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Ho-shik Kim - One of the best experts on this subject based on the ideXlab platform.
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Nutlin 3 induces bcl2a1 expression by activating elk1 through the mitochondrial p53 ros erk1 2 pathway
International Journal of Oncology, 2014Co-Authors: Sun-young Lee, Yun-jeong Choe, Seok Joon Shin, Hyun Chul Choi, Sug Hyung Lee, Ho-shik KimAbstract:Nutlin-3 which occupies the p53 binding pocket in HDM2, has been reported to activate apoptosis through both the transcriptional activity-dependent and -independent programs of p53. Transcription-independent apoptosis by Nutlin-3 is triggered by p53 which is translocated to mitochondria. However, we previously demonstrated that the Nutlin-3-induced mitochondrial translocation of p53 stimulates ERK1/2 activation, an anti-apoptosis signal, via mitochondrial ROS generation. We report on how Nutlin-3-stimulated ERK1/2 activity inhibits p53-induced apoptosis. Among the anti-apoptotic BCL2 family proteins, BCL2A1 expression was increased by Nutlin-3 at both the mRNA and protein levels, and this increase was prevented by the inhibition of ERK1/2. TEMPO, a ROS scavenger, and PFT-μ , a blocker of the mitochondrial translocation of p53, also inhibited BCL2A1 expression as well as ERK1/2 phosphorylation. In addition, Nutlin-3 stimulated phosphorylation of ELK1, which was prevented by all compounds that inhibited Nutlin-3-induced ERK1/2 such as U0126, PFT-μ and TEMPO. Moreover, an increase in BCL2A1 expression was weakened by the knockdown of ELK1. Finally, Nutlin-3-induced apoptosis was found to be potentiated by the knockdown of BCL2A1, as demonstrated by an increase of in hypo-diploidic cells and Annexin V-positive cells. Parallel to the increase in apoptotic cells, the knockdown of BCL2A1 augmented the cleavage of poly(ADP-ribose) polymerase-1. It is noteworthy that the augmented levels of apoptosis induced by the knockdown of BCL2A1 were comparable to those of apoptosis induced by U0126. Collectively, these results suggest that Nutlin-3-activated ERK1/2 may stimulate the transcription of BCL2A1 via the activation of ELK1, and BCL2A1 expression may contribute to the inhibitory effect of ERK1/2 on Nutlin-3-induced apoptosis, thereby constituting a negative feedback loop of p53-induced apoptosis.
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Nutlin-3 induces BCL2A1 expression by activating ELK1 through the mitochondrial p53-ROS-ERK1/2 pathway
International Journal of Oncology, 2014Co-Authors: Sun-young Lee, Yun-jeong Choe, Seok Joon Shin, Hyun Chul Choi, Sug Hyung Lee, Ho-shik KimAbstract:Nutlin-3 which occupies the p53 binding pocket in HDM2, has been reported to activate apoptosis through both the transcriptional activity-dependent and -independent programs of p53. Transcription-independent apoptosis by Nutlin-3 is triggered by p53 which is translocated to mitochondria. However, we previously demonstrated that the Nutlin-3-induced mitochondrial translocation of p53 stimulates ERK1/2 activation, an anti-apoptosis signal, via mitochondrial ROS generation. We report on how Nutlin-3-stimulated ERK1/2 activity inhibits p53-induced apoptosis. Among the anti-apoptotic BCL2 family proteins, BCL2A1 expression was increased by Nutlin-3 at both the mRNA and protein levels, and this increase was prevented by the inhibition of ERK1/2. TEMPO, a ROS scavenger, and PFT-μ , a blocker of the mitochondrial translocation of p53, also inhibited BCL2A1 expression as well as ERK1/2 phosphorylation. In addition, Nutlin-3 stimulated phosphorylation of ELK1, which was prevented by all compounds that inhibited Nutlin-3-induced ERK1/2 such as U0126, PFT-μ and TEMPO. Moreover, an increase in BCL2A1 expression was weakened by the knockdown of ELK1. Finally, Nutlin-3-induced apoptosis was found to be potentiated by the knockdown of BCL2A1, as demonstrated by an increase of in hypo-diploidic cells and Annexin V-positive cells. Parallel to the increase in apoptotic cells, the knockdown of BCL2A1 augmented the cleavage of poly(ADP-ribose) polymerase-1. It is noteworthy that the augmented levels of apoptosis induced by the knockdown of BCL2A1 were comparable to those of apoptosis induced by U0126. Collectively, these results suggest that Nutlin-3-activated ERK1/2 may stimulate the transcription of BCL2A1 via the activation of ELK1, and BCL2A1 expression may contribute to the inhibitory effect of ERK1/2 on Nutlin-3-induced apoptosis, thereby constituting a negative feedback loop of p53-induced apoptosis.
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Nutlin-3 induces HO-1 expression by activating JNK in a transcription-independent manner of p53
International Journal of Oncology, 2013Co-Authors: Yun-jeong Choe, Sun-young Lee, Seok Joon Shin, Ho-shik KimAbstract:A recent study reported that p53 can induce HO-1 by directly binding to the putative p53 responsive element in the HO-1 promoter. In this study, we report that Nutlin-3, a small molecule antagonist of HDM2, induces the transcription of HO-1 in a transcription-independent manner of p53. Nutlin-3 induced HO-1 expression at the level of transcription in human cancer cells such as U2OS and RKO cells. This induction of HO-1 did not occur in SAOS cells in which p53 was mutated and was prevented by knocking down the p53 protein using p53 siRNA transfection, but not by PFT-α, an inhibitor of the transcriptional activity of p53. Accompanying HO-1 expres- sion, Nutlin-3 stimulated the accumulation of ROS and the phosphorylation of MAPKs such as JNK, p38 MAPK and ERK1/2. Nutlin-3-induced HO-1 expression was suppressed by TEMPO, a ROS scavenger, and chemical inhibitors of JNK and p38 MAPK but not ERK1/2. In addition, Nutlin-3-induced phosphorylation of JNK but not p38 MAPK was inhibited by TEMPO. Notably, the levels of Nutlin-3-induced ROS were correlated with the mitochondrial translocation of p53 and this induction was prevented by PFT-μ, an inhibitor of the mito- chondrial translocation of p53. Consistent with the effect of the ROS scavenger and MAPK inhibitors, PFT-μ reduced HO-1 expression and the phosphorylation of JNK induced by Nutlin-3. In the experiments of analyzing cell death, the knockdown of HO-1 augmented Nutlin-3-induced apoptosis. Collectively, these results suggest that Nutlin-3 induces HO-1 expression via the activation of both JNK which is dependent on ROS generated by p53 translocated to the mitochondria and p38 MAPK which appears to be stimulated by a ROS-independent mechanism, and this HO-1 induction may inhibit Nutlin-3-induced apoptosis, constituting a negative feedback loop of p53-induced apoptosis.
