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Glyoxalase I inhibition induces apoptosis in irradiated MCF-7 cells via a novel mechanism involving Hsp27, p53 and NF-κB

Background: Glyoxalase I (GI) is a cellular defence enzyme involved in the detoxification of methylglyoxal (MG), a cytotoxic byproduct of glycolysis, and MG-derived advanced glycation end products (AGEs). Argpyrimidine (AP), one of the major AGEs coming from MG modifications of proteins arginines, is a pro-apoptotic agent. Radiotherapy is an important modality widely used in cancer treatment. Exposure of cells to ionising radiation (IR) results in a number of complex biological responses, including apoptosis. The present study was aimed at investigating whether, and through which mechanism, GI was involved in IR-induced apoptosis. Methods: Apoptosis, by TUNEL assay, transcript and protein levels or enzymatic activity, by RT–PCR, western blot and spectrophotometric methods, respectively, were evaluated in irradiated MCF-7 breast cancer cells, also in experiments with appropriate inhibitors or using small interfering RNA. Results: Ionising radiation induced a dramatic reactive oxygen species (ROS)-mediated inhibition of GI, leading to AP-modified Hsp27 protein accumulation that, in a mechanism involving p53 and NF-κB, triggered an apoptotic mitochondrial pathway. Inhibition of GI occurred at both functional and transcriptional levels, the latter occurring via ERK1/2 MAPK and ERα modulation. Conclusions: Glyoxalase I is involved in the IR-induced MCF-7 cell mitochondrial apoptotic pathway via a novel mechanism involving Hsp27, p53 and NF-κB.

Glyoxalase I (GI) is a key enzyme in the pathway leading to glutathione (GSH)-mediated detoxification of the side product of glycolysis methylglyoxal (MG) that, reacting with DNA and proteins, forms stable products called advanced glycation end products (AGEs) (Rabbani and Thornalley, 2012). Among them, argpyrimidine (AP) represents one of the major products deriving from MG modifications of protein arginine residues (Kim et al, 2012a), recently shown to induce apoptosis to cultured cells (Kim et al, 2012b;. Radiotherapy (RT) is an important modality widely used in cancer treatment, including breast cancer (BC). In early-stage breast cancer, whole breast RT is usually applied after conserving surgery; nevertheless, most tumour recurrences occur near the tumour bed, thus indicating that tolerable doses of external RT are often insufficient to achieve control of locally advanced BC. Therefore, intraoperative RT (IORT), a procedure in which a high dose of IR is precisely applied to the tumour bed during surgery (Calvo et al, 2013), has been clinically and epidemiologically suggested as a valid alternative to whole breast RT. However, in vitro research in such a field has been scarcely performed. In such a therapeutic tool ambit (IORT), the Italian intraoperative radiotherapy with electrons (ELIOT) trial appeared a promising feature in early BC, treated with breastconserving surgery (Veronesi et al, 2010).
Exposure of cells to ionising radiation (IR) results in a number of complex biological responses, including programmed cell death . Radiation-induced apoptosis is a phenomenon dependent on cell type, dose and time after irradiation (Urashima et al, 2006), and can occur through different mechanisms (Golden et al, 2012;Kwon et al, 2013). Certainly, reactive oxygen species (ROS) are crucial mediators of the apoptosis by IR in a variety of cells (Morales et al, 1998;Van Laethem et al, 2006). Reactive oxygen species are very transient species owing to their high reactivity that gradually leads to impose oxidative damage on biomolecules ultimately compromising cell viability, leading to activation of the programmed cell death machinery (Sinha et al, 2013). In particular, ROS are known to damage cells by peroxidising lipids, disrupting DNA and oxidising amino acids, thus inactivating specific enzymes directly or through oxidation of cofactors (Bolisetty and Jaimes, 2013). Moreover, they act as important signalling molecules regulating many signal-transduction pathways and modulating gene transcription (Forman and Torres, 2001). The ROS-induced apoptosis can occur through a wide variety of mechanisms and signalling pathway often depending on the considered cell model and different cell contexts (Soga et al, 2012;Zhang et al, 2012;Zhu et al, 2012). Biological systems have evolved an effective and complicated network of defence mechanisms that enable cells to cope with lethal oxidative environments. These defence mechanisms involve the tripeptide GSH and some antioxidant enzymes, as superoxide dismutase (SOD) that catalyses the dismutation of O2 .to H 2 O 2 and O 2 (McCord and Fridovich, 1969), catalase and peroxidases that remove hydrogen peroxide and hydroperoxides (Chance et al, 1979).
