International Journal of Radiation Biology, December 2014; 90(12): 1152–1161 © 2014 Informa UK, Ltd. ISSN 0955-3002 print / ISSN 1362-3095 online DOI: 10.3109/09553002.2014.934927
PARP inhibitor attenuated colony formation can be restored by MAP kinase inhibitors in different irradiated cancer cell lines Eniko Hocsak1,4, Anna Cseh1, Aliz Szabo1, Szabolcs Bellyei1,2, Eva Pozsgai1, Tamas Kalai3, Kalman Hideg3, Balazs Sumegi1,4,5 & Arpad Boronkai1,2 1Department of Biochemistry and Medical Chemistry, Medical School, 2Institution of Oncotherapy, Clinical Centre, and
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3Institution of Organic and Medicinal Chemistry, University of Pecs, 4Nuclear-Mitochondrial Interactions Research Group,
Hungarian Academy of Sciences, Budapest, and 5Szentagothai Research Centre, Pecs, Hungary
a new era of drug development. In recent years numerous molecular targets have been identified. At the same time, various more or less efficient blocking agents, like anti-EGFR (epidermal growth factor receptor) or anti-VEGF (vascular endothelial growth factor) monoclonal antibodies, inhibitors of tyrosine kinase domains of certain signal transduction elements (EGFR, BCR/ABL, EML4/ALK, etc.) have been developed. While these efforts are seeking to determine tumor-specific genetic aberrations (chronic myeloid leukaemia, gastrointestinal stromal tumour, non-smokers’ lung adenocarcinoma), intensive research has also been carried out to explore general signal transduction pathways, like PTEN-PI3K-Akt-mTOR, and RasRaf-Mek-Erk. Their possible function in tumor genesis, survival and progression is under investigation. In clinical practice, inhibitors are widely used against these latter cascade elements (everolimus, temsirolimus), and large number of further compounds are being developed or tested in clinical phase trials. Nevertheless, these inhibitors are rarely examined as a potential tool to improve the efficiency of simultaneous radiotherapy, or even if a research of this nature takes place, it will rarely yield synergistic or additive effect (Dedes et al. 2010). However, an intensely investigated target is the action of the poly(ADPribose) polymerase (PARP) in cancer cells. Relevant literature have reported several successful clinical trials on PARP inhibitors (Audeh et al. 2010, Gelmon et al. 2011, Yuan et al. 2011). At the same time, controversial results appeared in the literature concerning their role along with simultaneous chemotherapy (Drew et al. 2011, Gangopadhyay et al. 2011, Geng et al. 2011, Rios and Puhalla 2011). This paper presents evidence for the possible role of PARP enzyme in irradiation-related cellular restoring mechanisms, as well as the potential role of PARP inhibitor, HO3089, as a radiosensitizer in different cancer cell lines. Additionally, we suggest that the inhibition of signal
Abstract Purpose: Sensitizing cancer cells to irradiation is a major challenge in clinical oncology. We aimed to define the signal transduction pathways involved in poly(ADP-ribose) polymerase (PARP) inhibitor-induced radiosensitization in various mammalian cancer lines. Materials and methods: Clonogenic survival assays and Western blot examinations were performed following telecobalt irradiation of cancer cells in the presence or absence of various combinations of PARP- and selective mitogen-activated protein kinase (MAPK) inhibitors. Results: HO3089 resulted in significant cytotoxicity when combined with irradiation. In human U251 glioblastoma and A549 lung cancer cell lines, Erk1/2 and JNK/SAPK were found to mediate this effect of HO3089 since inhibitors of these kinases ameliorated it. In murine 4T1 breast cancer cell line, p38 MAPK rather than Erk1/2 or JNK/SAPK was identified as the main mediator of HO3089’s radiosensitizing effect. Besides the aforementioned changes in kinase signaling, we detected increased p53, unchanged Bax and decreased Bcl-2 expression in the A549 cell line. Conclusions: HO3089 sensitizes cancer cells to photon irradiation via proapoptotic processes where p53 plays a crucial role. Activation of MAPK pathways is regarded the consequence of irradiation-induced DNA damage, thus their inhibition can counteract the radiosenzitizing effect of the PARP inhibitor. Keywords: PARP, radiosensitizers, clonogenic cell survival, p53
Introduction Considering the fact that cytotoxic drugs commonly used in the last decades were unable to provide sufficient antitumor effect – even in combinations – in every cancer cell types, targeted therapy appeared heralding new aims and
Correspondence: Dr Arpad Boronkai, PhD, Department of Biochemistry and Medical Chemistry, Medical School, University of Pecs, 12 Szigeti Street, 7624, Pecs, Hungary. Tel: ⫹ 36 72536 080. Fax: ⫹ 36 72536-481. E-mail: [email protected] (Received 9 May 2013; revised 12 May 2014; accepted 9 June 2014)
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Cancer cell radiosensitization by PARP inhibitor 1153 transduction pathways involved have disadvantageous effect on the aforementioned process.
Materials and methods
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Cell and culture conditions Human A549 lung cancer, human U251 glioblastoma and murine 4T1 breast cancer cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). A549 cells were maintained in Minimum Essential Medium (MEM), while U251 and 4T1 cells in Roswell Park Memorial Institute (RPMI) 1640 medium (PAA Laboratories, Cölbe, Germany), both supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin mixture (Gibco/Invitrogen, Carlsbad, CA, USA). Cells were grown in an atmosphere containing 5% CO2 in a humidified 37°C incubator.
Drug and radiation treatment HO3089, a PARP inhibitor was synthesized by Professor Kalman Hideg (University of Pecs, Faculty of Medicine). Inhibitors (PD98059, SB203580, LY294002, JNK Inhibitor II) were purchased from Calbiochem (Darmstadt, Germany). Stock solutions were dissolved in dimethyl sulfoxide (DMSO) and diluted in culture medium to the specified final concentrations (HO3089 – 10 μM, PD98059 – 4 μM, SB203580 – 1.2 μM, LY294002 – 10 μM, JNK Inhibitor II – 1 μM) for cell treatment. A telecobalt external irradiation equipment (Theratron 780C, average photon energy of 1.25 MeV) was applied for irradiation of the cells with a dose of 2.0 or 4.0 Grays.
Clonogenic survival assay Cells were trypsinized and plated in triplicates into six-well plates at 500 cells/well. Cells were treated in combination with HO3089 and MAPK inhibitors 1 h prior to radiation exposure. The effect of individual chemicals and irradiation was assessed separately in parallel experiments. The efficacy of the MAPK inhibitors applied was tested by Western blot method, using phosphorylation specific anti-Erk1/2 (extracellular signal regulated kinase), anti-p38 MAPK and anti-SAPK/ JNK (stress activated protein kinase/Jun-kinase) primary antibody (Cell Signaling Technology, Danvers, MA, USA). All of the experiments were carried out along with the appropriate corresponding controls. After 7 days of incubation, the cells were washed and stained with crystal violet, and the colonies containing more than 50 cells were counted. The number of colonies were determined and expressed as the fraction of the number of colonies in untreated controls of each cell type.
Western blot analysis Cells were seeded and cultured overnight before chemicals were added to the medium in a concentration as indicated earlier. After the 24-h incubation period, the cells were harvested in cold lysis buffer (0.5 mM sodium metavanadate, 1 mM ethylenediaminetetraacetic acid [EDTA] and a protease inhibitor cocktail in phosphate-buffered saline, pH: 7.4). After cell disruption in a Teflon/glass homogenizer, the homogenate was pelleted, and protein content of the supernatant was measured by bicinchonicic acid reagent and
equalized for 1 mg/ml protein content in Laemmli sample buffer. Proteins (50 μg/lane) were separated in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% low-fat milk for 1 h at room temperature, then exposed to anti-Akt-1, phosphospecific anti-Akt-1 Ser473, anti-Bax, anti-Bcl-2, phospho-specific anti-Erk1/2 Thr202/Tyr204, phospho-specific anti-p38 MAPK Thr180/Tyr182 (Cell Signaling Technology, Danvers, MA, USA), anti-PARP-1, anti-PAR (Santa Cruz Biotechnology, Heidelberg, Germany), phospho-specific anti-SAPK/JNK Thr183/ Tyr185 (RandD Systems, Abingdon, UK), phospho-specific anti-p53 Ser15 (PromoKine, Heidelberg, Germany), anti-caspase-3, phospho-specific anti-Raf1 Ser338, anti-p21 (Thermo Scientific, Runcorn, UK) or anti-β actin (Sigma Aldrich Co, Budapest, Hungary) at 4°C overnight, in a dilution of 1:1000 in 5% bovine serum albumine, 1 ⫻ tris(hydroxymethyl) aminomethane buffered saline and 0.1% Tween20 solution. Appropriate horseradish peroxidase-conjugated anti-rabbit (1:3000, Bio-Rad, Budapest, Hungary), anti-mouse (1:5000, Sigma Aldrich Co, Budapest, Hungary) or anti-rat (1:5000, Enzo Life Sciences, Lörrach, Germany) secondary antibodies were applied for 1 h at room temperature. Peroxidase labeling was visualized by enhanced chemiluminescence (ECL) using ECL Western blot detection system (GE Healthcare, Freiburg, Germany). After scanning, pixel densities of bands were quantified by means of NIH ImageJ program. Pixel densities of bands of the same film were normalized to that of the loading control and expressed as a percentage of the respective untreated, unirradiated control samples. All experiments were performed at least three times.
