P53 Upregulated Modulator Of Apoptosis Expression Research Articles

Induction of breast cancer cell apoptosis by novel thiouracil-fused heterocyclic compounds through boosting of Bax/Bcl-2 ratio and DFT study

Breast cancer is a common public health disease causing mortality worldwide. Thus, providing novel chemotherapies that tackle breast cancer is of great interest. In this investigation, novel pyrido[2,3-d]pyrimidine derivatives 3,4,(6a-c),(8a,b),9–20 were synthesized and characterized using a variety of spectrum analyses. The geometric and thermal parameters of the novel thiouracil derivatives 3,4,6a,(8a,b),11,12,17,18, 19 were measured using density functional theory (DFT) via DFT/B3LYP/6–31 + G(d,p) basis set. All synthesized compounds were evaluated by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) method using MCF-7 and MDA-MB-231 breast cancerous cells, compound 17 had the maximum anticancer activity against both breast cancerous cells, recording the lowest half-maximal inhibitory concentration (IC50) values (56.712 μg/mL for MCF-7 cells and 48.743 μg/mL for MDA-MB-231 cells). The results were confirmed in terms of the intrinsic mechanism of apoptosis, where compound 17 had the highest percentage in the case of both cancer cells and recorded Bax (Bcl-2 associated X)/Bcl-2 (B-cell lymphoma 2) ratio 17.5 and 96.667 for MCF-7 and MDA-MB-231 cells, while compound 19 came after 17 in the ability for induction of apoptosis, where the Bax/Bcl-2 ratio was 15.789 and 44.273 for both cancerous cells, respectively. Also, compound 11 recorded a high Bax/Bcl-2 ratio for both cells. The safety of the synthesized compounds was applied on normal WI-38 cells, showing minimum cytotoxic effect with undetectable IC50. Compounds 17, 11, and 19 recorded a significant increase of p53 upregulated modulator of apoptosis (PUMA) expression levels in the cancerous cells. The DFT method was also used to establish a connection between the experimentally determined values of the present investigated compounds and their predicted quantum chemical parameters. It was concluded that Compounds 17, 11, and 19 had anti-breast cancer potential through the induction of apoptotic Bax/Bcl-2 and PUMA expression levels.

Abstract 17456: Inositol 1,4,5-Trisphosphate Receptors (ITP3Rs) and Activation of Cardiac Fibroblasts in Ischemia/Reperfusion Injury

Introduction: Inositol 1,4,5-trisphosphate receptors (ITP3Rs) are Ca 2+ release channels on the endoplasmic reticulum (ER). The role of ITP3Rs in cardiomyocytes has been previously investigated for hypertrophy and arrhythmias, however, the exact function in the activation of cardiac fibroblasts (FBs) is yet to be determined. The main goal of this project is to investigate the mechanisms linking FB ITP3Rs to post-ischemia/reperfusion (I/R) cardiac fibrosis. Hypothesis: We hypothesize that ITP3Rs activate FB via a direct transcriptional regulation of αSMA and other genes involved in myoFB activation. Indeed, myoFB differentiation and persistence is regulated by a balance between pro-and anti-apoptotic signaling pathways. Methods: We generated myoFB-specific ITP3Rs KO mice (ITP3R KO ) and isolated FBs as well as cardiomyocytes via collagenase digestion and performed immunoprecipitation (IP) assays showing that ITP3Rs bind p53 upregulated modulator of apoptosis (PUMA). Cytosolic levels of Cytochrome C (Cyt C) were assessed in myoFB isolated from areas remote from injury and scar areas 7 d after I/R. After having immunoprecipitated for ITP3R1 we blotted the isolated FBs for PUMA and, vice versa. PUMA levels were measured in cytosol, ER, and mitochondria of FBs isolated from regions remote from injury and scar areas 7 d post I/R. Results: Our data show reduced levels of cytosolic Cyt C following I/R, which was prevented by IP3Rs ablation, providing an explanation for the persistence of myoFBs in remote areas. We found that the mitochondrial expression of PUMA was significantly decreased - while ER levels were markedly increased - in myoFBs isolated from areas remote from I/R injury, indicating a sequestration of PUMA by ITP3Rs; this phenotype was rescued by ITP3Rs ablation. Instead, in myoFBs isolated from the scar areas, mitochondrial PUMA levels were increased both in ITP3R fl/fl and ITP3R KO mice, and no sequestration was observed, consistent with the absence of ITP3R upregulation. Conclusions: Using a specific Cre/lox KO model, we provide the first assessment of functional contribution of ITP3R to post-I/R activation of cardiac FBs and understanding the molecular mechanisms underlying excessive reactive fibrosis in the heart.