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Wilms' tumor gene 1 enhances Nutlin-3-induced apoptosis
Oncology Reports, 2013Co-Authors: Sun-young Lee, Yun-jeong Choe, Jik Young Park, Sang-sun Lee, Yoon-hyoung Kim, Seok Joon Shin, Yeun Jun Chung, Ho-shik KimAbstract:Nutlin-3, a human double minute 2 (HDM2) antagonist, induces cell cycle arrest or apoptosis by upregulating p53 in cancer cells. WT1, the product of Wilms' tumor gene 1, has been shown to interact with p53, but the effect of WT1 on Nutlin-3-induced apoptosis has yet to be examined. To address this issue, we analyzed the inhibitory effect of Nutlin-3 on cell growth as a function of Wt1 expression status using a Wt1-inducible U2OS cell line. In the absence of Wt1 expression, Nutlin-3 induced cell cycle arrest with marginal cytotoxicity. Furthermore, upon Wt1 expression, Nutlin-3 exerted a marked degree of cell death, as evidenced by the accumulation of hypo-diploid cells and LDH release. During cell death induction, cytochrome c was released into the cytosol, and caspase-9 and -3 were activated, suggesting that an intrinsic apoptotic pathway may be involved in this cell death. Consistent with this, z-VAD-Fmk, a pan-caspase inhibitor and the overexpression of BCL-XL attenuated the cell death. Nutlin-3 caused an increase in the mRNA levels of both BCL-XL and BAK, as well as their corresponding protein levels in mitochondria. In the presence of Wt1, Nutlin-3-induced BCL-XL expression was attenuated while the expression of Nutlin-3-induced BAK was potentiated. Collectively, these results suggest that WT1 potentiates Nutlin-3-induced apoptosis by downregulating the expression of BCL-XL while upregulating that of BAK, which leads to the activation of an intrinsic apoptotic pathway.
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erk1 2 activation mediated by the Nutlin 3 induced mitochondrial translocation of p53
International Journal of Oncology, 2013Co-Authors: Sun-young Lee, Seok Joon Shin, Ho-shik KimAbstract:Nutlin-3 is a small-molecule antagonist of murine double minute 2 (MDM2) that blocks its binding to p53, leading to an increase in p53 protein levels. The tumor suppressor p53 induces growth arrest or apoptosis in response to genotoxic stress. Along with its growth-suppressive effect, it has been reported that p53 stimulates the mitogen-activated protein kinase (MAPK) pathway via the upregulation of heparin- binding epidermal growth factor-like growth factor (HB-EGF), an epidermal growth factor receptor (EGFR) ligand, and discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor, at the transcription level. In this study, we propose a novel mechanism involved in the p53-induced MAPK activation. Nutlin-3 induced the phosphorylation of EGFR, MAPK/ERK kinase (MEK)1/2 and extracellular signal-regulated kinase (ERK)1/2 in U2OS human osteosarcoma cells harboring wild-type p53. This phosphorylation was completely inhibited by p53 siRNA, but not by pifithrin (PFT)-α, an inhibitor of the trans-criptional activity of p53. While the Nutlin-3-induced EGFR phosphorylation was prevented by the inactivation of ERK1/2, the Nutlin-3-induced MEK1/2-ERK1/2 phosphorylation was still observed in the cells in which EGFR phosphorylation was inhibited using EGFR siRNA and AG1478, an inhibitor of EGFR tyrosine kinase. Of note, Nutlin-3 caused the accumulation of mitochondrial reactive oxygen species (ROS) and this correlated with the mitochondrial translocation of p53. 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO), a ROS scavenger, prevented the phosphorylation of ERK1/2. PFT-μ, which prevented the mitochondrial localization of p53, suppressed ERK1/2 phosphorylation, as well as ROS accumulation. Finally, we analyzed the effect of ERK1/2 activation on Nutlin-3-induced apoptosis. The knockdown of MEK1/2 and ERK1/2 activity using U0126, a MEK inhibitor, or siRNAs, resulted in the enhancement of Nutlin-3-induced apoptosis. In addition, TEMPO and PFT-μ also promoted Nutlin-3-induced apoptosis. Taking the above findings into account, it can be concluded that Nutlin-3 activates ERK1/2 prior to EGFR phosphorylation via ROS generation following the mitochondrial translocation of p53 and that Nutlin-3-induced ERK1/2 activation may play a role in protecting U2OS cells from p53-dependent apoptosis, constituting a negative feedback loop for p53-induced apoptosis.
Giorgio Zauli - One of the best experts on this subject based on the ideXlab platform.
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Nanoparticles loaded with Nutlin-3 display cytotoxicity towards p53(wild-type) JVM-2 but not towards p53(mutated) BJAB leukemic cells.