Statistical analysis All data were expressed as mean ⫾ standard error of mean (SEM). Statistical analysis was performed by using twofactor analysis of variance with replication for correlations, and unpaired Student’s t-test for group comparisons. Differences with p values below 0.05 were considered as significant.
Results Effect of PARP inhibition and irradiation on colony formation in A549 cells PARP activity is indisposable in repairing irradiation-induced DNA damage. Therefore we tested the assumed synergism between PARP inhibition and irradiation by determining colony formation in the presence and absence of HO3089, a water soluble PARP inhibitor after irradiation. As we observed, the PARP inhibitor dramatically enhanced the photon irradiation effect since HO3089 pretreatment of A549 cells prior irradiation resulted in significant decrease in colony formation. Compared to untreated control group, irradiation by 2 Grays and 4 Grays without and with simultaneous HO3089 exposition shows a median of 60.98%, 20.73%, 25.61% and 4.88% colony counts, respectively. HO3089 pretreatment also resulted in significantly decreased colony formation ability in itself (68.29 % ⫾ SEM ***p ⬍ 0.001 compared to control) (Figure 1A).
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Figure 1. Effect of irradiation and HO3089 treatment on colony formation and PAR level in A549 cell line. (A) Clonogenic assay results are shown for A549 lung adenocarcinoma cells exposed or not to 2 or 4 Gy irradiation and/or PARP inhibitor (HO3089) as indicated underneath the diagram. Colony numbers in the percentages of untreated cells are presented as mean ⫾ SEM. *P ⬍ 0.05 compared to control values; #p ⬍ 0.05 compared to corresponding 2 or 4 Gy irradiated group; †p ⬍ 0.05 compared to HO3089 values. (B) PAR levels were detected by Western blotting from homogenates of cells treated as above. β-actin immunoreactivity was used to show even loading. Representative blots of three independent experiments are presented.
Efficacy of PARP inhibitor has been proved by Western blotting utilizing an anti-PAR primary antibody (Figure 1B).
Effect of PARP inhibition and irradiation on MAP kinases and pro- and antiapoptotic factors in A549 cell line As an attempt to identify mechanisms underlying HO3089’s radiosensitizing effect, we assessed early changes in protein expression and activation in A549 homogenates 24 h after irradiation (Figure 2). Significantly increased amount of the cleaved proapoptotic caspase-3 was observed in all cases except for the control. However, the amount of it did not show any notable differences when groups 3–6 were compared. Akt phosphorylation was elevated in irradiated cells, treated or not with HO3089. PARP inhibitor was unable to trigger detectable Akt activation by itself. Total Akt level, however, remained unchanged. Definite p53 activation was resulted by the PARP inhibitor exposition. Meanwhile, a dose-dependent enhanced phosphorylation of p53 was observed as the irradiation fraction doses were increased (246% and 623% increase, respectively). This effect was further augmented by the simultaneous administration of HO3089. Parallel with the results of caspase-3 activation, an elevated level of p21 phosphorylation appeared in irradiated cells irrespective of the fact whether they had been treated or not with HO3089. The proapoptotic signal transduction member, Bax, was found to be expressed in the same extent in all samples examined. On the other hand, antiapoptotic Bcl-2 was suggested to be an element playing an important
role in the irradiation mediated processes, i.e., its level decreased in all circumstances compared to control. HO3089 had no significant influence on the expression of Bcl-2. Detecting PARP enzyme levels, no considerable alterations could be seen among the samples examined. In A549 cell line Raf-1 was almost inactivated in control cells. At the same time, Raf-1 phosphorylation – activation – was detected in nearly the same extent in all other cases. Erk1/2 activation – similarly to its upstream regulator Raf-1 – was the consequence of HO3089 administration, such as irradiation alone. This phenomenon was self-evident in case higher fraction dose, 4 Gy irradiation, was applied (19% and 148% median increase, respectively). Combined treatment also resulted in undoubtedly elevated Erk1/2 activation (see the bar diagram). JNK/SAPK activation decreased in a dose-dependent manner in case irradiation had been applied (16% and 38% median decrease compared to control), which attenuated further in the presence of HO3089. Slightly increased activation of p38 MAPK was detected following irradiation (57% and 61% median elevation compared to control), which was enhanced further by the simultaneous application of HO3089.
Effect of PARP inhibition and irradiation on MAP kinases and pro- and antiapoptotic factors in 4T1 cell line To check whether the observed changes in MAPK activation and apoptosis regulating protein expression are universal among the cell lines investigated, we performed the above
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Cancer cell radiosensitization by PARP inhibitor 1155
Figure 2 (A) and (B). Western blot: Effect of HO3089 on steady-state level or phosphorylation state of the indicated signaling pathway members with or without 2 and 4 Gy irradiation in A549 cells. Different experimental combinations were tested, as shown in the figure. Protein expression was assessed following 24 hours of treatment by immunoblotting. β-actin immunoreactivity was used to show even loading. Representative blots of three independent experiments are presented. Pixel densities of the bands from three independent experiments are presented as mean ⫾ SEM for those members that were significantly affected by the treatments (*p ⬍ 0.05 compared to control values, #p ⬍ 0.05 compared to corresponding 2 or 4 Gy irradiated group, †p ⬍ 0.05 compared to HO3089 values). Data are shown in arbitrary units.
experiment on 4T1 cells. As we found (Figure 3), phosphop53 level was not affected by the PARP enzyme inhibitor HO3089 in itself; however, following irradiation the activation increased in a dose-dependent manner. Further significant elevation was detected in case of irradiated and PARP inhibited samples (91% and 121% elevation, respectively). There was no significant elevation in the Bcl-2 expression in case HO3089 exposition was applied separately. However, significant dose-dependent reduction was observed following irradiation (32% and 54% reduction as a fraction of the control value), which was synergistically influenced by the simultaneous PARP inhibition of the low dose irradiated cells. Single exposition by HO3089 did not have any significant effect on the Bax expression. Although significant increase was evident following irradiation, this effect was independent of both the dose applied and the simultaneous exposition to the PARP inhibitor (median elevation of 68%, 76%, 72%, and 75%, respectively). Erk1/2 presented significant increase only when HO3089 pretreated cells were irradiated (69% and 81% median change). JNK activation was a significant consequence of PARP inhibition or irradiation as well (respective medians of increase are of 30%, 36% and 63%). Coexposition resulted in synergistic augmentation of JNK phosphorylation. This effect was strongly significant in case higher fraction dose was applied (58% and 164% median elevation respectively, compared to untreated, unirradiated
control). Phospho-p38 MAPK level significantly increased both after single agent HO3089 treatment and irradiation, irrespectively of the dose applied (62% and 107% median increase). Simultaneous administration of HO3089 to photon irradiation resulted in the synergistic enhancement in p38 MAPK activation (109% and 206% median elevation as the fraction of untreated, unirradiated control).
Effect of MAPK inhibitors on PARP inhibitor-induced radiosensitizing in A549, 4T1 and U251 cells Inhibitory effect was confirmed for the kinase pathways examined (Figure 4), except for Akt, since the latter had been found to block the proliferation of the cells in colony formation assays entirely (data not shown). The Erk1/2 inhibitor PD98059 has been proven to extremely reduce Erk1/2 activation, although the level of the active molecule had been lower even initially in 4T1 cell lysates compared to that of A549 and U251 cells. JNK-inhibitor II significantly decreased JNK/SAPK activation in human-derived cell line lysates, while a total inhibition was achieved in 4T1 cell line. Initial level of this signal transduction element was higher in A549 and U251 cells than in the murine 4T1 cell line. SB203580, a well-known inhibitor of p38 MAPK phosphorylation, almost completely knocked out the activation of this signal cascade member in all cell cultures used. However, initial content of the active protein was higher in 4T1 cell line than those of
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Figure 3. Western blot: Effect of HO3089 on steady-state level or phosphorylation state of the indicated signaling pathway members with or without 2 and 4 Gy irradiation in 4T1 cells. Experimental conditions, methods and evaluation process are the same as described in the legend of Figure 2.
the other MAPK members examined. By using the selective inhibitors, we investigated their interactions with HO3089 in all three cell lines (Figure 5). Administration of MAPK inhibitors all resulted in altered ability of colony formation. This effect was found to be the most significant influence in case of Erk1/2 inhibition of A549 and U251 cells. 29% and 85.7% median elevation in colony numbers was detected as compared to control groups. P38 MAPK inhibitor was proven to be the most effective agent in the stimulation of the 4T1 cell line colony formation. Significant amelioration in colony formation ability was evoked by JNK inhibitor II in U251 glioblastoma cell line. However, Akt inhibition produced a total inability of colony formation (not shown in the Figure). HO3089 pretreatment has been shown to have inverse effect on A549 and U251 as compared to 4T1 cell proliferation.
Figure 4. Effect of MAPK inhibitors on the cell lines investigated. Phosphorylation state of the indicated MAPK was determined by Western blot analysis in A549, U251 and 4T1 cells in the presence and absence of appropriate inhibitors. β-actin was used as a loading control. Representative blots of three independent experiments are presented.