Interdependent Nuclear Co-Trafficking of ASPP1 and p53 Aggravates Cardiac Ischemia/Reperfusion Injury.

ASPP1 (apoptosis stimulating of p53 protein 1) is critical in regulating cell apoptosis as a cofactor of p53 to promote its transcriptional activity in the nucleus. However, whether cytoplasmic ASPP1 affects p53 nuclear trafficking and its role in cardiac diseases remains unknown. This study aims to explore the mechanism by which ASPP1 modulates p53 nuclear trafficking and the subsequent contribution to cardiac ischemia/reperfusion (I/R) injury. The immunofluorescent staining showed that under normal condition ASPP1 and p53 colocalized in the cytoplasm of neonatal mouse ventricular cardiomyocytes, while they were both upregulated and translocated to the nuclei upon hypoxia/reoxygenation treatment. The nuclear translocation of ASPP1 and p53 was interdependent, as knockdown of either ASPP1 or p53 attenuated nuclear translocation of the other one. Inhibition of importin-β1 resulted in the cytoplasmic sequestration of both p53 and ASPP1 in neonatal mouse ventricular cardiomyocytes with hypoxia/reoxygenation stimulation. Overexpression of ASPP1 potentiated, whereas knockdown of ASPP1 inhibited the expression of Bax (Bcl2-associated X), PUMA (p53 upregulated modulator of apoptosis), and Noxa, direct apoptosis-associated targets of p53. ASPP1 was also increased in the I/R myocardium. Cardiomyocyte-specific transgenic overexpression of ASPP1 aggravated I/R injury as indicated by increased infarct size and impaired cardiac function. Conversely, knockout of ASPP1 mitigated cardiac I/R injury. The same qualitative data were observed in neonatal mouse ventricular cardiomyocytes exposed to hypoxia/reoxygenation injury. Furthermore, inhibition of p53 significantly blunted the proapoptotic activity and detrimental effects of ASPP1 both in vitro and in vivo. Binding of ASPP1 to p53 triggers their nuclear cotranslocation via importin-β1 that eventually exacerbates cardiac I/R injury. The findings imply that interfering the expression of ASPP1 or the interaction between ASPP1 and p53 to block their nuclear trafficking represents an important therapeutic strategy for cardiac I/R injury.

Platycodin D induces apoptosis through JNK1/AP-1/PUMA pathway in non-small cell lung cancer cells: A new mechanism for an old compound.

Platycodin D, a triterpenoid monomer, has been shown to possess an anti-tumor effect on various types of cancer. Although Platycodin D has been reported to suppress tumorigenesis, the detailed underlying mechanism remains elusive. Platycodin D treatment significantly reduced the cell viability, decreased the number of colonies, impaired the mitochondrial function, and induced apoptosis in non-small cell lung cancer (NSCLC) cells. To understand the mechanism by which platycodin D induces apoptosis, the expression levels of apoptosis-related proteins were examined, and we found that the expression of PUMA (p53 upregulated modulator of apoptosis) was upregulated upon platycodin D treatment. Knockdown of PUMA resulted in attenuation of platycodin D-induced apoptosis, indicating that PUMA up-regulation is essential for platycodin D to induce apoptosis. The induction of PUMA expression by platycodin D treatment was through activation of AP-1 since mutation of AP-1 binding site in the PUMA promoter abolished the PUMA promoter activity. In addition, the chromatin immunoprecipitation further demonstrated that platycodin D promoted AP-1 binding to PUMA promoter. Moreover, knockdown of JNK1, but not JNK2, significantly abolished the phosphorylation of c-Jun at Ser63 (a component of AP-1), decreased the platycodin D-induced expression of PUMA and cleaved caspase 3, indicating that platycodin D inhibits JNK1/AP-1 signaling pathway. Furthermore, immunohistochemical staining studies showed that tumors from the mice treated with platycodin D activated JNK by translocation of JNK into nuclei, increased phosphorylation of JNK and c-Jun at Ser63 in nuclei, and boosted the PUMA expression. Taken together, our in vitro and in vivo data revealed a novel mechanism by which platycodin D up-regulates PUMA to induce apoptosis through JNK1/AP-1 axis in NSCLC.