The present study was aimed at investigating whether, and through which molecular mechanism, GI was involved in IR-induced apoptosis, possibly by the involvement of ROS, in human MCF-7 oestrogen-responsive early-stage BC cell line, exposed to a single dose of 21 Gy, as in the ELIOT trial (Veronesi et al, 2010). We demonstrated that IR-induced ROSmediated GI inhibition, leading to AP accumulation, triggered a mitochondrial apoptotic pathway via a novel mechanism involving heat shock protein 27 (Hsp27), p53 and nuclear factor-kB (NF-kB), and that GI inhibition occurred at both functional and transcriptional levels, the latter via p44/42 mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase 1/2 (ERK1/2)) and oestrogen receptor-a (ERa) involvement.

MATERIALS AND METHODS
Irradiation. The same dose used in the partial breast irradiation ELIOT trial, 21 Gy (Veronesi et al, 2010), was administered at a dose rate of 200 cGy min À 1 at room temperature using 6 MV photons produced by a linear accelerator (Clinac DBX, Varian Medical Systems, Palo Alto, CA, USA). A 5-mm-thick plexiglass spoiler was placed on the flasks or plates.
Cell culture and exposure. The MCF-7 human breast cancer cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured as per the manufacturer's recommendations at 37 1C and 5% CO 2 . Analyses were performed immediately (time T 0 ) and at 0.5, 24, 48 or 72 h post irradiation. During these latter periods, cells were incubated at 37 1C. As no significant biological effects were observed at T 0 time compared with unexposed cells, serving as control, results were reported only for all the rest of the time course. Untreated cells were incubated for the same time periods. Independent experiments were also performed by pretreating cells with N-acetyl-Lcysteine (NAC, 10 mM, for 0.5 h), a known antioxidant that increases cellular GSH levels, aminoguanidine bicarbonate (AG, 1 mM, for 2 h), a scavenger of free MG (Fleming et al, 2013) and MG-derived AGEs (Thornalley et al, 2000), the specific wild-type p53 inhibitor, pifithrin-a (PFT-a, 20 mM in DMSO, for 2 h), the specific NF-kB inhibitor BAY 11-7082 (10 mM in DMSO, for 0.5 h, Santa Cruz Biotechnology, Inc., Heidelberg, Germany), the ERa anti-oestrogen ICI 182,780 (100 nM in DMSO, for 4 h), ERK-1/2 inhibitor U-0126 (10 mM in DMSO, for 1 h), and then irradiated in the conditions above described. Unless otherwise stated, these reagents were from Sigma-Aldrich (Milan, Italy) and used at concentrations producing no significant toxicity to MCF-7 cells. Control cells for the experiments with DMSO-dissolved agents did not show any significant difference with respect to control cells in RPMI-1640 medium, and therefore all the relative treatments were compared with these latter controls.
Cell viability assays. Cell viability was evaluated by assessment of reduction of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) (Kanagasabai et al, 2010) or Cell Counting Kit-8 (CCK8) (Yi et al, 2013) and lactate dehydrogenase (LDH) protein amount in cell culture medium by LDH ELISA kit (Antibodiesonline, GmbH, Aachen, Germany). As to MTT and CCK8 assays, absorbance of cell survival was calculated relative to control cells that were set to 100%; for LDH protein amount in cell culture medium, basic values were provided.
GSH assay. Control and irradiated cells were suspended in 50 mM phosphate buffer containing 1 mM EDTA, pH 6.8, at the concentration of 10 7 cells per ml. Suspensions were then homogenised and centrifuged at 10 000 g for 15 min at 4 1C. Supernatants were collected, deproteinised with 2.5% metaphosphoric acid in a 1 : 1 ratio (v/v) and centrifuged at 2000 g for 5 min. The resulting extracts were used for glutathione assay. Reduced and oxidised glutathione intracellular concentrations were measured using Cayman's GSH Assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions.