Current Medicinal Chemistry, 2013Co-Authors: Rebecca Voltan, Paola Secchiero, Giorgio Zauli, Barbara Ruozi, L. Caruso, Flavio Forni, M. Palomba, Maria Angela VandelliAbstract:The small molecule Nutlin-3 is a potent antagonist of the murine double minute 2 (MDM2)/p53 interaction exhibiting promising therapeutic anti-cancer activity. Nutlin-3 has been proposed as an anti-neoplastic agent for the treatment of onco-hematological diseases characterized by a lower incidence of p53 mutation with respect to solid tumors. Indeed, based on its selective non-genotoxic p53 activation, Nutlin-3 might represent an alternative to the current cytotoxic chemotherapy. To overcome the poor bioavailability of Nutlin-3, we have assessed the potential efficacy of Nutlin-3 loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NP) against hematological malignancies. To test the specificity of the anti-leukemic activity, we have used leukemic cell lines characterized by different p53 status (JVM-2 and BJAB). NP loaded with Nutlin-3 (NP-Nutlin) were rapidly taken up by the leukemic cells and were as effective as native Nutlin-3 in promoting both induction of apoptosis and cell cycle arrest in p53 wild-type JVM-2 cells, but not in p53 mutated BJAB cells. Moreover, injection of NP-Nutlin, but not of free Nutlin-3, in a JVM-2-derived xenograft mouse model, reduced the subcutaneous tumor volume and promoted induction of apoptosis in the tumor mass. Overall, the chemical and structural characteristics of the NP-Nutlin-3, as well as their biological activity in vitro and in vivo, made them promising for further preclinical evaluations as potentially useful anti-leukemic carriers.
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The MDM2 inhibitor Nutlin-3 attenuates streptozotocin-induced diabetes mellitus and increases serum level of IL-12p40
Acta Diabetologica, 2013Co-Authors: Paola Secchiero, Elisabetta Melloni, Barbara Toffoli, Chiara Agnoletto, Lorenzo Monasta, Giorgio ZauliAbstract:Besides its well-established oncosuppressor activity, a key function of p53 in regulating metabolic pathways has been recently identified. Nevertheless, the role of p53 with respect to diabetes mellitus (DM) appears highly controversial. To address this issue, we have used the cis-imidazoline compound Nutlin-3, an inhibitor of MDM2/p53 interaction, which represents a potent and selective non-genotoxic activator of the p53 pathway both in in vivo and in vitro experimental settings. Experimental DM was induced by intraperitoneal injections of low concentrations of streptozotocin (STZ) in C57BL/6N mice (n = 20). A group of control vehicle-injected mice (n = 10) and of STZ-treated mice (n = 10) was co-injected with Nutlin-3. Mice co-injected with STZ + Nutlin-3 exhibited attenuated features of DM with respect to animals treated with STZ alone. Indeed, STZ + Nutlin-3-treated mice were characterized by significantly (p < 0.05) lower levels of hyperglycemia, reduced weight loss, and increased spleen weight. In addition, STZ alone promoted a marked decrease in the levels of several circulating cytokines, including interleukin-12 (IL-12)p40. On the other hand, co-injection of STZ + Nutlin-3 significantly (p < 0.01) counteracted IL-12p40 down-modulation. In vitro experiments performed on the RAW264.7 macrophagic cell line model, used as cellular source of IL-12p40, demonstrated that Nutlin-3 treatment increased IL-12p40 release, strongly suggesting a direct effect of Nutlin-3 on the immune system. Overall, these data demonstrate that systemic administration of Nutlin-3 ameliorates the severity of STZ-induced DM and increases the levels of circulating IL-12p40.
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The non-genotoxic activator of the p53 pathway Nutlin-3 shifts the balance between E2F7 and E2F1 transcription factors in leukemic cells.
Investigational New Drugs, 2012Co-Authors: M. G. Di Iasio, Giorgio ZauliAbstract:The effect of Nutlin-3, a small molecule inhibitor of the MDM2/p53 interaction, was investigated on the steady-state mRNA levels of the transcription factors E2F1 and E2F7 in a cohort of primary B-chronic lymphocytic leukemia (B-CLL) patient samples (n = 15) and normal peripheral blood mononuclear cells (PBMC). A 24-h treatment with Nutlin-3 significantly down-regulated E2F1 and promoted the concomitant up-regulation of E2F7 in both leukemic and normal cells. Our data suggest that the ability of Nutlin-3 to up-regulate E2F7 likely represents an important molecular determinant in the anti-proliferative activity of Nutlin-3.
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The Oncogene DEK Promotes Leukemic Cell Survival and Is Downregulated by both Nutlin-3 and Chlorambucil in B-Chronic Lymphocytic Leukemic Cells
Clinical Cancer Research, 2010Co-Authors: Paola Secchiero, M. G. Di Iasio, Elisabetta Melloni, Mario Tiribelli, Rebecca Voltan, Giorgio ZauliAbstract:Purpose: To characterize the role of the oncogene DEK in modulating the response to either Nutlin-3, a small-molecule inhibitor of the MDM2/p53 interaction, or chlorambucil in primary B-chronic lymphocytic leukemia (B-CLL) cells. Experimental Design: DEK mRNA and protein levels were evaluated in primary B-CLL samples ( n = 21), p53 wild-type SKW6.4, p53 mutated BJAB lymphoblastoid cell lines, and normal CD19 + B lymphocytes–treated Nutlin-3 or chlorambucil (10 μmol/L, each). Knocking down experiments with either p53 or DEK small interfering RNA (siRNA) were done to investigate the potential role of p53 in controlling the expression of DEK and the role of DEK in leukemic cell survival/apoptosis. Results: Both Nutlin-3 and chlorambucil downregulated DEK in primary B-CLL samples ( n = 21) and SKW6.4 but not in BJAB cells. Knocking down p53 attenuated the effect of Nutlin-3 on DEK expression, whereas knocking down DEK significantly increased both spontaneous and Nutlin-3–induced apoptosis. Conversely, counteracting DEK downmodulation by using p53 small interfering RNA reduced Nutlin-3–mediated apoptosis. On the other hand, Nutlin-3 potently induced p53 accumulation, but it did not affect DEK levels in normal CD19 + B lymphocytes. Conclusions: These data show that the downregulation of DEK in response to either Nutlin-3 or chlorambucil represents an important molecular determinant in the cytotoxic response of leukemic cells, and suggest that strategies aimed to downregulate DEK might improve the therapeutic potential of these drugs. Clin Cancer Res; 16(6); 1824–33
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Exposure of B cell chronic lymphocytic leukemia (B-CLL) cells to Nutlin-3 induces a characteristic gene expression profile, which correlates with Nutlin-3-mediated cytotoxicity.