While Erk1/2 inhibition resulted in significant improvement in cell viability in the former group (12% and 57% increase in the percentage of the controls, respectively), 4T1 cells reacted the other way around. Meanwhile, p38 MAPK inhibition achieved considerable improvement in 4T1 cells’ viability along with HO3089 exposition, while it was not seen in the other cell lines. JNK inhibitor significantly counteracted the PARP inhibitor-related cell survival deterioration or benefit in A549 and 4T1 cells, while it hardly influenced the U251 lines (Figure 5). After determining the interaction between the PARP- and the MAPK inhibitors, we utilized them to verify the findings presented in Figures 2 and 3 about the role of the respective kinase signaling pathways in mediating the radiosensitizing effect of the PARP inhibitor in the cell lines. In order to demonstrate the results more clearly – particularly in case of extreme deviations – the corresponding data are presented here as the log(10) values of the percentages representing the respective untreated, unirradiated cells (Figure 6). Application of irradiation resulted in definitely decreased colony formation ability in all cell lines. However, this effect was dramatically antagonized by Erk1/2 inhibitor in the human-derived samples. A significant improvement was seen in the colony formation in A549 and U251 cells compared to the appropriate irradiated groups with no Erk1/2 inhibitor. Similarly, significantly improved proliferation was observed in the groups above as compared to the respective irradiated samples in case JNK inhibitor II coexposition was applied. 4T1 cells, at the same time, did not react on this agent to its advantage. However, p38 MAPK inhibitor increased the number of colonies the most significantly in 4T1 irradiated cells, while leaving A549 cell types almost unchanged.
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Cancer cell radiosensitization by PARP inhibitor 1157
Figure 5. Effect of MAPK inhibitors and HO3089 on colony formation in A549, U251 and 4T1 cells. Cells were seeded into six-well plates (500 cells/ well), cultured for 7 days and treated with Erk1/2-, p38 MAPK- or JNK-inhibitor without or with HO3089. The plates were stained with crystal violet and the colonies were quantified. The treatment protocol is indicated underneath the diagram. Data from three independent experiments are expressed as mean ⫾ SEM. *p ⬍ 0.05 compared to the untreated control group, #p ⬍ 0.05 compared to the HO3089-treated group and †p ⬍ 0.05 compared to the corresponding MAPK inhibitor group.
HO3089 administration to the culture medium before irradiation significantly decreased proliferation in all cell lines examined. Erk1/2 inhibitor in this setting enhanced colony formation ability of A549 and U251 cells exceedingly. Significant improvement in cell viability also occured in
case of JNK/SAPK inhibition without exception. This latter effect was evident even in case of low fraction dose in A549 cells; however, it appeared in U251 and 4T1 cell lines only when high fraction dose was applied. Concerning the highest level of significance, proliferation of 4T1cells was
Figure 6. Effect of MAPK inhibitors and 2 or 4 Gy irradiation on colony formation in A549, U251 and 4T1 cells. Cells were seeded into six-well plates (500 cells/well), exposed or not to 2 or 4 Gy irradiation in the presence or absence of Erk1/2-, p38 MAPK- or JNK-inhibitor, then cultured for 7 days in the presence or absence of the inhibitors. The plates were stained with crystal violet and the colonies were quantified. The treatment protocol is indicated underneath the diagram. Data from three independent experiments are expressed as % of the untreated cells, mean ⫾ SEM. Note that the y axis is logarithmic. *P ⬍ 0.05 compared to the untreated control group and #p ⬍ 0.05 compared to the corresponding 2 or 4 Gy-irradiated group.
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strikingly improved again by p38 MAPK inhibitor exposition. Significant increase in colony numbers was found even in comparison to the 2 Gy-irradiated HO3089 pretreated 4T1 cell sample. Results seen with high fraction dose in p38 MAPK-inhibited 4T1 cells are even more dramatic. This value was found to be higher in the log(10) scale than that of the respective p38 MAPK-uninhibited samples (Figure 7).
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Discussion Several papers reported on the significance of PI3K-PTENAkt-mTOR, Ras-Raf-MEK-Erk1/2 or even JNK/SAPK and p38 MAPK pathway activation in the development and promotion of cancer cells, as well as the consequent sensitivity to proapoptotic agents, and cytotoxic effects influencing signal transduction (Kim et al. 2003, Gao et al. 2006, Lee et al. 2008a, 2008b, Affolter et al. 2010, Ma et al. 2011, Marampon et al. 2011). We are also aware of signal transduction element coding gene mutations having impact on anticancer therapy (McCubrey et al. 2011). Besides, the Ras-Raf-MEKErk1/2 cascade, which is predominantly known as a promoter of cell viability, differentiation, survival and tumour progression, can play an undisputable role in cell death and inhibition of proliferation in U251 cell line (Arai et al. 2004, Hagan et al. 2007, Lu et al. 2010, Tomiyama et al. 2010). This fact is of outstanding significance in cases like k-Ras
mutated malignancies – which we demonstrated with A549 cell line – since they are still considered as one of the major challenges in drug development. Still, JAK2 inhibition and Ras-1 kinase suppressor inhibition resulted in enhanced radiosensitivity of such cells (Xiao et al. 2010, Sun et al. 2011). Our results confirmed the significance of Erk1/2 and SAPK/JNK activation in U251 and A549 cell lines, while the p38 MAPK activation was associated with improved clonogenity of 4T1 cells. Several data obtained with regard to U251 cell line was found to be in line with those described by Tomiyama’s group (Tomiyama et al. 2010). However, we can report on the significance of Erk1/2 activation in photon irradiation-induced glioblastoma cell death as well. As far as the role of Erk1/2 concerned, its activation and actual impact on cell survival can vary depending on the irradiation dose applied (Khalil et al. 2011). Enhanced sensitivity of irradiated cancer cell lines to PARP inhibition has already been demonstrated (Daniel et al. 2010, Senra et al. 2011, Wang et al. 2011). This process is mediated by EGFR-Erk signaling (Hagan et al. 2007). An efficient PARP enzyme inhibitor, HO3089, applied frequently in our previous experiments (Alexy et al. 2004, Kalai et al. 2009) was used in present study. It had been shown to increase the activation of PI3K-Akt pathway and suppress JNK and p38 MAPK (Alexy et al. 2004, Kovacs et al. 2006, 2009,
Figure 7. Effect of HO3089, MAPK inhibitors and 2 or 4 Gy irradiation on colony formation in A549, U251 and 4T1 cells. Cells were seeded into six-well plates (500 cells/well), exposed or not to 2 or 4 Gy irradiation in the presence or absence of Erk1/2-, p38 MAPK- or JNK inhibitor and/or HO3089, then cultured for 7 days in the presence or absence of the aforementioned compounds. The plates were stained with crystal violet and the colonies were quantified. The treatment protocol is indicated underneath the diagram. Data from three independent experiments are expressed as % of the untreated cells, mean ⫾ SEM. Note that the y axis is logarithmic. *P ⬍ 0.05 compared to the untreated control group, #p ⬍ 0.05 compared to the HO3089-treated group and †p ⬍ 0.05 compared to the corresponding MAPK inhibitor group.
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Cancer cell radiosensitization by PARP inhibitor 1159 Mester et al. 2009). We have demonstrated in A549 cells that HO3089 activated Erk1/2 pathways, while it had only slight effects on Akt, JNK/SAPK or p38 MAPK activation if administered alone. Irradiation resulted in Akt phosphorylation – possibly through the ROS formation generated PTEN activation – which remained unchanged beside PARP inhibition. Extreme activation of the Raf-1 – Erk1/2 pathway seems to be responsible for the significant anticancer effect in A549 cell line (Figure 2). Similar cytotoxicity is attributable to the significant activation of p38 MAPK in the 4T1 cells (Figure 3). That is, we have to take into account those – currently not explored – molecular pathways which are activated following ionizing irradiation. Damage of the DNA strand can evoke several complex signaling mechanisms (like ATM kinase mediated pathways) which may also contribute to the activation of the Erk1/2 route in cell death processes. A common result with Senra et al. (2011) and Wang et al. (2011) is that extreme decrease in cell proliferation was observed in our study, regardless of cancer cell type, in case irradiation was anticipated by exposition to HO3089. As we have found, human A549 and U251 cells were negatively influenced by radiation and PARP inhibitor-induced Erk1/2 activation. Although the exact pathway has to be clarified, a possible role of the endoplasmic reticulum is suggested (Arai et al. 2004). On the contrary, the colony formation ability of 4T1 cells was significantly improved by the p38 MAPK suppressor SB203580. Furthermore, A549 and U251 cells sensitively reacted on JNK/SAPK inhibitor as well, having improved colony formation ability. Similarly to previous results in the literature (Gangopadhyay et al. 2011), caspase-3 has slight influence on PARP inhibitor-mediated cancer cell toxicity in our models as well. The reason why HO3089 resulted in reduced PARP-1 expression in A549 cells was not clarified within the confines of the current work. Similar experiments concerning cancer cells have already been published (Tang et al. 2013). Taking our results into consideration, it can be concluded, that the difference within the signal transduction pathway balance may serve as a target in approaching the sensitivity of various cancer cell types. Concerning the three cell lines examined, no exact conformity was explored in their responsiveness to the agents applied. On the one hand, it is a novel encouraging result regarding the sensitization of malignancies to irradiation while normal tissue resistance can be enhanced. On the other hand, these results emphasize the significance of cell type specific individual approach in anticancer drug development and treatment planning. The uncertain MAPK-related signaling mechanisms arising following irradiation may seriously contribute to the treatment outcome, thus their inhibition may result inversely; i.e., in the deterioration of anticancer effect, as it has already been published (Daniel et al. 2010, Dedes et al. 2010, Issaeva et al. 2010, Konstantinopoulos et al. 2010, Toshimitsu et al. 2010, Drew et al. 2011). We have also observed that photon irradiation with or without HO3089 exposition significantly suppressed Bcl-2 expression (Figures 2 and 3), which can be explained by the
observations that NF-kappaB activates the expression of Bcl-2 (Catz and Johnson 2003, Zhao et al. 2011). Inhibition of PARP suppresses the NF-kappaB activation (Oliver et al. 1999, Veres et al. 2004, Zerfaoui et al. 2010, Tang et al. 2013). DNA damaging can activate the endogenous mitochondrial apoptotic pathway of p53 (Brown and Attardi 2005). Thus, most of the anticancer agents, including irradiation, that act through DNA damage or stress-inducing mechanisms require wild type p53 for apoptosis (Erster and Moll 2004, Brown and Attardi 2005, Galluzzi et al. 2008). Actions of p53 permeabilize the outer mitochondrial membrane, and promote the release of proapoptotic factors. In addition, PARP inhibitor can promote the nuclear export of p53 (Abd Elmageed et al. 2012), therefore inhibition of PARP by HO3089 can facilitate the cytoplasmic transport of p53, resulting in a more effective activation of cell death. Focusing on the proapoptotic – antiapoptotic balance in our experimental model with the A549 cells, increased p53 activity – as a common result of irradiation and PARP inhibition, and constant Bax expression was found, while the Bcl-2 expression decreased considerably. These changes are undisputably attributable to the dramatic decrease in cell proliferation of 4T1 cells as well. Summarizing our results and referring to all the data from the articles cited, it can be concluded that the differences in the role and balance of common signal transduction pathways are able to influence the outcome of a cytotoxic effect. A complex antitumour intervention found to be useful in certain vertebrates, not necessarily associated with the same efficiency in human cases. Thus, it is worth to taking this aspect into account in drug development or introduction of a new compound into irradiation-based combined modality treatment to avoid undesired consequences.