Enzymatic activity assays for antioxidant defences. Control cells and IR-exposed cells were harvested and suspended at the concentration of 10 7 cells per ml in 10 mM phosphate buffer, pH 7.0, containing 1 mM dithiothreitol (for GI and GSH-Px) or 1 mM Triton X-100 (for CuZn-SOD) and 0.1 mM phenylmethanesulphonylfluoride (PMSF). Cell suspensions were then homogenised with a Potter-Helvehjem homogeniser, centrifuged at 13 000 g for 30 min and the resulting cell extracts used for enzymatic activities and protein content measurements. The GI activity was assayed according to Mannervik et al (1981, 297-301). The assay solution contained 0.1 M sodium-phosphate buffer, pH 7.2, 2 mM MG and 1 mM GSH. The reaction was monitored spectrophotometrically by following the increase of absorbance at 240 nm and 25 1C. One unit activity is defined as 1 mmol of S-D-lactoylglutathione produced per min. The Se-dependent GSH-Px activity was measured according to Paglia and Valentine (1967). The oxidation of NADPH was followed spectrophotometrically at 340 nm and 25 1C. One unit activity was defined as 1 mmol NADPH oxidised per min. Catalase activity was measured spectrophotometrically at 240 nm at 25 1C, according to Aebi (1974, 673-684), by following the rate of reduction of H 2 O 2 (10 mM). One unit activity is defined as 1 mmol H 2 O 2 reduced per min. The CuZn-SOD activity was assayed spectrophotometrically at 480 nm and 25 1C by its capability to inhibit the autoxidation of adrenaline according to Sun and Zigman (1978). One unit activity is defined as the amount of enzyme required to halve the rate of substrate oxidation.
Knockdown of GI with specific siRNA. Subconfluent MCF-7 cells were transiently transfected with a 100 nmol pool of four siRNAs targeting GI (si-GI) and nonspecific siRNA (si-C) (Dharmacon, Milan, Italy), using Lipofectamine 2000 (Invitrogen, Milan, Italy). After culturing for 72 h (maximum silencing effect in preliminary experiments), the culture medium was removed and cells were added with a fresh one containing new transfection complexes and then irradiated. The effect of silencing was evaluated analysing the specific protein knockdown by western blot using the specific Ab, and b-actin as loading control, and enzymatic activity, either 72 h before or after irradiation. In addition, a mock transfection was performed with all vehicles used except for the siRNA. In all transfection experiments, mock transfection and transfection with si-C did not affect all the studied parameters, providing results comparable with those obtained from untreated cells (data not shown).
Protein assay. Protein concentration was determined by BCA kit (Pierce, Rockford, IL, USA) (Shibuya et al, 1989), using bovine serum albumin as a standard.
Affinity purification of the 27 kDa AGE AP and amino acid sequence analysis. Purification and identification of AGE AP was performed according to Sakamoto et al (2002).
RNA isolation and cDNA synthesis. Total cellular RNA was isolated using TRIzol Reagent (Invitrogen). The cDNA was then synthesised from 1 mg of RNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (M-Medical, Milan, Italy).
The thermal cycling conditions were as follows: 1 cycle at 95 1C for 10 min, followed by 45 cycles at 95 1C for 20 s and 55 1C for 1 min. Data for comparative analysis of gene expression were obtained using the DDCt method (Livak and Schmittgen, 2001).
Statistical analysis. Results were expressed as the percentage of irradiated cells (control) and are representative of at least four independent experiments performed in quadruplicate. The data were expressed as mean ± s.d. and compared by Student's t-test.
The null hypothesis was rejected at the 5% significance level (Po0.05).

IR-induced cytotoxicity occurs via apoptosis in MCF-7 cells.
To study IR-induced cytotoxicity, viability was examined at various post-irradiation time points in MCF-7 cells exposed to a single dose (21 Gy) of IR. We found that IR treatment resulted in a significant time-dependent decrease of cell viability since 24 h post irradiation, compared with nonirradiated control cells, as shown by the decrease in viable cell percentage by MTT or CCK8 tests and increase in LDH protein release into the medium ( Figure 1A). It is known that IR-induced cell death has been identified in many cases as apoptosis (Urashima et al, 2006;Chen et al, 2013;Kwon et al, 2013). Therefore, to investigate the mechanism of cell death induced by IR in our study, apoptosis was investigated by DNA fragmentation assays. The TUNEL assay showed that the percentage of apoptotic cells measured after IR exposure, 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), significantly increased in a time-dependent manner compared with untreated cells ( Figure 1B). The DNA fragmentation into oligonucleosomes confirmed the apoptotic responses as evidenced by the typical DNA laddering ( Figure 1C).