Current Cancer Drug Targets, 2009Co-Authors: Giorgio Zauli, Paola Secchiero, M. G. Di Iasio, M. Dal Bo, D. Marconi, Riccardo Bomben, G. Del Poeta, Valter GatteiAbstract:By analyzing the cDNA obtained from 16 B-cell chronic lymphocytic leukemia (B-CLL) patient samples, we found that Nutlin-3, a small molecule inhibitor of MDM2/p53 interaction, induced a characteristic gene expression profile (GEP) signature in 13 out of 16 B-CLL samples. The lack of Nutlin-3-induced GEP signature in 3 out of 16 B-CLL samples was not due to p53 deletion and/or mutation, as demonstrated by FISH analysis and p53 sequencing. Of note, the 3 B-CLL samples in which Nutlin-3 did not elicit the GEP signature were also less susceptible to Nutlin-3-mediated cytotoxicity with respect to the remaining 13 B-CLL samples. However, the partial lack of response in these p53 wild-type B-CLL samples was not due to defects in the ability of Nutlin-3 to promote p53 induction, as confirmed by the rapid accumulation of p53 protein at Western blot analysis in response to Nutlin-3 in all samples examined. Upon exposure to Nutlin-3, the genes up-regulated with the highest score in the majority of B-CLL cells were all known p53-target genes, including genes involved in apoptotic pathways, such as FAS and BAX, as well as MDM2. Taken together, our data indicate that the ability of Nutlin-3 to induce a characteristic GEP signature correlates with its cytotoxic potential in p53 wild-type B-CLL cells. However, in some p53 wild-type B-CLL samples, the response to Nutlin-3 cannot be predicted on the basis of FISH analysis or p53 sequencing.
Sun-young Lee - One of the best experts on this subject based on the ideXlab platform.
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Nutlin 3 induces bcl2a1 expression by activating elk1 through the mitochondrial p53 ros erk1 2 pathway
International Journal of Oncology, 2014Co-Authors: Sun-young Lee, Yun-jeong Choe, Seok Joon Shin, Hyun Chul Choi, Sug Hyung Lee, Ho-shik KimAbstract:Nutlin-3 which occupies the p53 binding pocket in HDM2, has been reported to activate apoptosis through both the transcriptional activity-dependent and -independent programs of p53. Transcription-independent apoptosis by Nutlin-3 is triggered by p53 which is translocated to mitochondria. However, we previously demonstrated that the Nutlin-3-induced mitochondrial translocation of p53 stimulates ERK1/2 activation, an anti-apoptosis signal, via mitochondrial ROS generation. We report on how Nutlin-3-stimulated ERK1/2 activity inhibits p53-induced apoptosis. Among the anti-apoptotic BCL2 family proteins, BCL2A1 expression was increased by Nutlin-3 at both the mRNA and protein levels, and this increase was prevented by the inhibition of ERK1/2. TEMPO, a ROS scavenger, and PFT-μ , a blocker of the mitochondrial translocation of p53, also inhibited BCL2A1 expression as well as ERK1/2 phosphorylation. In addition, Nutlin-3 stimulated phosphorylation of ELK1, which was prevented by all compounds that inhibited Nutlin-3-induced ERK1/2 such as U0126, PFT-μ and TEMPO. Moreover, an increase in BCL2A1 expression was weakened by the knockdown of ELK1. Finally, Nutlin-3-induced apoptosis was found to be potentiated by the knockdown of BCL2A1, as demonstrated by an increase of in hypo-diploidic cells and Annexin V-positive cells. Parallel to the increase in apoptotic cells, the knockdown of BCL2A1 augmented the cleavage of poly(ADP-ribose) polymerase-1. It is noteworthy that the augmented levels of apoptosis induced by the knockdown of BCL2A1 were comparable to those of apoptosis induced by U0126. Collectively, these results suggest that Nutlin-3-activated ERK1/2 may stimulate the transcription of BCL2A1 via the activation of ELK1, and BCL2A1 expression may contribute to the inhibitory effect of ERK1/2 on Nutlin-3-induced apoptosis, thereby constituting a negative feedback loop of p53-induced apoptosis.
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Nutlin-3 induces BCL2A1 expression by activating ELK1 through the mitochondrial p53-ROS-ERK1/2 pathway
International Journal of Oncology, 2014Co-Authors: Sun-young Lee, Yun-jeong Choe, Seok Joon Shin, Hyun Chul Choi, Sug Hyung Lee, Ho-shik KimAbstract:Nutlin-3 which occupies the p53 binding pocket in HDM2, has been reported to activate apoptosis through both the transcriptional activity-dependent and -independent programs of p53. Transcription-independent apoptosis by Nutlin-3 is triggered by p53 which is translocated to mitochondria. However, we previously demonstrated that the Nutlin-3-induced mitochondrial translocation of p53 stimulates ERK1/2 activation, an anti-apoptosis signal, via mitochondrial ROS generation. We report on how Nutlin-3-stimulated ERK1/2 activity inhibits p53-induced apoptosis. Among the anti-apoptotic BCL2 family proteins, BCL2A1 expression was increased by Nutlin-3 at both the mRNA and protein levels, and this increase was prevented by the inhibition of ERK1/2. TEMPO, a ROS scavenger, and PFT-μ , a blocker of the mitochondrial translocation of p53, also inhibited BCL2A1 expression as well as ERK1/2 phosphorylation. In addition, Nutlin-3 stimulated phosphorylation of ELK1, which was prevented by all compounds that inhibited Nutlin-3-induced ERK1/2 such as U0126, PFT-μ and TEMPO. Moreover, an increase in BCL2A1 expression was weakened by the knockdown of ELK1. Finally, Nutlin-3-induced apoptosis was found to be potentiated by the knockdown of BCL2A1, as demonstrated by an increase of in hypo-diploidic cells and Annexin V-positive cells. Parallel to the increase in apoptotic cells, the knockdown of BCL2A1 augmented the cleavage of poly(ADP-ribose) polymerase-1. It is noteworthy that the augmented levels of apoptosis induced by the knockdown of BCL2A1 were comparable to those of apoptosis induced by U0126. Collectively, these results suggest that Nutlin-3-activated ERK1/2 may stimulate the transcription of BCL2A1 via the activation of ELK1, and BCL2A1 expression may contribute to the inhibitory effect of ERK1/2 on Nutlin-3-induced apoptosis, thereby constituting a negative feedback loop of p53-induced apoptosis.