Acknowledgements The authors would like to express their deepest appreciation to Professor Ferenc Gallyas (Department of Biochemistry and Medical Chemistry, Medical School, University of Pecs) and Gyorgy Cserna for their professional and linguistic comments and advice which contributed to the improvement of this manuscript.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by the Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences, Research Grants from AOK-KA-10-04-2011, AOK-KA-34039-1004/2010, AOK-KA-34039/KA-OTKA/11-04, AOK-KA-34039/10-24, 34039/ KA-OTKA/2011/11-17, OTKA-K-73738, SROP-4.2.2/B-10/ 1-2010-0029 (Supporting Scientific Training of Talented Youth at the University of Pécs), SROP-4.2.1.B-10/2/KONV2010-0002, Developing the South-Transdanubian Regional University Competitiveness and the Nuclear-Mitochondrial Interactions Research Group, Hungarian Academy of Sciences, PO Box 1000, H-1245 Budapest, Hungary.
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References Abd Elmageed ZY, Naura AS, Errami Y, Zerfaoui M. 2012. The poly(ADPribose) polymerases (PARPs): New roles in intracellular transport. Cell Signal 24:1–8. Affolter A , Fruth K, Brochhausen C, Schmidtmann I, Mann WJ, Brieger J. 2010. Activation of mitogen-activated protein kinase extracellular signal-related kinase in head and neck squamous cell carcinomas after irradiation as part of a rescue mechanism. Head Neck 33:1448–57. Alexy T, Toth A , Marton Z, Horvath B, Koltai K , Feher G, Kesmarky G, Kalai T, Hideg K, Sumegi B, et al. 2004. Inhibition of ADP-evoked platelet aggregation by selected poly(ADP-ribose) polymerase inhibitors. J Cardiovasc Pharmacol 43:423–431. Arai K , Lee SR, van Leyen K , Kurose H, Lo EH. 2004. Involvement of ERK MAP kinase in endoplasmic reticulum stress in SH-SY5Y human neuroblastoma cells. J Neurochem 89:232–9. Audeh MW, Carmichael J, Penson RT, Friedlander M, Powell B, Bell-McGuinn KM, Scott C, Weitzel JN, Oaknin A , Loman N, et al. 2010. Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer: A proof-of-concept trial. Lancet 376:245–251. Brown JM, Attardi LD. 2005. The role of apoptosis in cancer development and treatment response. Nat Rev Cancer 5:231–237. Catz SD, Johnson JL. 2003. BCL-2 in prostate cancer: A mini review. Apoptosis 8:29–37. Daniel RA , Rozanska AL, Mulligan EA , Drew Y, Thomas HD, Castelbuono DJ, Hostomsky Z, Plummer ER, Tweddle DA , Boddy AV, et al. 2010. Central nervous system penetration and enhancement of temozolomide activity in childhood medulloblastoma models by poly(ADP-ribose) polymerase inhibitor AG-014699. Br J Cancer 103:1588–1596. Dedes KJ, Wetterskog D, Mendes-Pereira AM, Natrajan R, Lambros MB, Geyer FC, Vatcheva R, Savage K , Mackay A , Lord CJ, et al. 2010. PTEN deficiency in endometrioid endometrial adenocarcinomas predicts sensitivity to PARP inhibitors. Sci Transl Med 2:53ra75. Drew Y, Mulligan EA , Vong WT, Thomas HD, Kahn S, Kyle S, Mukhopadhyay A , Los G, Hostomsky Z, Plummer ER, et al. 2011. Therapeutic potential of poly(ADP-ribose) polymerase inhibitor AG014699 in human cancers with mutated or methylated BRCA1 or BRCA2. J Natl Cancer Inst 103:334–346. Erster S, Moll UM. 2004. Stress-induced p53 runs a direct mitochondrial death program: Its role in physiologic and pathophysiologic stress responses in vivo. Cell Cycle 3:1492–1495. Galluzzi L, Morselli E, Kepp O, Tajeddine N, Kroemer G. 2008. Targeting p53 to mitochondria for cancer therapy. Cell Cycle 7: 1949–1955. Gangopadhyay NN, Luketich JD, Opest A , Visus C, Meyer EM, Landreneau R, Schuchert MJ. 2011. Inhibition of poly(ADP-ribose) polymerase (PARP) induces apoptosis in lung cancer cell lines. Cancer Invest 29:608–616. Gao L, Feng Y, Bowers R, Becker-Hapak M, Gardner J, Council L, Linette G, Zhao H, Cornelius LA . 2006. Ras-associated protein-1 regulates extracellular signal-regulated kinase activation and migration in melanoma cells: Two processes important to melanoma tumorigenesis and metastasis. Cancer Res 66:7880–7888. Gelmon KA , Tischkowitz M, Mackay H, Swenerton K , Robidoux A , Tonkin K, Hirte H, Huntsman D, Clemons M, Gilks B, et al. 2011. Olaparib in patients with recurrent high-grade serous or poorly differentiated ovarian carcinoma or triple-negative breast cancer: A phase 2, multicentre, open-label, non-randomised study. Lancet Oncol 12:852–861. Geng L, Huehls AM, Wagner JM, Huntoon CJ, Karnitz LM. 2011. Checkpoint signaling, base excision repair, and PARP promote survival of colon cancer cells treated with 5-fluorodeoxyuridine but not 5-fluorouracil. PLoS One 6:e28862. Hagan MP, Yacoub A , Dent P. 2007. Radiation-induced PARP activation is enhanced through EGFR-ERK signaling. J Cell Biochem 101: 1384–1393. Issaeva N, Thomas HD, Djureinovic T, Jaspers JE, Stoimenov I, Kyle S, Pedley N, Gottipati P, Zur R, Sleeth K , et al. 2010. 6-thioguanine selectively kills BRCA2-defective tumors and overcomes PARP inhibitor resistance. Cancer Res 70:6268–6276. Kalai T, Balog M, Szabo A , Gulyas G, Jeko J, Sumegi B, Hideg K . 2009. New poly(ADP-ribose) polymerase-1 inhibitors with antioxidant activity based on 4-carboxamidobenzimidazole-2-ylpyrroline and -tetrahydropyridine nitroxides and their precursors. J Med Chem 52:1619–1629.