Effect of IR on GI and the AGE AP. We recently reported that GI can trigger apoptosis through the control of MG-derived AP intracellular levels in human prostate cancer cell lines . To reveal the potential involvement of GI in the apoptotic cell death caused by treatment with IR in MCF-7 cells, we first studied the levels of GI enzymatic activity by spectrophotometric method. A significant inhibitory time-dependent effect was observed for GI after IR exposure, compared with control cells (Figure 2A). Mean values of GI activity, expressed as mmol min À 1 per mg protein were (mean ± s.d.) 0.75 ± 0.07 for control cells and 0.51 ± 0.04, 0.41 ± 0.01, 0.30 ± 0.02 or 0.17 ± 0.01 for cells post irradiation. As the biological activity of a protein can be regulated at post-translational level, changes in protein functional level might not be necessarily reflected by similar changes in protein abundance. We, therefore, measured GI protein level in MCF-7 cells after IR treatment by immunoblotting detection. Western blot revealed an equally pronounced decrease of GI at such a level compared with untreated cells ( Figure 2B). Next, to determine whether the decrease in GI protein was paralleled by a similar trend at GI transcript level, GI mRNA was examined. The qRT-PCR revealed a significant downregulation of GI mRNA levels occurring in a time-dependent manner after irradiation compared with unirradiated cells ( Figure 2C). We then examined the hypothesis that IR-induced GI inhibition was paralleled by increased GI-dependent MG-derived AP intracellular levels. To this end, immunodetection of AP, the AGE derived from spontaneous MG adduction of arginine residues (Kim et al, 2012a), was performed in total protein extracts from both control and irradiated MCF-7 cells. Western blot analysis, using a monoclonal antibody directed against AP, identified a single band of 27 kDa molecular weight. Densitometry analysis revealed that AP cellular levels were significantly increased in IR-treated as compared with control cells ( Figure 2D).

IR induces oxidative stress in MCF-7 cells.
It is known that the cytotoxic effects of radiation are mediated primarily through increased formation of hydroxyl radicals and ROS that, in turn, can damage proteins and trigger apoptosis in a variety of cells (Morales et al, 1998;Van Laethem et al, 2006;Na et al, 2012;Soga et al, 2012;Zhu et al, 2012;Zhang et al, 2012;Sinha et al, 2013;Zhang et al, 2013). Therefore, to investigate whether the observed IR-induced GI inhibition, paralleled by induction of apoptosis, was associated with ROS formation, the levels of intracellular H 2 O 2 were initially measured with DCF staining. Compared with control, irradiation caused a significant increase in H 2 O 2 levels starting from 0.5 h post irradiation ( Figure 3A). We further confirmed IR-induced ROS generation using another ROS detection dye, DHE for O 2 .-. Similarly, we found a marked increase in O 2 .generation after treatment with IR as compared with untreated cells ( Figure 3A). As known, ROS production may result from impairment in the function of the defence mechanisms that enable cells to cope with lethal oxidative environment. Therefore, we analysed the level of GSH as well as some of the major cellular antioxidant enzymes, such as SOD, that catalyse the dismutation of O2 À to H 2 O 2 and O 2 (McCord and Fridovich, 1969), catalase and GSH-Px that remove H 2 O 2 (Chance et al, 1979), finding that treatment with IR resulted in a significant inhibition of all the studied enzymes with respect to control cells ( Figure 3B). As indicative marker of oxidative damage to cells, the occurrence of lipid peroxidation was also evaluated. It is well established that oxidative stress in various cells usually leads to accumulation of potent, cytotoxic lipid peroxides such as MDA (Esterbauer et al, 1991). Exposure to IR significantly increased MDA content compared with unirradiated cells ( Figure 3C).

IR-induced apoptosis occurs via ROS-mediated inhibition of GI enzyme and AP accumulation in MCF-7 cells.
To determine whether the increase in intracellular ROS levels was directly related to the observed IR-triggered apoptotic cell death and concomitant GI inhibition/AP accumulation, we pretreated MCF-7 cells with the antioxidant NAC before exposing them to IR. A total of 10 mM NAC added to cells 0.5 h before irradiation significantly reduced apoptotic cell death ( Figure 4A) and reverted GI inhibition ( Figures 4B-D) or AP intracellular content ( Figure 4E), caused by IR, to the level of control cells. The NAC did not significantly affect the other studied enzymes playing a protective role against ROS, thus indicating a specific effect to GI (data not shown). The role of GI in triggering AP accumulation and apoptosis was explored by knocking down GI via siRNA methods. We first tested the efficiency of GI siRNA. As shown in Figure 4F MCF-7 cells transfected with GI siRNA showed inhibition of GI protein expression and enzymatic activity, either 72 h before or after irradiation, whereas the control siRNA had no effect. We found that blockade of GI by siRNA additionally increased AP accumulation ( Figure 4G) and apoptosis ( Figure 4H) but not ROS ( Figure 4I), confirming that ROS are upstream signal of GI/AP axis in the observed apoptosis induction by IR. Pretreatment with AG demonstrated the effect of such MG scavenger in directly triggering the apoptosis mediated by ROS ( Figure 4J). Aminoguanidine showed no effects on ROS and GI expression level (data not shown), again confirming the proposed mechanism by which IR induces apoptosis in MCF-7 via AP accumulation due to ROS-mediated GI inhibition.