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Nutlin-3 induces HO-1 expression by activating JNK in a transcription-independent manner of p53
International Journal of Oncology, 2013Co-Authors: Yun-jeong Choe, Sun-young Lee, Seok Joon Shin, Ho-shik KimAbstract:A recent study reported that p53 can induce HO-1 by directly binding to the putative p53 responsive element in the HO-1 promoter. In this study, we report that Nutlin-3, a small molecule antagonist of HDM2, induces the transcription of HO-1 in a transcription-independent manner of p53. Nutlin-3 induced HO-1 expression at the level of transcription in human cancer cells such as U2OS and RKO cells. This induction of HO-1 did not occur in SAOS cells in which p53 was mutated and was prevented by knocking down the p53 protein using p53 siRNA transfection, but not by PFT-α, an inhibitor of the transcriptional activity of p53. Accompanying HO-1 expres- sion, Nutlin-3 stimulated the accumulation of ROS and the phosphorylation of MAPKs such as JNK, p38 MAPK and ERK1/2. Nutlin-3-induced HO-1 expression was suppressed by TEMPO, a ROS scavenger, and chemical inhibitors of JNK and p38 MAPK but not ERK1/2. In addition, Nutlin-3-induced phosphorylation of JNK but not p38 MAPK was inhibited by TEMPO. Notably, the levels of Nutlin-3-induced ROS were correlated with the mitochondrial translocation of p53 and this induction was prevented by PFT-μ, an inhibitor of the mito- chondrial translocation of p53. Consistent with the effect of the ROS scavenger and MAPK inhibitors, PFT-μ reduced HO-1 expression and the phosphorylation of JNK induced by Nutlin-3. In the experiments of analyzing cell death, the knockdown of HO-1 augmented Nutlin-3-induced apoptosis. Collectively, these results suggest that Nutlin-3 induces HO-1 expression via the activation of both JNK which is dependent on ROS generated by p53 translocated to the mitochondria and p38 MAPK which appears to be stimulated by a ROS-independent mechanism, and this HO-1 induction may inhibit Nutlin-3-induced apoptosis, constituting a negative feedback loop of p53-induced apoptosis.
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Wilms' tumor gene 1 enhances Nutlin-3-induced apoptosis
Oncology Reports, 2013Co-Authors: Sun-young Lee, Yun-jeong Choe, Jik Young Park, Sang-sun Lee, Yoon-hyoung Kim, Seok Joon Shin, Yeun Jun Chung, Ho-shik KimAbstract:Nutlin-3, a human double minute 2 (HDM2) antagonist, induces cell cycle arrest or apoptosis by upregulating p53 in cancer cells. WT1, the product of Wilms' tumor gene 1, has been shown to interact with p53, but the effect of WT1 on Nutlin-3-induced apoptosis has yet to be examined. To address this issue, we analyzed the inhibitory effect of Nutlin-3 on cell growth as a function of Wt1 expression status using a Wt1-inducible U2OS cell line. In the absence of Wt1 expression, Nutlin-3 induced cell cycle arrest with marginal cytotoxicity. Furthermore, upon Wt1 expression, Nutlin-3 exerted a marked degree of cell death, as evidenced by the accumulation of hypo-diploid cells and LDH release. During cell death induction, cytochrome c was released into the cytosol, and caspase-9 and -3 were activated, suggesting that an intrinsic apoptotic pathway may be involved in this cell death. Consistent with this, z-VAD-Fmk, a pan-caspase inhibitor and the overexpression of BCL-XL attenuated the cell death. Nutlin-3 caused an increase in the mRNA levels of both BCL-XL and BAK, as well as their corresponding protein levels in mitochondria. In the presence of Wt1, Nutlin-3-induced BCL-XL expression was attenuated while the expression of Nutlin-3-induced BAK was potentiated. Collectively, these results suggest that WT1 potentiates Nutlin-3-induced apoptosis by downregulating the expression of BCL-XL while upregulating that of BAK, which leads to the activation of an intrinsic apoptotic pathway.
