Flow cytometry evaluation of HeLa S3 cell death induced by gamma-radiation

In this study we followed the effects of radiation on human uterin cervix HeLa S3 cells viability, morphology and DNA structure 2-96 hours after treatment with 2-10 Gy from 60Co gamma radiation source. Staining of cells with Annexin V-FITC and propidium iodide showed very low degree of radiation-induced apoptosis. The prevailing form of HeLa S3 cell death according to flow-cytometry, DNA fragmentation and fluorescent microscopy, was necrosis. The gamma-radiation dose necessary to induce 50% of necrosis (termed DD50) was twice higher compared to dose that induced 50% inhibition of cell proliferation (LD50). These in vitro data suggested, that the increase in radiation dose might eradicate tumor cells, rather than just control their proliferation and growth.

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A Novel High-Throughput Technique for Identifying Monoclonal Antibodies capable of Death Receptor Induced Apoptosis

Journal of Cell Death ◽

10.4137/jcd.s3660 ◽

2009 ◽

Vol 2 ◽

pp. JCD.S3660

Author(s):

Hang Fai Kwok ◽

Julie A. Gormley ◽

Christopher J. Scott ◽

James A. Johnston ◽

Shane A. Olwill

Keyword(s):

Cell Death ◽

High Throughput ◽

High Throughput Screening ◽

False Negative ◽

Death Receptor ◽

Annexin V ◽

Fas Receptor ◽

Induced Apoptosis

The study of death receptor family induced apoptosis has gained momentum in recent years with the knowledge that therapeutic antibodies targeting DR4 and DR5 (death receptor's 4 and 5) have proved efficacious in multiple clinical trials. The therapeutic rationale is based on targeting and amplifying a tumour tissues normal cell death programme (apoptosis). While advances in the targeting of DR4 and DR5 have been successful the search for an agonistic antibody to another family member, the Fas receptor, has proven more elusive. This is partly due to the differing in vitro and in vivo characteristics of individual antibodies. In order to induce Fas targeted cell death an antibody must be capable of binding to and trimerising the receptor. It has been shown that antibodies capable of performing this function in vivo, with the assistance of tumour associated cells, do not always induce apoptosis in vitro. As a result the use of current methodologies to detect functional antibodies in vitro may have dismissed potential therapeutic candidates ('false negative'). Here we report a novel high throughput screening technique which artificially cross-links antibodies bound to the Fas receptor. By combining this process with Annexin-V and Prodidium Iodide (PI) staining we can select for antibodies which have the potential to induce apoptosis in vivo.

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In Vitro Evaluation of Apoptosis and Cell Death of Neuroblastoma Cell Lines Exposed to Busulfan.

Blood ◽

10.1182/blood.v106.11.4459.4459 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4459-4459

Author(s):

Morris Kletzel ◽

Sarah C. Tallman ◽

Marie Olszewski ◽

Wei Huang

Keyword(s):

Cell Death ◽

Cell Viability ◽

Cell Lines ◽

Neuroblastoma Cell ◽

Annexin V ◽

Resistant Cell ◽

Primary Tumors ◽

Induced Apoptosis ◽

Neuroblastoma Cell Lines

Abstract Objective: While busulfan is a commonly used chemotherapeutic agent in the treatment of many hematological diseases, its effectiveness against neuroblastoma is still in question. This study aims to assess the degree of apoptosis and cell death in neuroblastoma cell lines and primary neuroblastoma tumors when exposed to varying doses of busulfan. Materials and Methods: Cultures from established cell lines SKN-SH, SKN-DOX-R, IMR-5, and NGP (n=4), as well as cultures from primary tumors (n=2) were seeded at 106 cells/ml in RPMI640 supplemented with 10% fetal bovine serum (FBS) and transferred to 24-well plates, where cells were exposed to 1ml of busulfan at 0, 0.001, 0.005, 0.01, 0.05, and 0.1mg/ml per well. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hours. Wells were sacrificed after 0, 6, 24, 48 and 72 hours and tested with Annexin V and PI; 10,000 events were measured by flow cytometry. The percentage of apoptotic and dead cells was plotted in a graph and a t-test was performed against the untreated control. Results: After 24 hours, there was a significant decrease in cell viability of each dose when compared to the control untreated cells (p<0.005). 24 Hour % Cell Viability for Varying Doses of Busulfan (mg/ml) Dose 0 Dose 0.001 Dose 0.005 Dose 0.01 Dose 0.05 Dose 0.1 Mean 66.1 44.4 40.3 40.7 37.7 39 SEM 5.56 5.17 5.96 6.17 6.03 5.60 Median 65 33.5 38 39 37 31 Range 39 to 97 14 to 87 4 to 89 6 to 93 4 to 77 5 to 88 The overall mean decrease in cell viability when compared to the control was 25.7%. However, there were only modest differences in effectiveness when comparing the doses, with an average of only 5–7% difference between doses. Further, there was much variability between the different cell lines, some with changes in apoptosis and cell death of over 50%, while other lines showed no changes at all. Limited differences were seen after 6 hours, and after 72 hours any effect of busulfan was masked by cell death due to other factors, as seen through increased cell death in untreated cells. Conclusion: Busulfan induced apoptosis and cell death in vitro in neuroblastoma cell lines at a mean of 76.43% for non-resistant lines, 59.33% for primary tumors and 35% for resistant cell lines (at middle dose 0.01mg/ml). The resistance of certain cell lines confirms the difficulties of treating multi-drug resistant cells in often heterogeneous neuroblastoma tumors. That some cell lines were responsive shows the potential of using busulfan to treat neuroblastoma in the future.