IR-induced ROS-mediated GI inhibition, leading to argpyrimidine accumulation, triggers a mitochondrial apoptotic pathway involving NF-kB. It is known that AGEs can act as pro-apoptotic agents (Kim et al, 2012b; by downregulating the anti-apoptotic protein Bcl-2 (Honda et al, 2001;Yamagishi et al, 2002) and upregulating the pro-apoptotic Bax protein (Alikhani et al, 2005) in different cellular models, and that the specific AGE AP drives a mitochondrial apoptotic pathway in prostate cancer cell lines . As our results showed an IR-induced GI inhibition-dependent increase in AP levels, we next investigated whether the observed IR-driven apoptosis occurred via a mitochondrial pathway also in caspase-3-deficient (Santha et al, 2013;Charalambous et al, 2013) MCF-7 cells. To this aim, the levels of the anti-apoptotic Bcl-2 or Bcl-XL or the pro-apoptotic Bax proteins, Cyt c release into the cytosol and the activation of the final executioner caspase-7 (Santha et al, 2013;Charalambous et al, 2013), occurring in a mitochondrial apoptotic pathway (Kaufmann and Hengartner 2001), were analysed. As shown in Figure 5A, we found a significant decrease in the levels of the anti-apoptotic Bcl-2 or Bcl-XL proteins paralleled by a marked increase in the levels of the pro-apoptotic Bax protein in irradiated cells compared with controls. Concomitantly, Cyt c release into the cytosol, as well as the activation of caspase-7, were observed ( Figure 5B). We previously found that AP-driven mitochondrial apoptotic pathway occurred via NF-kB downregulation in prostate cancer cell lines . As known, NF-kB is a family of transcription factors present in the cytosol in an inactive state complexed with the inhibitory IkB proteins. Activation occurs via phosphorylation of IkBa at Ser32 and Ser36 followed by proteasome-mediated degradation that results in the release and nuclear translocation of active NF-kB (Baldwin, 2001). It has been described that NF-kB is able to upregulate Bcl-2 and Bcl-XL antiapoptotic genes (Baeuerle and Baltimore, 1988;Gasparian et al, 2002;Suh et al, 2002;Shukla and Gupta, 2004) and is constitutively activated in various human tumours (Bondong et al, 2012;Kim et al, 2012;Ranuncolo et al, 2012) and cancer cell lines (Lindholm et al, 2000;Ludwig et al, 2001;Hwang et al, 2012), including MCF-7 cells (Sovak et al, 1997). Therefore, we hypothesised that AP accumulation following IR-induced ROS-mediated GI inhibition might involve NF-kB signalling pathway via NF-kB desensitisation. In fact, a marked reduction in NF-kB activation was observed in irradiated compared with control MCF-7 cells, as indicated by the decrease in serine 32-phosphorylated IkBa and the increase in total IkBa levels ( Figure 5C). The use of the monoclonal antibody that detects endogenous levels of serine 32-phosphorylated IkBa is an excellent marker of NF-kB activation (Baeuerle and Baltimore, 1988), as phosphorylation of IkBa at Ser32 is essential for the release of active NF-kB. The effect of IR was completely abolished by pretreatment with MG-scavenging agent AG, proving the direct involvement of AP in the intrinsic NF-kB-mediated apoptotic pathway ( Figures 5A-C). Pretreatment with NAC reverted NF-kB-mediated mitochondrial apoptotic activation pattern induced by IR (data not shown).
