polymorphism rs2736654 causing the Ala111Glu substitution modulates enzyme activity – implications for autism. Autism Research 2011;4:262–70. Bento CF, Marques F, Fernandes R, Pereira P. Methylglyoxal alters the function and stability of critical components of the protein quality control. PLoS One 2010;5:e13007. Bidère N, Senik A. Caspase-independent apoptotic pathways in T lymphocytes: a minireview. Apoptosis 2001;6:371–5. Cabello CM, Bair III WB, Bause AS, Wondrak GT. Antimelanoma activity of the redox dye DCPIP (2,6-dichlorophenolindophenol) is antagonized by NQO1. Biochemical Pharmacology 2009;78:344–54. Cantero AV, Portero-Otín M, Ayala V, Auge N, Sanson M, Elbaz M, et al. Methylglyoxal induces advanced glycation end product (AGEs) formation and dysfunction of PDGF receptor-beta: implications for diabetic atherosclerosis. FASEB Journal 2007;21:3096–106. Castoldi AF, Barni S, Turin I, Gandini C, Manzo L. Early acute necrosis, delayed apoptosis and cytoskeletal breakdown in cultured cerebellar granule neurons exposed to methylmercury. Journal of Neuroscience Research 2000;59: 775–87.

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