Khalil A , Morgan RN, Adams BR, Golding SE, Dever SM, Rosenberg E, Povirk LF, Valerie K. 2011. ATM-dependent ERK signaling via AKT in response to DNA double-strand breaks. Cell Cycle 10:481–491. Kim DS, Kim SY, Lee JE, Kwon SB, Joo YH, Youn SW, Park KC. 2003. Sphingosine-1-phosphate-induced ERK activation protects human melanocytes from UVB-induced apoptosis. Arch Pharm Res 26: 739–746. Konstantinopoulos PA , Spentzos D, Karlan BY, Taniguchi T, Fountzilas E, Francoeur N, Levine DA , Cannistra SA . 2010. Gene expression profile of BRCAness that correlates with responsiveness to chemotherapy and with outcome in patients with epithelial ovarian cancer. J Clin Oncol 28:3555–3561. Kovacs K , Toth A , Deres P, Kalai T, Hideg K , Gallyas F Jr, Sumegi B. 2006. Critical role of PI3-kinase/Akt activation in the PARP inhibitor induced heart function recovery during ischemia-reperfusion. Biochem Pharmacol 71:441–452. Kovacs K , Hanto K , Bognar Z, Tapodi A , Bognar E, Kiss GN, Szabo A , Rappai G, Kiss T, Sumegi B, et al. 2009. Prevalent role of Akt and ERK activation in cardioprotective effect of Ca(2⫹) channel- and betaadrenergic receptor blockers. Mol Cell Biochem 321:155–164. Lee KB, Kim KR, Huh TL, Lee YM. 2008a. Proton induces apoptosis of hypoxic tumor cells by the p53-dependent and p38/JNK MAPK signaling pathways. Int J Oncol 33:1247–1256. Lee KB, Lee JS, Park JW, Huh TL, Lee YM. 2008b. Low energy proton beam induces tumor cell apoptosis through reactive oxygen species and activation of caspases. Exp Mol Med 40:118–129. Lu K, Liang CL, Liliang PC, Yang CH, Cho CL, Weng HC, Tsai YD, Wang KW, Chen HJ. 2010. Inhibition of extracellular signalregulated kinases 1/2 provides neuroprotection in spinal cord ischemia/reperfusion injury in rats: Relationship with the nuclear factor-kappaB-regulated anti-apoptotic mechanisms. J Neurochem 114:237–246. Ma ZC, Hong Q, Wang YG, Tan HL, Xiao CR, Liang QD, Wang DG, Gao Y. 2011. Ferulic acid protects lymphocytes from radiation-predisposed oxidative stress through extracellular regulated kinase. Int J Radiat Biol 87:130–140. Marampon F, Gravina GL, Di Rocco A , Bonfili P, Di Staso M, Fardella C, Polidoro L, Ciccarelli C, Festuccia C, Popov VM, et al. 2011. MEK/ERK inhibitor U0126 increases the radiosensitivity of rhabdomyosarcoma cells in vitro and in vivo by downregulating growth and DNA repair signals. Mol Cancer Ther 10:159–168. McCubrey JA , Steelman LS, Kempf CR, Chappell WH, Abrams SL, Stivala F, Malaponte G, Nicoletti F, Libra M, Basecke J, et al. 2011. Therapeutic resistance resulting from mutations in Raf/MEK/ ERK and PI3K/PTEN/Akt/mTOR signaling pathways. J Cell Physiol 226:2762–2781. Mester L, Szabo A , Atlasz T, Szabadfi K , Reglodi D, Kiss P, Racz B, Tamas A , Gallyas F Jr, Sumegi B, et al. 2009. Protection against chronic hypoperfusion-induced retinal neurodegeneration by PARP inhibition via activation of PI-3-kinase Akt pathway and suppression of JNK and p38 MAP kinases. Neurotox Res 16:68–76. Oliver FJ, Menissier-de Murcia J, Nacci C, Decker P, Andriantsitohaina R, Muller S, de la Rubia G, Stoclet JC, de Murcia G. 1999. Resistance to endotoxic shock as a consequence of defective NF-kappaB activation in poly (ADP-ribose) polymerase-1 deficient mice. EMBO J 18:4446–4454. Rios J, Puhalla S. 2011. PARP inhibitors in breast cancer: BRCA and beyond. Oncology (Williston Park) 25:1014–1025. Senra JM, Telfer BA , Cherry KE, McCrudden CM, Hirst DG, O’Connor MJ, Wedge SR, Stratford IJ. 2011. Inhibition of PARP-1 by olaparib (AZD2281) increases the radiosensitivity of a lung tumor xenograft. Mol Cancer Ther 10:1949–1958. Sun Y, Moretti L, Giacalone NJ, Schleicher S, Speirs CK , Carbone DP, Lu B. 2011. Inhibition of JAK2 signaling by TG101209 enhances radiotherapy in lung cancer models. J Thorac Oncol 6:699–706. Tang Y, Wang YL, Yang L, Xu JX, Xiong W, Xiao M, Li M. 2013. Inhibition of arginine ADP-ribosyltransferase 1 reduces the expression of poly(ADP-ribose) polymerase-1 in colon carcinoma. Int J Mol Med 32:130–136. Tomiyama A , Tachibana K, Suzuki K, Seino S, Sunayama J, Matsuda KI, Sato A , Matsumoto Y, Nomiya T, Nemoto K, et al. 2010. MEKERK-dependent multiple caspase activation by mitochondrial proapoptotic Bcl-2 family proteins is essential for heavy ion irradiation-induced glioma cell death. Cell Death Dis 1:e60. Toshimitsu H, Yoshimoto Y, Augustine CK, Padussis JC, Yoo JS, Angelica Selim M, Pruitt SK , Friedman HS, Ali-Osman F, Tyler DS. 2010. Inhibition of poly(ADP-ribose) polymerase enhances the effect of chemotherapy in an animal model of regional therapy for
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the treatment of advanced extremity malignant melanoma. Ann Surg Oncol 17:2247–2254. Veres B, Radnai B, Gallyas F, Jr., Varbiro G, Berente Z, Osz E, Sumegi B. 2004. Regulation of kinase cascades and transcription factors by a poly(ADP-ribose) polymerase-1 inhibitor, 4-hydroxyquinazoline, in lipopolysaccharide-induced inflammation in mice. J Pharmacol Exp Ther 310:247–255. Wang L, Mason KA , Ang KK , Buchholz T, Valdecanas D, Mathur A , Buser-Doepner C, Toniatti C, Milas L. 2011. MK-4827, a PARP-1/-2 inhibitor, strongly enhances response of human lung and breast cancer xenografts to radiation. Invest New Drugs 30:2113–20. Xiao H, Zhang Q, Shen J, Bindokas V, Xing HR. 2010. Pharmacologic inactivation of kinase suppressor of Ras1 sensitizes epidermal
growth factor receptor and oncogenic Ras-dependent tumors to ionizing radiation treatment. Mol Cancer Ther 9:2724–2736. Yuan Y, Liao YM, Hsueh CT, Mirshahidi HR. 2011. Novel targeted therapeutics: Inhibitors of MDM2, ALK and PARP. J Hematol Oncol 4:16. Zerfaoui M, Errami Y, Naura AS, Suzuki Y, Kim H, Ju J, Liu T, Hans CP, Kim JG, Abd Elmageed ZY, et al. 2010. Poly(ADP-ribose) polymerase-1 is a determining factor in Crm1-mediated nuclear export and retention of p65 NF-kappa B upon TLR4 stimulation. J Immunol 185:1894–1902. Zhao X, Ning Q, Sun X, Tian D. 2011. Pokemon reduces Bcl-2 expression through NF-kappa Bp65: A possible mechanism of hepatocellular carcinoma. Asian Pac J Trop Med 4:492–497.
PARP inhibitor attenuated colony formation can be restored by MAP kinase inhibitors in different irradiated cancer cell lines Eniko Hocsak1,4, Anna Cseh1, Aliz Szabo1, Szabolcs Bellyei1,2, Eva Pozsgai1, Tamas Kalai3, Kalman Hideg3, Balazs Sumegi1,4,5 & Arpad Boronkai1,2 1Department of Biochemistry and Medical Chemistry, Medical School, 2Institution of Oncotherapy, Clinical Centre, and
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3Institution of Organic and Medicinal Chemistry, University of Pecs, 4Nuclear-Mitochondrial Interactions Research Group,
Hungarian Academy of Sciences, Budapest, and 5Szentagothai Research Centre, Pecs, Hungary
a new era of drug development. In recent years numerous molecular targets have been identified. At the same time, various more or less efficient blocking agents, like anti-EGFR (epidermal growth factor receptor) or anti-VEGF (vascular endothelial growth factor) monoclonal antibodies, inhibitors of tyrosine kinase domains of certain signal transduction elements (EGFR, BCR/ABL, EML4/ALK, etc.) have been developed. While these efforts are seeking to determine tumor-specific genetic aberrations (chronic myeloid leukaemia, gastrointestinal stromal tumour, non-smokers’ lung adenocarcinoma), intensive research has also been carried out to explore general signal transduction pathways, like PTEN-PI3K-Akt-mTOR, and RasRaf-Mek-Erk. Their possible function in tumor genesis, survival and progression is under investigation. In clinical practice, inhibitors are widely used against these latter cascade elements (everolimus, temsirolimus), and large number of further compounds are being developed or tested in clinical phase trials. Nevertheless, these inhibitors are rarely examined as a potential tool to improve the efficiency of simultaneous radiotherapy, or even if a research of this nature takes place, it will rarely yield synergistic or additive effect (Dedes et al. 2010). However, an intensely investigated target is the action of the poly(ADPribose) polymerase (PARP) in cancer cells. Relevant literature have reported several successful clinical trials on PARP inhibitors (Audeh et al. 2010, Gelmon et al. 2011, Yuan et al. 2011). At the same time, controversial results appeared in the literature concerning their role along with simultaneous chemotherapy (Drew et al. 2011, Gangopadhyay et al. 2011, Geng et al. 2011, Rios and Puhalla 2011). This paper presents evidence for the possible role of PARP enzyme in irradiation-related cellular restoring mechanisms, as well as the potential role of PARP inhibitor, HO3089, as a radiosensitizer in different cancer cell lines. Additionally, we suggest that the inhibition of signal
Abstract Purpose: Sensitizing cancer cells to irradiation is a major challenge in clinical oncology. We aimed to define the signal transduction pathways involved in poly(ADP-ribose) polymerase (PARP) inhibitor-induced radiosensitization in various mammalian cancer lines. Materials and methods: Clonogenic survival assays and Western blot examinations were performed following telecobalt irradiation of cancer cells in the presence or absence of various combinations of PARP- and selective mitogen-activated protein kinase (MAPK) inhibitors. Results: HO3089 resulted in significant cytotoxicity when combined with irradiation. In human U251 glioblastoma and A549 lung cancer cell lines, Erk1/2 and JNK/SAPK were found to mediate this effect of HO3089 since inhibitors of these kinases ameliorated it. In murine 4T1 breast cancer cell line, p38 MAPK rather than Erk1/2 or JNK/SAPK was identified as the main mediator of HO3089’s radiosensitizing effect. Besides the aforementioned changes in kinase signaling, we detected increased p53, unchanged Bax and decreased Bcl-2 expression in the A549 cell line. Conclusions: HO3089 sensitizes cancer cells to photon irradiation via proapoptotic processes where p53 plays a crucial role. Activation of MAPK pathways is regarded the consequence of irradiation-induced DNA damage, thus their inhibition can counteract the radiosenzitizing effect of the PARP inhibitor. Keywords: PARP, radiosensitizers, clonogenic cell survival, p53
Introduction Considering the fact that cytotoxic drugs commonly used in the last decades were unable to provide sufficient antitumor effect – even in combinations – in every cancer cell types, targeted therapy appeared heralding new aims and
Correspondence: Dr Arpad Boronkai, PhD, Department of Biochemistry and Medical Chemistry, Medical School, University of Pecs, 12 Szigeti Street, 7624, Pecs, Hungary. Tel: ⫹ 36 72536 080. Fax: ⫹ 36 72536-481. E-mail: [email protected] (Received 9 May 2013; revised 12 May 2014; accepted 9 June 2014)
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Cancer cell radiosensitization by PARP inhibitor 1153 transduction pathways involved have disadvantageous effect on the aforementioned process.