Involvement of Hsp27 and p53 in the IR-induced ROS-mediated mitochondrial apoptotic pathway. To investigate the mechanism by which AP drove the observed mitochondrium-dependent apoptosis, we identified the detected 27 kDa AP protein in cells 72 h post irradiation, where the maximum AP band intensity was observed. To this aim the 27 kDa AP protein was purified by immunoaffinity chromatography using the anti-AP mAb. Single column fractions, containing the eluted proteins, were individually subjected to SDS-PAGE followed by Coomassie Blue staining. As shown in Figure 6A, one column fraction presented a very marked stained band, containing a 27-kDa polypeptide. Other protein bands were also present in the other fractions of the immunoaffinity elution, showing, however, a by far less staining (data not shown). To determine the identity of the 27 kDa protein, it was digested and resolved as individual peptides by HPLC. As a result, the internal peptides identified with human Hsp27 upon comparison with standard sequencing databases in the public domain (BLAST) ( Figure 6B). The Hsp27 is known to accelerate proteasomal degradation of p53 in cancer cells (O'Callaghan-Sunol et al, 2007) and, as demonstrated in a recent research, a negative correlation between Hsp27 and p53 in UV-irradiated MCF-7 cells exists, in a way that Hsp27 potentiates the degradation of p53 post irradiation (Kanagasabai et al, 2010). Moreover, in the same MCF-7 cell line, an inverse correlation has been recently shown between Hsp27, p53 and NF-kB; in particular, the absence of Hsp27 results in accumulation of p53 that, in turn, triggers the NF-kB-dependent signalling pathway (Kanagasabai et al, 2011). Therefore, we assumed that after irradiation, the observed increase in APmodified Hsp27 protein, along with the concomitant reduction in NF-kB activation, might be paralleled by a decrease in p53 levels. Indeed, we found significantly reduced p53 protein levels starting from 48 h and peaking at 72 h post irradiation compared with unirradiated cells ( Figure 6C). Pretreatment with AG completely reversed IR-induced p53 downregulation, indicating a direct action by AP-modified Hsp27 on p53 modulation ( Figure 6C). The role of p53 in inducing NF-kB desensitisation was verified via a pharmacological approach by using a specific wild-type p53 inhibitor, PFT-a. Pifithrin-a is a small molecule that binds to the DNA-binding domain of p53, thereby inhibiting its transcriptional activity (Wang and Sun, 2010). Western blot analysis revealed that pretreatment with PFT-a significantly potentiated IR-induced NF-kB desensitisation as shown by p-IkBa and IkBa expression level that resulted undetectable or enhanced, respectively ( Figure 6D). In parallel, pretreatment with PFT-a significantly increased the number of apoptotic cells ( Figure 6E) but did not affect AP levels (data not shown). Finally, to prove the involvement of NF-kB in triggering IR-induced apoptosis, the specific NF-kB inhibitor BAY 11-7082 that diminishes the activation of NF-kB by preventing phosphorylation of its inhibitory IkB-a protein was used. Figure 6E shows that NF-kB complete inactivation again significantly potentiated apoptosis ( Figure 6E) but had no effect on AP or p53 levels (data not shown).
IR-induced ROS-mediated GI mRNA downregulation occurs through the involvement of ERa and ERK1/2 MAPK. As we found that ROS can even modulate GI gene expression at mRNA level ( Figure 4C), we attempted to reveal the molecular mechanism of the observed ROS-mediated GI downregulation by investigating the possible involvement of ERa and ERK1/2 signalling. In fact, it has been shown that ROS can induce post-translational Erk1/2dependent phosphorylation of ERa at serine 118, leading to ERa downregulation in MCF-7 (Weitsman et al, 2009). Moreover, we previously found that oestrogens positively modulate GI transcript levels in MCF-7 cell, possibly through the interaction of their receptors with degenerated oestrogen response element (ERE) motives, identified in GI promoter by bioinformatics methods (Rulli et al, 2006). Hence, we reasonably assumed that the observed GI transcriptional downregulation could be consequent to the ROS-induced Erk1/2-mediated downregulation of ERa. Indeed, we found that irradiation, associated with ROS production, significantly changed the expression profile of phosphorylated serine 118 (p-ser118) ERa, ERa as well as Erk1/2. In particular, a marked increase in phosphorylation of serine 118 occurred, paralleled by a significant decrease in the level of total ERa and concurrent activation of Erk1/2 over the same period post irradiation ( Figure 7A). Pretreatment with NAC abrogated such effects, proving the direct involvement of ROS ( Figure 7A). To validate the involvement of ERK1/2 signalling on p-ERa and ERa protein level, or GI mRNA expression, cells were exposed to the specific ERK 1/2 inhibitor, U-0126. As shown in Figure 7B, the effect of IR was completely abolished in the presence of U-0126 ( Figure 7B). Western blot analysis of p-Erk1/2 proved the biochemistry evidence of the inhibitory action of U-0126 on ERK1/2 activity ( Figure 7B). The inhibitor U-0126 did not affect ROS accumulation (data not shown), confirming that such reactive species act upstream of ERK-1/2 in negatively modulating GI. To prove that ERa was directly involved in the downregulation of GI expression at the mRNA level, ICI 182,780, an ERa-degrading anti-oestrogen (Osborne et al, 2004;Chen et al, 2009), was used. Inhibition of ERa by ICI 182,780 ( Figure 7C) potentiated IR-induced GI mRNA inhibition ( Figure 7C), indicating its direct effect on GI. The ICI 182,780 did not affect either ROS accumulation or Erk-1/2 activation (data not shown), thus indeed corroborating that ERa receptor acts downstream of ROS/Erk-1/2 axis in downregulating GI mRNA level.