Effects of PUMA knockout on the apoptosis of H9C2 cardiomyocytes induced by high glucose

To investigate the effects of p53 upregulated modulator of apoptosis (PUMA) on the apoptosis of H9C2 cardiomyocytes induced by high glucose and its mechanisms. H9C2 cardiomyocytes were treated with 5.5mmol/L (control group) or 35 mmol/L glucose (HG group) for 6 h, 12 h, 24 h or 48 h respectively to induce apoptosis, each group sets 5 multiple wells. Apoptosis was tested by TUNEL assay. PUMA mRNA was measured by RT-PCR and protein expression was measured by Western blot assay. The mitochondrial membrane potential was detected by JC-1 method. The expressions of cleaved caspase-3 and cytochrome C (Cyt C) protein in mitochondria and cytoplasm were determined by Western blot assay. H9C2 cardiomyocytes were randomly divided into four groups, control group (5.5 mmol/L), HG (35 mmol/L) group, HG+si-scramble group(si-scramble treatment for 24 h, then 35 mmol/L high glucose treatment for 24 h) and HG-si-PUMA group (si-PUMA treatment for 24 h, then 35mmol/L high glucose treatment for 24 h). Si-PUMA was transfected into cardiomyocytes and the effects of PUMA on high glucose-induced apoptosis were studied. Compared with the control group, high glucose increased cardiomyocyte apoptosis and enhanced PUMA mRNA and protein expressions significantly (P＜0.05 or P＜0.01). Cell injury and increased PUMA expression were time-dependent and there was no significant difference between the high glucose 24 h group and the high glucose 48h group. The following experiment used high glucose 24 h as the stimulation time. The cardiomyocytes transfected with si-PUMA to inhibit PUMA expression had decreased apoptotic rate and cleaved caspase-3, increased mitochondria membrane potential and decreased Cyt C release (P＜0.05 or P＜0.01). There were no significant differences between the HG+si-scramble group and the high glucose group (P＞0.05). PUMA mediates high glucose-induced cardiomyocyte apoptosis suggesting PUMA may be an important target gene of diabetic cardiomyopathy.

Abstract 110: Elevation of pro-apoptotic protein, PUMA, contributes to the growth suppression of lung cancer by platycodin D

Abstract Lung cancer is the most devastating disease and leading cause of cancer death in men and women. Platycodin D is one of the major bioactive components of the Platycodon grandiflorum, a herb that is frequently used for the treatment of lung diseases in Chinese medicine. Platycodin D is a triterpenoid monomer with activities of antioxidant, anti-obesity, and anti-inflammatory. Studies have also shown that platycodin D has an anti-tumor effect on various types of cancer. However, the mechanism by which platycodin D suppresses tumorigenesis remains elusive and some experimental results are controversial. In this study, we aim to understand the effect of platycodin D on the growth of non-small lung cancer cells (NSCLC) and the underlying mechanism. We found that NSCLC cells (H1299, H2030, and A549 cells) treated with platycodin D significantly reduced the cell viability, decreased the number of colonies, impaired the mitochondrial function, and induced apoptosis. To understand the mechanism, the protein levels of the Bcl-2 protein family were examined and p53 upregulated modulator of apoptosis (PUMA) was found elevated by platycodin D treatment. The induction of PUMA expression by platycodin D treatment was through activation of the c-Jun N-terminal kinase (JNK) and AP-1 dependent transcription since JNK inhibitor or over-expression of dominant negative c-Jun abolished the elevated PUMA expression. Moreover, knockdown of JNK1, but not JNK2, significantly decreased the level of platycodin D-induced PUMA and cleaved caspase 3, indicating the essential role of JNK1 in platycodin D-induced apoptosis. Furthermore, platycodin D efficiently suppressed the tumor growth in the H1299 xenograft. Taken together, activation of JNK1 by platycodin D is essential for induction of apoptosis in suppression of growth of NSCLC, providing a new mechanism of how platylcodin D suppresses NSCLC. Citation Format: Qing Wang, Hsin-Sheng Yang. Elevation of pro-apoptotic protein, PUMA, contributes to the growth suppression of lung cancer by platycodin D [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 110.

Effects of trans-(±)-kusunokinin on chemosensitive and chemoresistant ovarian cancer cells.