Materials and methods
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Cell and culture conditions Human A549 lung cancer, human U251 glioblastoma and murine 4T1 breast cancer cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). A549 cells were maintained in Minimum Essential Medium (MEM), while U251 and 4T1 cells in Roswell Park Memorial Institute (RPMI) 1640 medium (PAA Laboratories, Cölbe, Germany), both supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin mixture (Gibco/Invitrogen, Carlsbad, CA, USA). Cells were grown in an atmosphere containing 5% CO2 in a humidified 37°C incubator.
Drug and radiation treatment HO3089, a PARP inhibitor was synthesized by Professor Kalman Hideg (University of Pecs, Faculty of Medicine). Inhibitors (PD98059, SB203580, LY294002, JNK Inhibitor II) were purchased from Calbiochem (Darmstadt, Germany). Stock solutions were dissolved in dimethyl sulfoxide (DMSO) and diluted in culture medium to the specified final concentrations (HO3089 – 10 μM, PD98059 – 4 μM, SB203580 – 1.2 μM, LY294002 – 10 μM, JNK Inhibitor II – 1 μM) for cell treatment. A telecobalt external irradiation equipment (Theratron 780C, average photon energy of 1.25 MeV) was applied for irradiation of the cells with a dose of 2.0 or 4.0 Grays.
Clonogenic survival assay Cells were trypsinized and plated in triplicates into six-well plates at 500 cells/well. Cells were treated in combination with HO3089 and MAPK inhibitors 1 h prior to radiation exposure. The effect of individual chemicals and irradiation was assessed separately in parallel experiments. The efficacy of the MAPK inhibitors applied was tested by Western blot method, using phosphorylation specific anti-Erk1/2 (extracellular signal regulated kinase), anti-p38 MAPK and anti-SAPK/ JNK (stress activated protein kinase/Jun-kinase) primary antibody (Cell Signaling Technology, Danvers, MA, USA). All of the experiments were carried out along with the appropriate corresponding controls. After 7 days of incubation, the cells were washed and stained with crystal violet, and the colonies containing more than 50 cells were counted. The number of colonies were determined and expressed as the fraction of the number of colonies in untreated controls of each cell type.
Western blot analysis Cells were seeded and cultured overnight before chemicals were added to the medium in a concentration as indicated earlier. After the 24-h incubation period, the cells were harvested in cold lysis buffer (0.5 mM sodium metavanadate, 1 mM ethylenediaminetetraacetic acid [EDTA] and a protease inhibitor cocktail in phosphate-buffered saline, pH: 7.4). After cell disruption in a Teflon/glass homogenizer, the homogenate was pelleted, and protein content of the supernatant was measured by bicinchonicic acid reagent and
equalized for 1 mg/ml protein content in Laemmli sample buffer. Proteins (50 μg/lane) were separated in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% low-fat milk for 1 h at room temperature, then exposed to anti-Akt-1, phosphospecific anti-Akt-1 Ser473, anti-Bax, anti-Bcl-2, phospho-specific anti-Erk1/2 Thr202/Tyr204, phospho-specific anti-p38 MAPK Thr180/Tyr182 (Cell Signaling Technology, Danvers, MA, USA), anti-PARP-1, anti-PAR (Santa Cruz Biotechnology, Heidelberg, Germany), phospho-specific anti-SAPK/JNK Thr183/ Tyr185 (RandD Systems, Abingdon, UK), phospho-specific anti-p53 Ser15 (PromoKine, Heidelberg, Germany), anti-caspase-3, phospho-specific anti-Raf1 Ser338, anti-p21 (Thermo Scientific, Runcorn, UK) or anti-β actin (Sigma Aldrich Co, Budapest, Hungary) at 4°C overnight, in a dilution of 1:1000 in 5% bovine serum albumine, 1 ⫻ tris(hydroxymethyl) aminomethane buffered saline and 0.1% Tween20 solution. Appropriate horseradish peroxidase-conjugated anti-rabbit (1:3000, Bio-Rad, Budapest, Hungary), anti-mouse (1:5000, Sigma Aldrich Co, Budapest, Hungary) or anti-rat (1:5000, Enzo Life Sciences, Lörrach, Germany) secondary antibodies were applied for 1 h at room temperature. Peroxidase labeling was visualized by enhanced chemiluminescence (ECL) using ECL Western blot detection system (GE Healthcare, Freiburg, Germany). After scanning, pixel densities of bands were quantified by means of NIH ImageJ program. Pixel densities of bands of the same film were normalized to that of the loading control and expressed as a percentage of the respective untreated, unirradiated control samples. All experiments were performed at least three times.
Statistical analysis All data were expressed as mean ⫾ standard error of mean (SEM). Statistical analysis was performed by using twofactor analysis of variance with replication for correlations, and unpaired Student’s t-test for group comparisons. Differences with p values below 0.05 were considered as significant.
Results Effect of PARP inhibition and irradiation on colony formation in A549 cells PARP activity is indisposable in repairing irradiation-induced DNA damage. Therefore we tested the assumed synergism between PARP inhibition and irradiation by determining colony formation in the presence and absence of HO3089, a water soluble PARP inhibitor after irradiation. As we observed, the PARP inhibitor dramatically enhanced the photon irradiation effect since HO3089 pretreatment of A549 cells prior irradiation resulted in significant decrease in colony formation. Compared to untreated control group, irradiation by 2 Grays and 4 Grays without and with simultaneous HO3089 exposition shows a median of 60.98%, 20.73%, 25.61% and 4.88% colony counts, respectively. HO3089 pretreatment also resulted in significantly decreased colony formation ability in itself (68.29 % ⫾ SEM ***p ⬍ 0.001 compared to control) (Figure 1A).
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Figure 1. Effect of irradiation and HO3089 treatment on colony formation and PAR level in A549 cell line. (A) Clonogenic assay results are shown for A549 lung adenocarcinoma cells exposed or not to 2 or 4 Gy irradiation and/or PARP inhibitor (HO3089) as indicated underneath the diagram. Colony numbers in the percentages of untreated cells are presented as mean ⫾ SEM. *P ⬍ 0.05 compared to control values; #p ⬍ 0.05 compared to corresponding 2 or 4 Gy irradiated group; †p ⬍ 0.05 compared to HO3089 values. (B) PAR levels were detected by Western blotting from homogenates of cells treated as above. β-actin immunoreactivity was used to show even loading. Representative blots of three independent experiments are presented.
Efficacy of PARP inhibitor has been proved by Western blotting utilizing an anti-PAR primary antibody (Figure 1B).