DISCUSSION
Glyoxalase I represents the major scavenging enzyme of MG, a potent precursor of AGEs (Rabbani and Thornalley, 2012). Among AGEs, the pro-apoptotic AP represents one of the major products deriving from MG modification of protein arginine residues (Kim et al, 2012a). The present study was aimed at investigating whether, and through which mechanism, GI was involved in IR-induced apoptosis in MCF-7 human early-stage breast cancer cell line exposed to a single dose of 21 Gy, as in the ELIOT trial (Veronesi et al, 2010). In line with published experimental work where IRinduced cell death has been identified in many cases as apoptosis (Urashima et al, 2006;Golden et al, 2012;Chen et al, 2013;Kwon et al, 2013), our results also showed that the IR used in this study was able to activate a programmed cell death pathway in MCF-7 cells. By using si-GI transient transfection and MG/AP scavenging agent, AG, we next demonstrated that IR-induced apoptosis was driven by GI downregulation-dependent AP intracellular accumulation. We recently demonstrated that AP triggers a mitochondrial apoptotic pathway, by modifying the expression of molecules typically involved in such apoptotic mechanism, through NF-kB signalling desensitisation in other human cancer cell lines . In agreement with these observations and as proved by AG pretreatment experiments, the increased level of intracellular AP induced by IR also drove a similar activation pattern in irradiated caspase-3-deficient MCF-7 cells (Santha et al, 2013;Charalambous et al, 2013) compared with untreated controls, as indicated by the decrease in the anti-apoptotic Bcl-2 or Bcl-XL or increase in the pro-apoptotic Bax proteins levels, Cyt c release and procaspase-7 activation, typically involved in the mitochondrial apoptotic pathway (Kaufmann and Hengartner 2001;Charalambous et al, 2013;Santha et al, 2013) and, accordingly, reduced NF-kB activity. Therefore, our initial data indicated that IR induces a NF-kB signalling-mediated mitochondrial apoptotic pathway triggered by GI inhibition-driven AP accumulation. In the attempt to understand how AP accumulation triggered an intrinsic apoptotic pathway moved by NF-kB desensitisation, we identified the AP-modified 27 kDa polypeptide, interestingly finding it to be Hsp27. It is known that HSP family are agents mainly protective against programmed cell death. However, in particular conditions, some of these proteins may promote apoptosis (Kazmierczuk and Kilianska, 2010), as observed for the identified AP (Hsp27) in this study. In addition to AP-modified Hsp27, we observed other polypeptides that formed AP by immunoaffinity purification (data not shown). Therefore, the possibility that other proteins may play some role in the same context cannot be ruled out. The Hsp27 is known to accelerate proteasomal degradation of p53 in cancer cells (O'Callaghan-Sunol et al, 2007) and, as demonstrated in a recent research, a negative correlation between Hsp27 and p53 in UV irradiated MCF-7 cells exists in a way that Hsp27 potentiates the degradation of p53 post irradiation (Kanagasabai et al, 2010). Moreover, in the same MCF-7 cell line, an inverse correlation has been recently shown between Hsp27, p53 and NF-kB; in particular, the absence of Hsp27 resulted in accumulation of p53 that, in turn, activated NF-kB signalling pathway (Kanagasabai et al, 2011). Therefore, we assumed that after irradiation, the observed increase in AP-modified Hsp27 protein might be paralleled by a decrease in p53 levels, leading to the observed concomitant NF-kB desensitisation. Indeed, we found that p53 protein levels were not only significantly reduced in irradiated compared with unirradiated cells, but also that AG completely reverted them, indicating a direct effect of AP on p53 in driving IR-induced apoptosis. It is broadly accepted that p53 is a master tumour suppressor protein promoting apoptosis in many cellular systems. However, it has also been reported that caspase-mediated apoptosis does not always require p53 (Miyake et al, 2012;Yeh et al, 2012;Amir et al, 2013). Here, we showed that IR-induced apoptosis, via GI inhibition and AP-modified Hsp27 protein accumulation, occurred  in a mechanism independent of the canonical pro-apoptotic pathway induced by p53 but involving the Hsp27-dependent desensitisation of p53, leading to NF-kB nearly complete