Ovarian cancer ranks eighth in cancer incidence and mortality among women worldwide. Cisplatin-based chemotherapy is commonly used for patients with ovarian cancer. However, the clinical efficacy of cisplatin is limited due to the occurrence of adverse side effects and development of cancer chemoresistance during treatment. Trans-(±)-kusunokinin has been previously reported to inhibit cell proliferation and induce cell apoptosis in various cancer cell types, including breast, colon and cholangiocarcinoma. However, the potential effects of (±)-kusunokinin on ovarian cancer remains unknown. In the present study, chemosensitive ovarian cancer cell line A2780 and chemoresistant ovarian cancer cell lines A2780cis, SKOV-3 and OVCAR-3 were treated with trans-(±)-kusunokinin to investigate its potential effects. MTT, colony formation, apoptosis and multi-caspase assays were used to determine cytotoxicity, the ability of single cells to form colonies, induction of apoptosis and multi-caspase activity, respectively. Moreover, western blot analysis was performed to determine the proteins level of topoisomerase II, cyclin D1, CDK1, Bax and p53-upregulated modulator of apoptosis (PUMA). The results demonstrated that trans-(±)-kusunokinin exhibited the strongest cytotoxicity against A2780cis cells with an IC50 value of 3.4 µM whilst also reducing the colony formation of A2780 and A2780cis cells. Trans-(±)-kusunokinin also induced the cells to undergo apoptosis and increased multi-caspase activity in A2780 and A2780cis cells. This compound significantly downregulated topoisomerase II, cyclin D1 and CDK1 expression, but upregulated Bax and PUMA expression in both A2780 and A2780cis cells. In conclusion, trans-(±)-kusunokinin suppressed ovarian cancer cells through the inhibition of colony formation, cell proliferation and the induction of apoptosis. This pure compound could be a potential targeted therapy for ovarian cancer treatment in the future. However, studies in an animal model and clinical trial need to be performed to support the efficacy and safety of this new treatment.

Cisatracurium besilate enhances the TRAIL-induced apoptosis of gastric cancer cells via p53 signaling

ABSTRACT Cisatracurium besilate is the most commonly used non-depolarizing muscle relaxant in general anesthesia and in intensive care units. Studies have indicated that the proliferation of gastric cancer (GC) cells can be restrained by cisatracurium besilate. The present study aimed to investigate the mechanism underlying the role of cisatracurium besilate in TNF-related apoptosis-inducing ligand (TRAIL)-induced GC. The AGS cell line was exposed to cisatracurium besilate, and then cell viability, colony formation and apoptosis were assessed by performing Cell Counting Kit-8, colony formation, TUNEL and Western blot assays, respectively. Furthermore, the expression levels of p53 and p53 upregulated modulator of apoptosis (PUMA) were measured by Western blotting to determine the effect of cisatracurium besilate on p53/PUMA signaling. After co-treatment with p53 inhibitor, cisatracurium besilate and pifithrin-α/TRAIL, cell apoptosis was detected. Finally, cisatracurium besilate and pifithrin-α were used to co-treat TRAIL-induced AGS cells followed by apoptosis detection. Cisatracurium besilate treatment restrained the proliferation and promoted the apoptosis of AGS cells. Cisatracurium besilate also promoted the expression of p53 and PUMA in AGS cells. Furthermore, TRAIL induced the apoptosis of AGS cells, which was aggravated by cisatracurium besilate treatment. However, pifithrin-α reversed the synergistic effects of cisatracurium besilate and TRAIL on the activities of AGS cells. Therefore, the present study suggested that cisatracurium besilate enhanced the TRAIL-induced apoptosis of GC cells via p53 signaling, and the synergistic effects of cisatracurium besilate and TRAIL may achieve maximal therapeutic efficacy in GC management.

Up-regulation of PUMA caused the activation of p53 phosphorylation and acetylation, enhancing the interaction between PUMA and Bcl-X and mediating arsenic-induced apoptosis

Arsenic is a toxic metalloid vastly dispersed all over the occupational environments, manifesting multiple adverse health issues related to apoptosis. PUMA (p53 up-regulated modulator of apoptosis) is a crucial member of the Bcl-2 protein family and plays a key role in pro-apoptosis. The purpose of this work was to determine whether inorganic arsenic (NaAsO2) and its metabolites influenced the expression of PUMA in vivo and vitro, followed by investigating the mechanisms. RNA was extracted from serum and used to determine the expression of PUMA in vivo. The urine samples performed arsenic speciation analysis. This trial tested three-dose proportions in two cell lines (A549: 20, 40, 60 μM/L; 16HBE: 1.5, 3.0, 4.5 μM/L), respectively. According to the results of qRT-PCR and western blotting, NaAsO2 caused the overexpression of PUMA, not its metabolites. Furthermore, NaAsO2 induced phosphorylation of p53 at Ser315, 376, 392, and Thr55, and acetylation of p53 at K370, 382 with a dose-response relationship, suggesting the contribution of PUMA up-regulation to p53 phosphorylation and acetylation. CCK-8, JC-1 (5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetramethylbenzimi-dazolylcarbocyanine iodide), Hoechst33342/PI and the caspase3 and PARP1 blots were utilized to reveal apoptosis responding to NaAsO2 exposure. The co-immunoprecipitation assay showed that the interaction between PUMA and Bcl-X enhanced in intensity responding to NaAsO2 exposure, disrupting the complexes of Bcl-X with other pro-survival Bcl-2-related proteins. To our knowledge, we first reported that NaAsO2 activated phosphorylation of p53 at Ser315, 376, and Thr55, as well as acetylation of p53 at K370.