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erk1 2 activation mediated by the Nutlin 3 induced mitochondrial translocation of p53
International Journal of Oncology, 2013Co-Authors: Sun-young Lee, Seok Joon Shin, Ho-shik KimAbstract:Nutlin-3 is a small-molecule antagonist of murine double minute 2 (MDM2) that blocks its binding to p53, leading to an increase in p53 protein levels. The tumor suppressor p53 induces growth arrest or apoptosis in response to genotoxic stress. Along with its growth-suppressive effect, it has been reported that p53 stimulates the mitogen-activated protein kinase (MAPK) pathway via the upregulation of heparin- binding epidermal growth factor-like growth factor (HB-EGF), an epidermal growth factor receptor (EGFR) ligand, and discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor, at the transcription level. In this study, we propose a novel mechanism involved in the p53-induced MAPK activation. Nutlin-3 induced the phosphorylation of EGFR, MAPK/ERK kinase (MEK)1/2 and extracellular signal-regulated kinase (ERK)1/2 in U2OS human osteosarcoma cells harboring wild-type p53. This phosphorylation was completely inhibited by p53 siRNA, but not by pifithrin (PFT)-α, an inhibitor of the trans-criptional activity of p53. While the Nutlin-3-induced EGFR phosphorylation was prevented by the inactivation of ERK1/2, the Nutlin-3-induced MEK1/2-ERK1/2 phosphorylation was still observed in the cells in which EGFR phosphorylation was inhibited using EGFR siRNA and AG1478, an inhibitor of EGFR tyrosine kinase. Of note, Nutlin-3 caused the accumulation of mitochondrial reactive oxygen species (ROS) and this correlated with the mitochondrial translocation of p53. 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO), a ROS scavenger, prevented the phosphorylation of ERK1/2. PFT-μ, which prevented the mitochondrial localization of p53, suppressed ERK1/2 phosphorylation, as well as ROS accumulation. Finally, we analyzed the effect of ERK1/2 activation on Nutlin-3-induced apoptosis. The knockdown of MEK1/2 and ERK1/2 activity using U0126, a MEK inhibitor, or siRNAs, resulted in the enhancement of Nutlin-3-induced apoptosis. In addition, TEMPO and PFT-μ also promoted Nutlin-3-induced apoptosis. Taking the above findings into account, it can be concluded that Nutlin-3 activates ERK1/2 prior to EGFR phosphorylation via ROS generation following the mitochondrial translocation of p53 and that Nutlin-3-induced ERK1/2 activation may play a role in protecting U2OS cells from p53-dependent apoptosis, constituting a negative feedback loop for p53-induced apoptosis.
Sanjeeb K. Sahoo - One of the best experts on this subject based on the ideXlab platform.
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retraction notice to epithelial cell adhesion molecule targeted Nutlin 3a loaded immunonanoparticles for cancer therapy acta biomaterialia 7 2011 355 369
Acta Biomaterialia, 2011Co-Authors: Manasi Das, Sanjeeb K. SahooAbstract:Recently much attention has been given to the anti-cancer drug Nutlin-3a, an antagonist of murine double minute 2 (MDM2) that actively inhibits p53-MDM2 interaction. Reactivating p53 function by Nutlin-3a thus provides a promising therapeutic strategy for the treatment of cancer. Although Nutlin-3a seems a potential candidate in restoring p53 activity, it has many lacunae, toxicity, poor bioavailability, nonspecific delivery, and most importantly it is a substrate of multidrug resistance protein. The objective of the present study is to prepare and characterize Nutlin-3a loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs), surface functionalized with epithelial cell adhesion molecule (EpCAM) antibody, with an aim to deliver encapsulated drug in a targeted manner to its site of action and to enhance its therapeutic efficacy many times over. The enhanced cellular uptake of EpCAM antibody conjugated Nutlin-3a loaded NPs (EpCAM-Nutlin-3a-NPs) over native nulin-3a, Nutlin-3a loaded NPs (Nutlin-3a-NPs) in HCT116 and A549 cells substantiate the targeting potentiality of conjugated system. IC₅₀ values depicted superior antiproliferative activity of EpCAM-Nutlin-3a-NPs over Nutlin-3a-NPs and native Nutlin-3a in the above studied cell lines. Cell cycle arrest, loss of mitochondrial membrane potential and apoptosis induced by above formulation were confirmed by flow cytometry. Expression of p53, p21, EpCAM, and C-myc proteins involved in cell cycle regulation and apoptosis were investigated by western blotting. The above investigation indicates the enhanced therapeutic ability of EpCAM-Nutlin-3a-NPs compared to Nutlin-3a or Nutlin-3a-NPs. Thus, our results suggest that EpCAM-Nutlin-3a-NPs could be a potentially useful drug carrier system for targeted delivery of potent anti-cancer drug Nutlin-3a for cancer therapy.
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Retraction notice to “Epithelial cell adhesion molecule targeted Nutlin-3a loaded immunonanoparticles for cancer therapy” [Acta Biomaterialia 7 (2011) 355–369]
Acta biomaterialia, 2010Co-Authors: Manasi Das, Sanjeeb K. SahooAbstract:Recently much attention has been given to the anti-cancer drug Nutlin-3a, an antagonist of murine double minute 2 (MDM2) that actively inhibits p53-MDM2 interaction. Reactivating p53 function by Nutlin-3a thus provides a promising therapeutic strategy for the treatment of cancer. Although Nutlin-3a seems a potential candidate in restoring p53 activity, it has many lacunae, toxicity, poor bioavailability, nonspecific delivery, and most importantly it is a substrate of multidrug resistance protein. The objective of the present study is to prepare and characterize Nutlin-3a loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs), surface functionalized with epithelial cell adhesion molecule (EpCAM) antibody, with an aim to deliver encapsulated drug in a targeted manner to its site of action and to enhance its therapeutic efficacy many times over. The enhanced cellular uptake of EpCAM antibody conjugated Nutlin-3a loaded NPs (EpCAM-Nutlin-3a-NPs) over native nulin-3a, Nutlin-3a loaded NPs (Nutlin-3a-NPs) in HCT116 and A549 cells substantiate the targeting potentiality of conjugated system. IC₅₀ values depicted superior antiproliferative activity of EpCAM-Nutlin-3a-NPs over Nutlin-3a-NPs and native Nutlin-3a in the above studied cell lines. Cell cycle arrest, loss of mitochondrial membrane potential and apoptosis induced by above formulation were confirmed by flow cytometry. Expression of p53, p21, EpCAM, and C-myc proteins involved in cell cycle regulation and apoptosis were investigated by western blotting. The above investigation indicates the enhanced therapeutic ability of EpCAM-Nutlin-3a-NPs compared to Nutlin-3a or Nutlin-3a-NPs. Thus, our results suggest that EpCAM-Nutlin-3a-NPs could be a potentially useful drug carrier system for targeted delivery of potent anti-cancer drug Nutlin-3a for cancer therapy.
Paola Secchiero - One of the best experts on this subject based on the ideXlab platform.
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Nanoparticles loaded with Nutlin-3 display cytotoxicity towards p53(wild-type) JVM-2 but not towards p53(mutated) BJAB leukemic cells.