Effect of PARP inhibition and irradiation on MAP kinases and pro- and antiapoptotic factors in A549 cell line As an attempt to identify mechanisms underlying HO3089’s radiosensitizing effect, we assessed early changes in protein expression and activation in A549 homogenates 24 h after irradiation (Figure 2). Significantly increased amount of the cleaved proapoptotic caspase-3 was observed in all cases except for the control. However, the amount of it did not show any notable differences when groups 3–6 were compared. Akt phosphorylation was elevated in irradiated cells, treated or not with HO3089. PARP inhibitor was unable to trigger detectable Akt activation by itself. Total Akt level, however, remained unchanged. Definite p53 activation was resulted by the PARP inhibitor exposition. Meanwhile, a dose-dependent enhanced phosphorylation of p53 was observed as the irradiation fraction doses were increased (246% and 623% increase, respectively). This effect was further augmented by the simultaneous administration of HO3089. Parallel with the results of caspase-3 activation, an elevated level of p21 phosphorylation appeared in irradiated cells irrespective of the fact whether they had been treated or not with HO3089. The proapoptotic signal transduction member, Bax, was found to be expressed in the same extent in all samples examined. On the other hand, antiapoptotic Bcl-2 was suggested to be an element playing an important
role in the irradiation mediated processes, i.e., its level decreased in all circumstances compared to control. HO3089 had no significant influence on the expression of Bcl-2. Detecting PARP enzyme levels, no considerable alterations could be seen among the samples examined. In A549 cell line Raf-1 was almost inactivated in control cells. At the same time, Raf-1 phosphorylation – activation – was detected in nearly the same extent in all other cases. Erk1/2 activation – similarly to its upstream regulator Raf-1 – was the consequence of HO3089 administration, such as irradiation alone. This phenomenon was self-evident in case higher fraction dose, 4 Gy irradiation, was applied (19% and 148% median increase, respectively). Combined treatment also resulted in undoubtedly elevated Erk1/2 activation (see the bar diagram). JNK/SAPK activation decreased in a dose-dependent manner in case irradiation had been applied (16% and 38% median decrease compared to control), which attenuated further in the presence of HO3089. Slightly increased activation of p38 MAPK was detected following irradiation (57% and 61% median elevation compared to control), which was enhanced further by the simultaneous application of HO3089.
Effect of PARP inhibition and irradiation on MAP kinases and pro- and antiapoptotic factors in 4T1 cell line To check whether the observed changes in MAPK activation and apoptosis regulating protein expression are universal among the cell lines investigated, we performed the above
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Cancer cell radiosensitization by PARP inhibitor 1155
Figure 2 (A) and (B). Western blot: Effect of HO3089 on steady-state level or phosphorylation state of the indicated signaling pathway members with or without 2 and 4 Gy irradiation in A549 cells. Different experimental combinations were tested, as shown in the figure. Protein expression was assessed following 24 hours of treatment by immunoblotting. β-actin immunoreactivity was used to show even loading. Representative blots of three independent experiments are presented. Pixel densities of the bands from three independent experiments are presented as mean ⫾ SEM for those members that were significantly affected by the treatments (*p ⬍ 0.05 compared to control values, #p ⬍ 0.05 compared to corresponding 2 or 4 Gy irradiated group, †p ⬍ 0.05 compared to HO3089 values). Data are shown in arbitrary units.
experiment on 4T1 cells. As we found (Figure 3), phosphop53 level was not affected by the PARP enzyme inhibitor HO3089 in itself; however, following irradiation the activation increased in a dose-dependent manner. Further significant elevation was detected in case of irradiated and PARP inhibited samples (91% and 121% elevation, respectively). There was no significant elevation in the Bcl-2 expression in case HO3089 exposition was applied separately. However, significant dose-dependent reduction was observed following irradiation (32% and 54% reduction as a fraction of the control value), which was synergistically influenced by the simultaneous PARP inhibition of the low dose irradiated cells. Single exposition by HO3089 did not have any significant effect on the Bax expression. Although significant increase was evident following irradiation, this effect was independent of both the dose applied and the simultaneous exposition to the PARP inhibitor (median elevation of 68%, 76%, 72%, and 75%, respectively). Erk1/2 presented significant increase only when HO3089 pretreated cells were irradiated (69% and 81% median change). JNK activation was a significant consequence of PARP inhibition or irradiation as well (respective medians of increase are of 30%, 36% and 63%). Coexposition resulted in synergistic augmentation of JNK phosphorylation. This effect was strongly significant in case higher fraction dose was applied (58% and 164% median elevation respectively, compared to untreated, unirradiated
control). Phospho-p38 MAPK level significantly increased both after single agent HO3089 treatment and irradiation, irrespectively of the dose applied (62% and 107% median increase). Simultaneous administration of HO3089 to photon irradiation resulted in the synergistic enhancement in p38 MAPK activation (109% and 206% median elevation as the fraction of untreated, unirradiated control).
Effect of MAPK inhibitors on PARP inhibitor-induced radiosensitizing in A549, 4T1 and U251 cells Inhibitory effect was confirmed for the kinase pathways examined (Figure 4), except for Akt, since the latter had been found to block the proliferation of the cells in colony formation assays entirely (data not shown). The Erk1/2 inhibitor PD98059 has been proven to extremely reduce Erk1/2 activation, although the level of the active molecule had been lower even initially in 4T1 cell lysates compared to that of A549 and U251 cells. JNK-inhibitor II significantly decreased JNK/SAPK activation in human-derived cell line lysates, while a total inhibition was achieved in 4T1 cell line. Initial level of this signal transduction element was higher in A549 and U251 cells than in the murine 4T1 cell line. SB203580, a well-known inhibitor of p38 MAPK phosphorylation, almost completely knocked out the activation of this signal cascade member in all cell cultures used. However, initial content of the active protein was higher in 4T1 cell line than those of
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Figure 3. Western blot: Effect of HO3089 on steady-state level or phosphorylation state of the indicated signaling pathway members with or without 2 and 4 Gy irradiation in 4T1 cells. Experimental conditions, methods and evaluation process are the same as described in the legend of Figure 2.
the other MAPK members examined. By using the selective inhibitors, we investigated their interactions with HO3089 in all three cell lines (Figure 5). Administration of MAPK inhibitors all resulted in altered ability of colony formation. This effect was found to be the most significant influence in case of Erk1/2 inhibition of A549 and U251 cells. 29% and 85.7% median elevation in colony numbers was detected as compared to control groups. P38 MAPK inhibitor was proven to be the most effective agent in the stimulation of the 4T1 cell line colony formation. Significant amelioration in colony formation ability was evoked by JNK inhibitor II in U251 glioblastoma cell line. However, Akt inhibition produced a total inability of colony formation (not shown in the Figure). HO3089 pretreatment has been shown to have inverse effect on A549 and U251 as compared to 4T1 cell proliferation.
Figure 4. Effect of MAPK inhibitors on the cell lines investigated. Phosphorylation state of the indicated MAPK was determined by Western blot analysis in A549, U251 and 4T1 cells in the presence and absence of appropriate inhibitors. β-actin was used as a loading control. Representative blots of three independent experiments are presented.
While Erk1/2 inhibition resulted in significant improvement in cell viability in the former group (12% and 57% increase in the percentage of the controls, respectively), 4T1 cells reacted the other way around. Meanwhile, p38 MAPK inhibition achieved considerable improvement in 4T1 cells’ viability along with HO3089 exposition, while it was not seen in the other cell lines. JNK inhibitor significantly counteracted the PARP inhibitor-related cell survival deterioration or benefit in A549 and 4T1 cells, while it hardly influenced the U251 lines (Figure 5). After determining the interaction between the PARP- and the MAPK inhibitors, we utilized them to verify the findings presented in Figures 2 and 3 about the role of the respective kinase signaling pathways in mediating the radiosensitizing effect of the PARP inhibitor in the cell lines. In order to demonstrate the results more clearly – particularly in case of extreme deviations – the corresponding data are presented here as the log(10) values of the percentages representing the respective untreated, unirradiated cells (Figure 6). Application of irradiation resulted in definitely decreased colony formation ability in all cell lines. However, this effect was dramatically antagonized by Erk1/2 inhibitor in the human-derived samples. A significant improvement was seen in the colony formation in A549 and U251 cells compared to the appropriate irradiated groups with no Erk1/2 inhibitor. Similarly, significantly improved proliferation was observed in the groups above as compared to the respective irradiated samples in case JNK inhibitor II coexposition was applied. 4T1 cells, at the same time, did not react on this agent to its advantage. However, p38 MAPK inhibitor increased the number of colonies the most significantly in 4T1 irradiated cells, while leaving A549 cell types almost unchanged.
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Cancer cell radiosensitization by PARP inhibitor 1157
Figure 5. Effect of MAPK inhibitors and HO3089 on colony formation in A549, U251 and 4T1 cells. Cells were seeded into six-well plates (500 cells/ well), cultured for 7 days and treated with Erk1/2-, p38 MAPK- or JNK-inhibitor without or with HO3089. The plates were stained with crystal violet and the colonies were quantified. The treatment protocol is indicated underneath the diagram. Data from three independent experiments are expressed as mean ⫾ SEM. *p ⬍ 0.05 compared to the untreated control group, #p ⬍ 0.05 compared to the HO3089-treated group and †p ⬍ 0.05 compared to the corresponding MAPK inhibitor group.
HO3089 administration to the culture medium before irradiation significantly decreased proliferation in all cell lines examined. Erk1/2 inhibitor in this setting enhanced colony formation ability of A549 and U251 cells exceedingly. Significant improvement in cell viability also occured in
case of JNK/SAPK inhibition without exception. This latter effect was evident even in case of low fraction dose in A549 cells; however, it appeared in U251 and 4T1 cell lines only when high fraction dose was applied. Concerning the highest level of significance, proliferation of 4T1cells was
Figure 6. Effect of MAPK inhibitors and 2 or 4 Gy irradiation on colony formation in A549, U251 and 4T1 cells. Cells were seeded into six-well plates (500 cells/well), exposed or not to 2 or 4 Gy irradiation in the presence or absence of Erk1/2-, p38 MAPK- or JNK-inhibitor, then cultured for 7 days in the presence or absence of the inhibitors. The plates were stained with crystal violet and the colonies were quantified. The treatment protocol is indicated underneath the diagram. Data from three independent experiments are expressed as % of the untreated cells, mean ⫾ SEM. Note that the y axis is logarithmic. *P ⬍ 0.05 compared to the untreated control group and #p ⬍ 0.05 compared to the corresponding 2 or 4 Gy-irradiated group.