Expression of p53 up-regulated modulator of apoptosis (PUMA) in non-melanoma skin cancer of long-term immunosuppressed solid organ transplant recipients compared to immunocompetent patients.

The risk of UV radiation (UVR)-induced non-melanoma skin cancer (NMSC) is dramatically increased in immunosuppressed organ transplant recipients compared to immunocompetent patients. In the skin, p53 up-regulated modulator of apoptosis (PUMA) is a central regulator of apoptosis in response to UVR damage and immune response regulation. Data on the expression of PUMA in patients with NMSC relative to immune status is limited To study differences in the expression and distribution of PUMA in cutaneous SCC and BCC by immunohistochemistry between immunocompetent patients and organ transplant recipients, and the effect of CsA-containing immunosuppressive maintenance regimens on this expression. PUMA expression in SCC (n = 34) and BCC (n = 20) was analysed comparatively by immunohistochemical staining in matched cohorts of 27 immunocompetent patients and 27 organ transplant recipients SCC and BCC showed unequivocal positive PUMA expression, however, there was no significant difference in NMSC between organ transplant recipients and immunocompetent patients. A 17% reduction in staining score for PUMA in SCC, but not in BCC, of organ transplant recipients treated with a cyclosporin (CsA)-containing regimen was noted compared to organ transplant recipients without chronic CsA intake (p = 0.0381) PUMA expression in SCC, but not BCC, is significantly reduced by CsA-containing therapy, suggesting a disturbance of apoptosis by iatrogenic immunosuppression with a divergent impact on SCC and BCC.

Expression and inhibitory effects of p53-upregulated modulator of apoptosis in gallbladder carcinoma.

The p53-upregulated modulator of apoptosis (PUMA) has been reported to be involved in various types of cancer. However, its potential biological role in gallbladder carcinoma (GBC) has not been fully elucidated. The present study aimed to determine the expression levels of PUMA and its biological effects on GBC. The mRNA and protein expression levels of PUMA in GBC tissues and cell lines were measured using reverse transcription-quantitative PCR and western blotting, respectively. The effects of PUMA overexpression on cell viability, proliferation and invasive ability were determined in vitro using the MTT, colony formation and Transwell invasion assays, respectively. The apoptotic rates were detected using the Annexin V-FITC apoptosis detection kit. Furthermore, follow-up of patients with GBC was performed to identify the association between PUMA expression levels and GBC prognosis. The results of the present study demonstrated that the expression levels of PUMA were significantly lower in the GBC tissues and cell lines compared with those in adjacent normal gallbladder tissues and normal gallbladder cells, respectively. Further experiments indicated that overexpression of PUMA inhibited the viability, proliferation and invasive ability of GBC cells compared with those in the control-transfected GBC cells. In addition, overexpression of PUMA significantly promoted apoptosis in GBC cells. Furthermore, overexpression of PUMA inhibited epithelial-mesenchymal transition, and promoted Bax upregulation and Bcl-2 downregulation compared with those in the control group. Low PUMA expression levels were associated with a short overall survival time in patients with GBC. In conclusions, PUMA may act as a tumor suppressor in GBC and may serve as a potential novel treatment target for human GBC.

RETRACTED: Depleting microRNA-183-3p improves renal tubulointerstitial fibrosis after acute kidney injury via SIRT1/PUMA/FOXO3a deacetylation

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. Concern was raised about the reliability of the Western blot results in Figs. 2E, and 3C, which appear to have the same eyebrow shaped phenotype as many other publications tabulated here (https://docs.google.com/spreadsheets/d/149EjFXVxpwkBXYJOnOHb6RhAqT4a2llhj9LM60MBffM/edit#gid=0). The journal requested the corresponding author comment on these concerns and provide the raw data. However the authors were not able to satisfactorily fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Recombinant human bone morphogenetic protein 2 and 7 inhibit the degeneration of intervertebral discs by blocking the Puma-dependent apoptotic signaling.