suppression, as proved by using the specific p53 inhibitor PFT-a as well as NF-kB inhibitor BAY 11-7082. Therefore, in the present work, we not only propose a novel mechanism in IR-induced apoptosis involving GI, Hsp27, p53 and NF-kB, but also reveal a novel noncanonical role of p53 among apoptotic mechanisms in irradiated MCF-7 cells. It is known that IR induces the production of ROS that play an important causative role in apoptotic cell death (Morales et al, 1998;Van Laethem et al, 2006) and that ROS may arise following antioxidant defence functional depletion. Therefore, after observing an effective reduction in the biological activity of some of the major antioxidant cellular defences, an increase in ROS and in MDA, a marker of lipid oxidative damage, we demonstrated that IR-induced GI inhibition, leading to Hsp27-p53-NF-kB pathway activation, triggering a mitochondrial apoptotic pathway, was mediated by ROS. In fact, NAC pretreatment reversed the IRinduced effect on the above-mentioned proteins as well as on those participating in the mitochondrial apoptotic mechanism. Besides, ROS are very reactive species imposing oxidative damage on indispensable biomolecules such as proteins, ultimately compromising cell viability and leading to activation of the programmed cell death machinery (Sinha et al, 2013). We also found that ROS negatively modulated GI at mRNA and protein levels, thus suggesting that the observed decrease in GI activity might also result from decreased protein expression as evident from western blot analysis for GI protein. In the attempt to reveal the molecular mechanism underlying ROS-dependent GI mRNA downregulation, ERa and ERK1/2 signalling were studied. In fact, it has been shown that ROS can induce post-translational Erk1/2-dependent phosphorylation of ERa at serine 118, leading to ERa downregulation in MCF-7 (Weitsman et al, 2009). Moreover, we previously found that oestrogens positively modulated GI transcript levels in MCF-7 cell, possibly through the interaction of their receptors with degenerated ERE motives, identified in GI promoter by bioinformatics methods (Rulli et al, 2006). We found that irradiation significantly changed the expression profile of phosphorylated serine 118 ERa, ERa as well as Erk1/2. In particular, a marked increase in phosphorylation of serine 118 occurred, paralleled by a significant decrease in the level of total ERa and concurrent activation of Erk1/2 over the same period post irradiation. Pretreatment with NAC, Erk1/2-specific inhibitor U-0126 and ERa anti-oestrogen ICI 182,780 accordingly changed such effects, proving the direct involvement of ROS/Erk1/2/ERa axis in GI mRNA negative control. Therefore, we found that IR-induced ROS-mediated GI downregulation occurred through the involvement of ERa and ERK1/2 MAPK, modulation that, to the best of our knowledge, it was never pointed out before.
In early breast cancer patients, standard treatment is conserving surgery followed by whole breast RT. Such treatment significantly reduces ipsilateral recurrence and increases survival (Veronesi et al, 2010). As most tumour recurrences occur near the tumour bed (Veronesi et al, 2010), studies were designed with the aim to shorten the duration of whole breast RT using partial breast irradiation, that is, irradiation of the excisional cavity plus 1-2 cm margins. Intraoperative RT with electrons is one of the techniques used to administer the treatment. In particular, the Italian ELIOT trial appeared a promising feature in early breast cancer treated with breast-conserving surgery, reducing the exposure of normal tissues to radiations and shortening the radiation course from 6 weeks to one single session (Veronesi et al, 2010). Our in vitro results, demonstrating that the single 21 Gy irradiation dose used in the ELIOT trial efficiently induces apoptosis in human ERpositive MCF-7 cells, appear to support the observed effectiveness of such a therapy in patients with a favourable biopathological characterisation, as hormone receptor expression.
In conclusion, our findings show that IR-induced ROSmediated GI inhibition results in a mitochondrial apoptotic pathway via Hsp27, p53 and NF-kB, providing a novel mechanism in 21 Gy single dose irradiated MCF-7 cell apoptosis, and significantly contributing to the scarcely investigated in vitro research in such a field.