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strikingly improved again by p38 MAPK inhibitor exposition. Significant increase in colony numbers was found even in comparison to the 2 Gy-irradiated HO3089 pretreated 4T1 cell sample. Results seen with high fraction dose in p38 MAPK-inhibited 4T1 cells are even more dramatic. This value was found to be higher in the log(10) scale than that of the respective p38 MAPK-uninhibited samples (Figure 7).
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Discussion Several papers reported on the significance of PI3K-PTENAkt-mTOR, Ras-Raf-MEK-Erk1/2 or even JNK/SAPK and p38 MAPK pathway activation in the development and promotion of cancer cells, as well as the consequent sensitivity to proapoptotic agents, and cytotoxic effects influencing signal transduction (Kim et al. 2003, Gao et al. 2006, Lee et al. 2008a, 2008b, Affolter et al. 2010, Ma et al. 2011, Marampon et al. 2011). We are also aware of signal transduction element coding gene mutations having impact on anticancer therapy (McCubrey et al. 2011). Besides, the Ras-Raf-MEKErk1/2 cascade, which is predominantly known as a promoter of cell viability, differentiation, survival and tumour progression, can play an undisputable role in cell death and inhibition of proliferation in U251 cell line (Arai et al. 2004, Hagan et al. 2007, Lu et al. 2010, Tomiyama et al. 2010). This fact is of outstanding significance in cases like k-Ras
mutated malignancies – which we demonstrated with A549 cell line – since they are still considered as one of the major challenges in drug development. Still, JAK2 inhibition and Ras-1 kinase suppressor inhibition resulted in enhanced radiosensitivity of such cells (Xiao et al. 2010, Sun et al. 2011). Our results confirmed the significance of Erk1/2 and SAPK/JNK activation in U251 and A549 cell lines, while the p38 MAPK activation was associated with improved clonogenity of 4T1 cells. Several data obtained with regard to U251 cell line was found to be in line with those described by Tomiyama’s group (Tomiyama et al. 2010). However, we can report on the significance of Erk1/2 activation in photon irradiation-induced glioblastoma cell death as well. As far as the role of Erk1/2 concerned, its activation and actual impact on cell survival can vary depending on the irradiation dose applied (Khalil et al. 2011). Enhanced sensitivity of irradiated cancer cell lines to PARP inhibition has already been demonstrated (Daniel et al. 2010, Senra et al. 2011, Wang et al. 2011). This process is mediated by EGFR-Erk signaling (Hagan et al. 2007). An efficient PARP enzyme inhibitor, HO3089, applied frequently in our previous experiments (Alexy et al. 2004, Kalai et al. 2009) was used in present study. It had been shown to increase the activation of PI3K-Akt pathway and suppress JNK and p38 MAPK (Alexy et al. 2004, Kovacs et al. 2006, 2009,
Figure 7. Effect of HO3089, MAPK inhibitors and 2 or 4 Gy irradiation on colony formation in A549, U251 and 4T1 cells. Cells were seeded into six-well plates (500 cells/well), exposed or not to 2 or 4 Gy irradiation in the presence or absence of Erk1/2-, p38 MAPK- or JNK inhibitor and/or HO3089, then cultured for 7 days in the presence or absence of the aforementioned compounds. The plates were stained with crystal violet and the colonies were quantified. The treatment protocol is indicated underneath the diagram. Data from three independent experiments are expressed as % of the untreated cells, mean ⫾ SEM. Note that the y axis is logarithmic. *P ⬍ 0.05 compared to the untreated control group, #p ⬍ 0.05 compared to the HO3089-treated group and †p ⬍ 0.05 compared to the corresponding MAPK inhibitor group.
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Cancer cell radiosensitization by PARP inhibitor 1159 Mester et al. 2009). We have demonstrated in A549 cells that HO3089 activated Erk1/2 pathways, while it had only slight effects on Akt, JNK/SAPK or p38 MAPK activation if administered alone. Irradiation resulted in Akt phosphorylation – possibly through the ROS formation generated PTEN activation – which remained unchanged beside PARP inhibition. Extreme activation of the Raf-1 – Erk1/2 pathway seems to be responsible for the significant anticancer effect in A549 cell line (Figure 2). Similar cytotoxicity is attributable to the significant activation of p38 MAPK in the 4T1 cells (Figure 3). That is, we have to take into account those – currently not explored – molecular pathways which are activated following ionizing irradiation. Damage of the DNA strand can evoke several complex signaling mechanisms (like ATM kinase mediated pathways) which may also contribute to the activation of the Erk1/2 route in cell death processes. A common result with Senra et al. (2011) and Wang et al. (2011) is that extreme decrease in cell proliferation was observed in our study, regardless of cancer cell type, in case irradiation was anticipated by exposition to HO3089. As we have found, human A549 and U251 cells were negatively influenced by radiation and PARP inhibitor-induced Erk1/2 activation. Although the exact pathway has to be clarified, a possible role of the endoplasmic reticulum is suggested (Arai et al. 2004). On the contrary, the colony formation ability of 4T1 cells was significantly improved by the p38 MAPK suppressor SB203580. Furthermore, A549 and U251 cells sensitively reacted on JNK/SAPK inhibitor as well, having improved colony formation ability. Similarly to previous results in the literature (Gangopadhyay et al. 2011), caspase-3 has slight influence on PARP inhibitor-mediated cancer cell toxicity in our models as well. The reason why HO3089 resulted in reduced PARP-1 expression in A549 cells was not clarified within the confines of the current work. Similar experiments concerning cancer cells have already been published (Tang et al. 2013). Taking our results into consideration, it can be concluded, that the difference within the signal transduction pathway balance may serve as a target in approaching the sensitivity of various cancer cell types. Concerning the three cell lines examined, no exact conformity was explored in their responsiveness to the agents applied. On the one hand, it is a novel encouraging result regarding the sensitization of malignancies to irradiation while normal tissue resistance can be enhanced. On the other hand, these results emphasize the significance of cell type specific individual approach in anticancer drug development and treatment planning. The uncertain MAPK-related signaling mechanisms arising following irradiation may seriously contribute to the treatment outcome, thus their inhibition may result inversely; i.e., in the deterioration of anticancer effect, as it has already been published (Daniel et al. 2010, Dedes et al. 2010, Issaeva et al. 2010, Konstantinopoulos et al. 2010, Toshimitsu et al. 2010, Drew et al. 2011). We have also observed that photon irradiation with or without HO3089 exposition significantly suppressed Bcl-2 expression (Figures 2 and 3), which can be explained by the
observations that NF-kappaB activates the expression of Bcl-2 (Catz and Johnson 2003, Zhao et al. 2011). Inhibition of PARP suppresses the NF-kappaB activation (Oliver et al. 1999, Veres et al. 2004, Zerfaoui et al. 2010, Tang et al. 2013). DNA damaging can activate the endogenous mitochondrial apoptotic pathway of p53 (Brown and Attardi 2005). Thus, most of the anticancer agents, including irradiation, that act through DNA damage or stress-inducing mechanisms require wild type p53 for apoptosis (Erster and Moll 2004, Brown and Attardi 2005, Galluzzi et al. 2008). Actions of p53 permeabilize the outer mitochondrial membrane, and promote the release of proapoptotic factors. In addition, PARP inhibitor can promote the nuclear export of p53 (Abd Elmageed et al. 2012), therefore inhibition of PARP by HO3089 can facilitate the cytoplasmic transport of p53, resulting in a more effective activation of cell death. Focusing on the proapoptotic – antiapoptotic balance in our experimental model with the A549 cells, increased p53 activity – as a common result of irradiation and PARP inhibition, and constant Bax expression was found, while the Bcl-2 expression decreased considerably. These changes are undisputably attributable to the dramatic decrease in cell proliferation of 4T1 cells as well. Summarizing our results and referring to all the data from the articles cited, it can be concluded that the differences in the role and balance of common signal transduction pathways are able to influence the outcome of a cytotoxic effect. A complex antitumour intervention found to be useful in certain vertebrates, not necessarily associated with the same efficiency in human cases. Thus, it is worth to taking this aspect into account in drug development or introduction of a new compound into irradiation-based combined modality treatment to avoid undesired consequences.
Acknowledgements The authors would like to express their deepest appreciation to Professor Ferenc Gallyas (Department of Biochemistry and Medical Chemistry, Medical School, University of Pecs) and Gyorgy Cserna for their professional and linguistic comments and advice which contributed to the improvement of this manuscript.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by the Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences, Research Grants from AOK-KA-10-04-2011, AOK-KA-34039-1004/2010, AOK-KA-34039/KA-OTKA/11-04, AOK-KA-34039/10-24, 34039/ KA-OTKA/2011/11-17, OTKA-K-73738, SROP-4.2.2/B-10/ 1-2010-0029 (Supporting Scientific Training of Talented Youth at the University of Pécs), SROP-4.2.1.B-10/2/KONV2010-0002, Developing the South-Transdanubian Regional University Competitiveness and the Nuclear-Mitochondrial Interactions Research Group, Hungarian Academy of Sciences, PO Box 1000, H-1245 Budapest, Hungary.
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