Recombinant human bone morphogenetic proteins (rhBMPs) can stimulate bone formation and growth in the treatment of spinal fusions and nonunions. However, it is still unclear whether rhBMPs function in the prevention of intervertebral disc degeneration (IDD). Here, we discovered that BMP levels were decreased in IDD patients, which impaired the BMP/Smad (Mothers against decapentaplegic homologs) signaling. Conducting a microarray assay in Smad4-knockdown cells, we found that expression of PUMA (p53-upregulated modulator of apoptosis) was significantly induced. The molecular analysis revealed that Smad4 recruited HDAC1 (histone deacetylase 1) and the phosphorylated Smad1/5/8 to dock on the promoter of PUMA to repress its expression. The impairment of BMP/Smad signaling in IDD patients caused the significant induction of Puma-dependent apoptosis and resulted in the pathogenesis of IDD. In vitro knockdown of BMP receptors (BMPR1a and BMPR2) in nucleus pulposus (NP) cells could mimic the molecular changes of BMP/Smad signaling and Puma-dependent apoptotic signaling that were observed in IDD patients. Exposing NP cells to RITA (reactivating p53 and inducing tumor apoptosis) small molecule and rhBMP2 (or rhBMP7), we observed that rhBMP2/7 could significantly decrease protein levels of Puma and its downstream proapoptotic molecules, blocking cell apoptosis. Importantly, administration of rhBMPs in aged rats could inhibit the occurrence of IDD. Our results provide a link between BMP/Smad signaling and Puma-dependent apoptotic signaling, revealing a new mechanism of how BMPs contribute to IDD pathogenesis and providing evidence that rhBMPs may decrease apoptosis and improve the outcome of IDD.

Conditioned medium from adipose-derived stem cells attenuates ischemia/reperfusion-induced cardiac injury through the microRNA-221/222/PUMA/ETS-1 pathway.

Rationale: Cardiovascular diseases, such as myocardial infarction (MI), are the leading causes of death worldwide. Reperfusion therapy is the common standard treatment for MI. However, myocardial ischemia/reperfusion (I/R) causes cardiomyocyte injury, including apoptosis and fibrosis. We aimed to investigate the effects of conditioned medium from adipose-derived stem cells (ADSC-CM) on apoptosis and fibrosis in I/R-treated hearts and hypoxia/reoxygenation (H/R)-treated cardiomyocytes and the underlying mechanisms.Methods: ADSC-CM was collected from ADSCs. The effects of intramuscular injection of ADSC-CM on cardiac function, cardiac apoptosis, and fibrosis examined by echocardiography, Evans blue/TTC staining, TUNEL assay, and Masson's trichrome staining in I/R-treated mice. We also examined the effects of ADSC-CM on apoptosis and fibrosis in H/R-treated H9c2 cells by annexin V/PI flow cytometry, TUNEL assay, and immunocytochemistry.Results: ADSC-CM treatment significantly reduced heart damage and fibrosis of I/R-treated mice and H/R-treated cardiomyocytes. In addition, the expression of apoptosis-related proteins, such as p53 upregulated modulator of apoptosis (PUMA), p-p53 and B-cell lymphoma 2 (BCL2), as well as the fibrosis-related proteins ETS-1, fibronectin and collagen 3, were significantly reduced by ADSC-CM treatment. Moreover, we demonstrated that ADSC-CM contains a large amount of miR-221/222, which can target and regulate PUMA or ETS-1 protein levels. Furthermore, the knockdown of PUMA and ETS-1 decreased the induction of apoptosis and fibrosis, respectively. MiR-221/222 overexpression achieved similar results. We also observed that cardiac I/R markedly increased apoptosis and fibrosis in miR-221/222 knockout (KO) mice, while ADSC-CM decreased these effects. The increased phosphorylation of p38 and NF‐κB not only mediated myocardial apoptosis through the PUMA/p53/BCL2 pathway but also regulated fibrosis through the ETS-1/fibronectin/collagen 3 pathway.Conclusions: Overall, our results show that ADSC-CM attenuates cardiac apoptosis and fibrosis by reducing PUMA and ETS-1 expression, respectively. The protective effect is mediated via the miR-221/222/p38/NF-κB pathway.

Role of the SIRT1/p53 regulatory axis in oxidative stress‑mediated granulosa cell apoptosis.

Oxidative stress has been suggested to induce granulosa cell apoptosis, which contributes to follicular atresia. However, the mechanism via which oxidative stress mediates granulosa cell apoptosis remains elusive. Therefore, the aim of this study was to elucidate the molecular mechanisms regulating oxidative stress‑induced granulosa cell apoptosis. The present study demonstrated that reactive oxygen species induced by H2O2 resulted in human granulosa COV434 cell apoptosis via the regulation of sirtuin1 (SIRT1)‑mediated p53 activity. Endogenous SIRT1 expression was alleviated by H2O2 treatment of COV434 cells in a time‑dependent manner. In addition, knockdown or inhibition of SIRT1 promoted H2O2‑induced poly(ADP‑ribose) polymerase (PARP) cleavage and p53 acetylation, which led to an increase in COV434 cell apoptosis. Treatment with H2O2 enhanced the expression levels of the p53‑dependent proteins, p53‑upregulated modulator of apoptosis (PUMA) and phorbol‑12‑myristate‑13‑acetate‑induced protein1 (PMAIP1), as well as those of p53; however, knockdown of p53 decreased cleaved PARP, PUMA and PMAIP1 expression levels induced by H2O2 treatment. Moreover, knockdown of PUMA or PMAIP1 attenuated the H2O2 induction of PARP cleavage and COV434 cell apoptosis. In conclusion, the present findings suggested that H2O2‑induced oxidative stress causes granulosa COV434 cell apoptosis via the upregulation of p53 activity by SIRT1 suppression, indicating a mechanistic role of the SIRT1/p53 axis in H2O2‑induced granulosa cell apoptosis.