Current Medicinal Chemistry, 2013Co-Authors: Rebecca Voltan, Paola Secchiero, Giorgio Zauli, Barbara Ruozi, L. Caruso, Flavio Forni, M. Palomba, Maria Angela VandelliAbstract:The small molecule Nutlin-3 is a potent antagonist of the murine double minute 2 (MDM2)/p53 interaction exhibiting promising therapeutic anti-cancer activity. Nutlin-3 has been proposed as an anti-neoplastic agent for the treatment of onco-hematological diseases characterized by a lower incidence of p53 mutation with respect to solid tumors. Indeed, based on its selective non-genotoxic p53 activation, Nutlin-3 might represent an alternative to the current cytotoxic chemotherapy. To overcome the poor bioavailability of Nutlin-3, we have assessed the potential efficacy of Nutlin-3 loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NP) against hematological malignancies. To test the specificity of the anti-leukemic activity, we have used leukemic cell lines characterized by different p53 status (JVM-2 and BJAB). NP loaded with Nutlin-3 (NP-Nutlin) were rapidly taken up by the leukemic cells and were as effective as native Nutlin-3 in promoting both induction of apoptosis and cell cycle arrest in p53 wild-type JVM-2 cells, but not in p53 mutated BJAB cells. Moreover, injection of NP-Nutlin, but not of free Nutlin-3, in a JVM-2-derived xenograft mouse model, reduced the subcutaneous tumor volume and promoted induction of apoptosis in the tumor mass. Overall, the chemical and structural characteristics of the NP-Nutlin-3, as well as their biological activity in vitro and in vivo, made them promising for further preclinical evaluations as potentially useful anti-leukemic carriers.
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The MDM2 inhibitor Nutlin-3 attenuates streptozotocin-induced diabetes mellitus and increases serum level of IL-12p40
Acta Diabetologica, 2013Co-Authors: Paola Secchiero, Elisabetta Melloni, Barbara Toffoli, Chiara Agnoletto, Lorenzo Monasta, Giorgio ZauliAbstract:Besides its well-established oncosuppressor activity, a key function of p53 in regulating metabolic pathways has been recently identified. Nevertheless, the role of p53 with respect to diabetes mellitus (DM) appears highly controversial. To address this issue, we have used the cis-imidazoline compound Nutlin-3, an inhibitor of MDM2/p53 interaction, which represents a potent and selective non-genotoxic activator of the p53 pathway both in in vivo and in vitro experimental settings. Experimental DM was induced by intraperitoneal injections of low concentrations of streptozotocin (STZ) in C57BL/6N mice (n = 20). A group of control vehicle-injected mice (n = 10) and of STZ-treated mice (n = 10) was co-injected with Nutlin-3. Mice co-injected with STZ + Nutlin-3 exhibited attenuated features of DM with respect to animals treated with STZ alone. Indeed, STZ + Nutlin-3-treated mice were characterized by significantly (p < 0.05) lower levels of hyperglycemia, reduced weight loss, and increased spleen weight. In addition, STZ alone promoted a marked decrease in the levels of several circulating cytokines, including interleukin-12 (IL-12)p40. On the other hand, co-injection of STZ + Nutlin-3 significantly (p < 0.01) counteracted IL-12p40 down-modulation. In vitro experiments performed on the RAW264.7 macrophagic cell line model, used as cellular source of IL-12p40, demonstrated that Nutlin-3 treatment increased IL-12p40 release, strongly suggesting a direct effect of Nutlin-3 on the immune system. Overall, these data demonstrate that systemic administration of Nutlin-3 ameliorates the severity of STZ-induced DM and increases the levels of circulating IL-12p40.
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The Oncogene DEK Promotes Leukemic Cell Survival and Is Downregulated by both Nutlin-3 and Chlorambucil in B-Chronic Lymphocytic Leukemic Cells
Clinical Cancer Research, 2010Co-Authors: Paola Secchiero, M. G. Di Iasio, Elisabetta Melloni, Mario Tiribelli, Rebecca Voltan, Giorgio ZauliAbstract:Purpose: To characterize the role of the oncogene DEK in modulating the response to either Nutlin-3, a small-molecule inhibitor of the MDM2/p53 interaction, or chlorambucil in primary B-chronic lymphocytic leukemia (B-CLL) cells. Experimental Design: DEK mRNA and protein levels were evaluated in primary B-CLL samples ( n = 21), p53 wild-type SKW6.4, p53 mutated BJAB lymphoblastoid cell lines, and normal CD19 + B lymphocytes–treated Nutlin-3 or chlorambucil (10 μmol/L, each). Knocking down experiments with either p53 or DEK small interfering RNA (siRNA) were done to investigate the potential role of p53 in controlling the expression of DEK and the role of DEK in leukemic cell survival/apoptosis. Results: Both Nutlin-3 and chlorambucil downregulated DEK in primary B-CLL samples ( n = 21) and SKW6.4 but not in BJAB cells. Knocking down p53 attenuated the effect of Nutlin-3 on DEK expression, whereas knocking down DEK significantly increased both spontaneous and Nutlin-3–induced apoptosis. Conversely, counteracting DEK downmodulation by using p53 small interfering RNA reduced Nutlin-3–mediated apoptosis. On the other hand, Nutlin-3 potently induced p53 accumulation, but it did not affect DEK levels in normal CD19 + B lymphocytes. Conclusions: These data show that the downregulation of DEK in response to either Nutlin-3 or chlorambucil represents an important molecular determinant in the cytotoxic response of leukemic cells, and suggest that strategies aimed to downregulate DEK might improve the therapeutic potential of these drugs. Clin Cancer Res; 16(6); 1824–33
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B-Cell Chronic Lymphocytic Leukemia Cells Exposed to the Non-Genotoxic p53 Activator Nutlin-3 Are Characterized by a Specific Gene Expression Signature.