Follicle Stimulating Hormone Inhibits the Expression of p53 Up-Regulated Modulator of Apoptosis Induced by Reactive Oxygen Species Through PI3K/Akt in Mouse Granulosa Cells.

In mammalian ovaries, follicular atresia occurs periodically and destroys almost all the follicles in the ovary. Follicle-stimulating hormone (FSH) acts as the primary survival factor during follicular atresia by preventing apoptosis in granulosa cells (GCs). Many studies have demonstrated that oxidative stress-induced apoptosis is a main cause of follicular atresia. Reactive oxygen species (ROS)-induced GCs apoptosis is regulated by a variety of signaling pathways involving numerous genes and transcription factors. Therefore, we examined whether FSH inhibits the expression of p53 up-regulated modulator of apoptosis (PUMA) induced by reactive oxygen species (ROS) through phosphoinositide 3-kinase (PI3K) / protein kinase B (AKT) in mouse GCs. In vivo study: thirty-two-mice were randomly assigned to four groups and given FSH. We found that FSH can inhibit the 3-nitropropionic acid (3-NP) induced apoptosis and PUMA expression in mRNA level. Moreover, In vitro experiment, we found that FSH can inhibit the H(2)O(2)-induced apoptosis and PUMA expression in mRNA level. Additionally, we also found that PI3K/AKT inhibitor LY294002 abolished the downregulation of PUMA mRNA by FSH in vitro, In conclusion, FSH inhibit the expression of PUMA induced by ROS through PI3K/AKT pathway in vivo and vitro.

INOS/NO is required for IRF1 activation in response to liver ischemia-reperfusion in mice

BackgroundIschemia and reperfusion (I/R) induces cytokines, and up-regulates inducible nitric oxide synthase (iNOS), interferon regulatory factor-1(IRF1) and p53 up-regulated modulator of apoptosis (PUMA), which contribute to cell death and tissue injury. However, the mechanisms that I/R induces IRF1-PUMA through iNOS/NO is still unknown.MethodsIschemia was induced by occluding structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes for 60 min, and reperfusion was initiated by removal of the clamp. Induction of iNOS, IRF1 and PUMA in response to I/R were analyzed. I/R induced IRF1 and PUMA expression were compared between iNOS wild-type and iNOS knockout (KO) mice. Human iNOS gene transfected-cells were used to determine iNOS/NO signals targeting IRF1. To test whether HDAC2 was involved in the mediation of iNOS/NO-induced IRF1 transcriptional activities and its target gene (PUMA and p21) expression, NO donors were used in vitro and in vivo.ResultsIRF1 nuclear translocation and PUMA transcription elevation were markedly induced following I/R in the liver of iNOS wild-type mice compared with that in knock-out mice. Furthermore, I/R induced hepatic HDAC2 expression and activation, and decreased H3AcK9 expression in iNOS wild-type mice, but not in the knock-out mice. Mechanistically, over-expression of human iNOS gene increased IRF1 transcriptional activity and PUMA expression, while iNOS inhibitor L-NIL reversed these effects. Cytokine-induced PUMA through IRF1 was p53 dependent. IRF1 and p53 synergistically up-regulated PUMA expression. iNOS/NO-induced HDAC2 mediated histone H3 deacetylation and promoted IRF1 transcriptional activity. Moreover, treating the cells with romidepsin, an HDAC1/2 inhibitor decreased NO-induced IRF1 and PUMA expression.ConclusionsThis study demonstrates a novel mechanism that iNOS/NO is required for IRF1/PUMA signaling through a positive-feedback loop between iNOS and IRF1, in which HDAC2-mediated histone modification is involved to up-regulate IRF1 in response to I/R in mice.