Blood, 2009Co-Authors: Michele Dal-bo, Paola Secchiero, Riccardo Bomben, Massimo Degan, D. Benedetti, Antonella Zucchetto, Daniela Marconi, Gabriele Pozzato, Gianluca Gaidano, G. Del PoetaAbstract:Abstract 4374 Introduction p53 plays a key role in determining the clinical features of B cell chronic lymphocytic leukemia (CLL). Disruption of p53 by point mutations, deletion at 17p13, or both, occurs in a fraction of cases at diagnosis and predicts poor survival and chemorefractoriness. In cells with functional p53, p53 activity is inhibited through interaction with MDM2. In fact, p53 can be activated upon exposure of cells to inhibitors of p53/MDM2 interaction, like Nutlins. Exposure of CLL cells to Nutlin-3 is effective in raising the levels of p53 protein with subsequent induction of cell cycle arrest and/or apoptosis, independently of the most relevant prognostic markers. The aim of the present study was to analyze the gene expression profile (GEP) induced by Nutlin-3 exposure in primary CLL cells from p53 wt and p53 del/mut cases. Patients and methods purified cells from 24 PB CLL samples, all characterized for IGHV mutational status, CD38 and ZAP-70 and p53 mutations (16 p53 wt CLL, 8 p53 del/mut CLL of which 6 with del17p13 and p53 mutations, 1 with del17p13 alone, and 1 with p53 mutations alone), were exposed to 10 mM Nutlin-3 for 24 hours. GEP was performed using a dual labelling strategy; the differential expression of the below reported genes were validated by quantitative real-time PCR. Results i) signature of Nutlin-3 exposure in p53 wt CLL: 144 differentially expressed genes (143 up-regulated, 1 down-regulated) were correlated with response to Nutlin-3. Among the over-expressed genes, several genes were related to apoptosis (e.g. BAX , BBC3 , E124 , IKIP , FAS, LRDD, FLJ11259, TRIAP1, GADD45, TP53INP1, ISG20L1 , ZMAT3 , TNFRS10C , TNFRSF10B/TRAIL- R2), while other genes (e.g. MDM2, CDKN1A, PCNA ) were up-regulated by Nutlin-3 as a part of a negative feed-back mechanism. Of note, this signature was not shared by 3/16 p53 wt cases (identified as “non-responder” p53 wt CLL) and 7/8 p53 del/mut cases (identified as “non-responder” p53 del/mut CLL); consistently, cells from these cases were also significantly resistant to the in-vitro cytotoxic effects of Nutlin-3; ii) signature of Nutlin-3 “non-responder” p53 wt CLL: by comparing the constitutive GEP of 13 “responder” versus 3 “non-responder” p53 wt CLL, we obtained 278 differentially expressed genes, 149 up-regulated and 129 down-regulated in “non-responder” p53 wt CLL. Among up-regulated genes, we focused on MDM4/MDMX, a gene whose product was known to have an inhibitor activity of p53-dependent transcription and to form Nutlin-3 resistant complexes with p53. Among down-regulated genes, validations were made for BIRC4BP , whose product is known to act as an antagonist of the anti-apoptotic protein XIAP; iii) signature of Nutlin-3 “non-responder” p53 del/mut CLL: by comparing the constitutive GEP of 13 “responder” versus 7 “non-responder” p53 del/mut cases, we obtained 72 differentially expressed genes, 26 up-regulated and 46 down-regulated (31/46 located at the 17p segment) in “non-responder” p53 del/mut CLL. Validations were made for several genes whose products display pro-apoptotic activities (e.g. PSMB6 , RPL26 and ZBTB4 , located at 17p segment, and GNAZ located at chromosome 22) among down-regulated genes, and ARHGDIA , whose gene product displays anti-apoptotic activities and mediates cellular resistance to chemotherapeutic agents, among up-regulated genes. Notably, CLL cells (n=43) displayed constitutively higher levels of MDM4/MDMX (p ARHGDIA (p=0.0002) transcripts than purified normal B cells (n=15), irrespectively to the major biologic prognosticators. Conclusions specific gene-sets and GEP were documented to be associated with response or resistance to Nutlin-3 exposure in p53 wt or p53 del/mut CLL. These findings may help to identify novel molecular targets for CLL therapy. Disclosures: No relevant conflicts of interest to declare.
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Exposure of B cell chronic lymphocytic leukemia (B-CLL) cells to Nutlin-3 induces a characteristic gene expression profile, which correlates with Nutlin-3-mediated cytotoxicity.
Current Cancer Drug Targets, 2009Co-Authors: Giorgio Zauli, Paola Secchiero, M. G. Di Iasio, M. Dal Bo, D. Marconi, Riccardo Bomben, G. Del Poeta, Valter GatteiAbstract:By analyzing the cDNA obtained from 16 B-cell chronic lymphocytic leukemia (B-CLL) patient samples, we found that Nutlin-3, a small molecule inhibitor of MDM2/p53 interaction, induced a characteristic gene expression profile (GEP) signature in 13 out of 16 B-CLL samples. The lack of Nutlin-3-induced GEP signature in 3 out of 16 B-CLL samples was not due to p53 deletion and/or mutation, as demonstrated by FISH analysis and p53 sequencing. Of note, the 3 B-CLL samples in which Nutlin-3 did not elicit the GEP signature were also less susceptible to Nutlin-3-mediated cytotoxicity with respect to the remaining 13 B-CLL samples. However, the partial lack of response in these p53 wild-type B-CLL samples was not due to defects in the ability of Nutlin-3 to promote p53 induction, as confirmed by the rapid accumulation of p53 protein at Western blot analysis in response to Nutlin-3 in all samples examined. Upon exposure to Nutlin-3, the genes up-regulated with the highest score in the majority of B-CLL cells were all known p53-target genes, including genes involved in apoptotic pathways, such as FAS and BAX, as well as MDM2. Taken together, our data indicate that the ability of Nutlin-3 to induce a characteristic GEP signature correlates with its cytotoxic potential in p53 wild-type B-CLL cells. However, in some p53 wild-type B-CLL samples, the response to Nutlin-3 cannot be predicted on the basis of FISH analysis or p53 sequencing.