RETRACTED: NF-κB inhibits the occurrence of type 1 diabetes through microRNA-150-dependent PUMA degradation

Type 1 diabetes (T1D) is the most common autoimmune disease that affects a global scale. Accumulating evidence has indicated, nuclear factor kappa B (NF-κB) and some microRNAs (miRNAs) as important biomarkers participating in the development of T1D. Thus, we aimed to determine the role of NF-κB and miR-150 in the development of T1D and to unravel the molecular mechanism. Non-obese diabetic mice were used for the T1D model establishment by injecting with streptozotocin. Besides, pancreatic islet β cells, separated from T1D mice, were induced by interferon-γ and tumor necrosis factor-α for 3days to mimic T1D damage. The expression of NF-κB p65, miR-150, and p53 up-regulated modulator of apoptosis (PUMA) was evaluated by RT-qPCR, while the expression of PUMA, p65, and apoptotic proteins in pancreatic islet β cells were determined by western blot analysis. Besides, inflammatory factors IL-17A, IL-2, IFN-γ, and IL-4 were detected by ELISA. The relationship among NF-κB, miR-150, and PUMA was analyzed by the dual-luciferase reporter gene, chromatin- and RNA-immunoprecipitation assays, respectively. Restoration of NF-κB reduced the incidence of T1D in mice. Over-expressed NF-κB inhibited the release of inflammatory factors and apoptosis in pancreatic islet β cells. PUMA was confirmed to be a potential target gene of miR-150. miR-150 suppressed PUMA to inhibit the T1D-induced inflammation and β cell apoptosis whereas NF-κB activated the miR-150 expression by binding to the miR-150 promoter, thereby preventing the T1D-induced inflammation and β cell apoptosis. NF-κB/miR-150/PUMA may serve as potential therapeutic targets for T1D.

MiR-22 protect PC12 from ischemia/reperfusion-induced injury by targeting p53 upregulated modulator of apoptosis (PUMA)

ABSTRACT MicroRNAs have been implicated as critical regulatory molecules in many cerebrovascular diseases. Recent studies demonstrated miR-22 might provide a potential neuroprotective effect. However, the neuroprotective effect of miR-22 in ischemia/reperfusion (I/R) injury has not been thoroughly elucidated. In this study, the PC12 cells were subjected to 4 h oxygen and glucose deprivation (I) and 24 h reoxygenation (R). The PC12 cells were pre-transfected with miR-22 or anti-miR-22 or siRNA-mediated downregulation of p53-upregulated-modulator-of-apoptosis (PUMA)(PUMA siRNA) or their controls at 24 h prior to exposure to I/R. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot were employed to analyze mRNA and protein expression. PI and Annexin V assays and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to quantify the rate of apoptosis. We found that miR-22 expression was significantly downregulated in the PC12 cells subjected to I/R. Loss of function of miR-22 increased PC12 apoptosis after I/R, and overexpression of miR-22 decreases PC12 apoptosis after I/R. PUMA protein was upregulated in the I/R group as compared with the sham group. The increased PUMA protein expression and apoptosis induced by I/R was reversed by transfection with PUMA siRNA. We concluded that I/R enhanced apoptosis and PUMA expression in PC12 cells via downregulation of miR-22. Enhanced miR-22 expression reversed both PUMA expression and apoptosis induced by I/R in PC12 cells. miR-22/PUMA axis has important implications for their clinical applications.

Targeting survivin sensitizes cervical cancer cells to radiation treatment

ABSTRACT Survivin is an inhibitor of apoptosis protein that functions to inhibit apoptosis, promote proliferation, and enhance invasion. It is selectively up-regulated in many human tumors and implicated in cellular radiation response through its role in apoptosis, cell division, and DNA damage response. This study aimed to investigate the effect and mechanisms of targeting survivin radiosensitivity in cervical cancer C33A cells. Here, the authors designed a small interfering RNA (siRNA) or plasmid-based small hairpin RNA (shRNA) targeting survivin and tested its effects on radiosensitivity to ionizing radiation (IR) treatment of C33A cells in vitro, as well as on the tumorigenicity of C33A cells in nude mice in vivo. Transient transfection of survivin siRNA into C33A cells suppressed survivin expression, induced cell apoptosis and G2/M arrest and reduced cell proliferation, clone formation ability after IR, followed by p53 upregulated modulator of apoptosis (PUMA) upregulation. But, transient transfection of survivin siRNA alone has no significant effect on cell growth and apoptosis. To confirm that PUMA upregulation is necessary for survivin silencing -induced radiosensitivity to IR treatment, the effect of targeting PUMA in survivin sliencing cells was observed. The results showed that targeting PUMA in survivin sliencing cells rescued C33A cells’ radioresistance. Furthermore, knocking down survivin expression combined with IR treatment significantly slowed tumor growth and promoted tumor cell apoptosis in C33A xenografted tumors. It was concluded that survivin played a role in radiotherapy resistance. Targeting survivin increased the radiosensitivity of C33A cells through induction of PUMA expression.