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MicroRNA-29c Increases the Chemosensitivity of Pancreatic Cancer Cells by Inhibiting USP22 Mediated Autophagy

Cellular Physiology and Biochemistry ◽

10.1159/000490027 ◽

2018 ◽

Vol 47 (2) ◽

pp. 747-758 ◽

Cited By ~ 26

Author(s):

Limin Huang ◽

Chaoquan Hu ◽

Hui Cao ◽

Xiaoliang Wu ◽

Rongpin Wang ◽

...

Keyword(s):

Pancreatic Cancer ◽

Flow Cytometry ◽

Annexin V ◽

Luciferase Reporter ◽

Pancreatic Cancer Cells ◽

Reporter Assays ◽

Direct Target ◽

Induced Apoptosis

Background/Aims: Pancreatic cancer (PC) is an aggressive malignancy with a poor survival rate. Despite advances in the treatment of PC, the efficacy of therapy is limited by the development of chemoresistance. Here, we examined the role of microRNA-29c (miR-29c) and the involvement of autophagy and apoptosis in the chemoresistance of PC cells in vivo and in vitro. Methods: We employed qRT-PCR, western blot and immunofluorescence to examine the expression level of miR-29c, USP22 and autophagy relative protein. In addition, we used MTT assay to detect cell proliferation and transwell assay to measure migration and invasiveness. The apoptosis was determined using annexin V-FITC/PI apoptosis detection kit by flow cytometry. Luciferase reporter assays confirmed the relationship between USP22 and miR-29c. Results: miR-29c overexpression in the PC cell line PANC-1 enhanced the effect of gemcitabine on decreasing cell viability and inducing apoptosis and inhibited autophagy, as shown by western blotting, immunofluorescence staining, colony formation assays, and flow cytometry. Ubiquitin specific peptidase (USP)-22, a deubiquitinating enzyme known to induce autophagy and promote PC cell survival, was identified as a direct target of miR-29c. USP22 knockdown experiments indicated that USP22 suppresses gemcitabine-induced apoptosis by promoting autophagy, thereby increasing the chemoresistance of PC cells. Luciferase reporter assays confirmed that USP22 is a direct target of miR-29c. A xenograft mouse model demonstrated that miR-29c increases the chemosensitivity of PC in vivo by downregulating USP22, leading to the inhibition of autophagy and induction of apoptosis. Conclusions: Taken together, these findings reveal a potential mechanism underlying the chemoresistance of PC cells mediated by the regulation of USP22-mediated autophagy by miR-29c, suggesting potential targets and therapeutic strategies in PC.

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In-Vitro Treatment of Chronic Lymphocytic Leukemia (CLL) Cells with Fludarabine, Etoposide and Alemtuzumab (Campath-1H) Reveals Different Rates and Mechanisms of Cell Death.

Blood ◽

10.1182/blood.v106.11.2112.2112 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2112-2112 ◽

Cited By ~ 1

Author(s):

Dirk Winkler ◽

Christof Schneider ◽

Daniela Nitsch ◽

Annett Habermann ◽

Hartmut Doehner ◽

...

Keyword(s):

Cell Death ◽

High Risk ◽

Caspase 3 ◽

Annexin V ◽

Chemotherapeutic Agents ◽

Cross Linking ◽

Serum Free ◽

Induced Apoptosis ◽

In Vitro Treatment

Abstract Chemotherapeutic agents such as fludarabine, etoposide and the monoclonal anti-CD52 antibody alemtuzumab induce cell death and clinical responses in CLL. However, the mechanisms by which these processes occur are not well understood. In an effort to gain better insight into these mechanisms CLL cells from 21 patients were collected and individually treated with fludarabine 500μM (n=19) and etoposide 60 μM (n=19) for 24 and 48 hours respectively, and 24 hours with alemtuzumab 10 μg/ml ± cross-linking F(ab’)2 fragments. Each sample treated with alemtuzumab was also cultured with and without serum as a source of complement. Enrichment for B-cells was also done in 4 cases using negative selection with anti-CD2 and anti-CD14 magnetic beads. Of 18 cases investigated 10 were unmutated VH and 6 of 19 had del11q and/or del17p. A FACS analysis with double staining for Annexin V/7AAD was used to measure rates of cell death and caspase-3 activation. Results: treatment with fludarabine and etoposide induced apoptosis in all cases. However, rates of apoptosis decreased in cells from patients with genetically high-risk CLL, and these cells showed higher caspase-3 activation in response to fludarabine. Response to alemtuzumab was significantly dependent on presence of serum in the culture: 8% Annexin-V/7AAD-positive cells in serum-free cultures vs 53% in cultures with serum. However, addition of F(ab’)2 fragments increased the percentage of Annexin-V/7AAD-positive cells even in serum-free cultures: 61% with serum vs 25% without serum. Response to alemtuzumab was found to be independent of the genetic subgroup of the case. Notably, treatment with alemtuzumab in serum cultures did not produce cells that stained Annexin-positive/7AAD-negative, a typical feature of early apoptosis, whereas treatment with fludarabine, etoposide and alemtuzumab in serum-free medium resulted in a significant number of Annexin-positive/7AAD-negative cells. This was also observed in CD19+ purified cultures. In the presence of serum alemtuzumab did not induce caspase-3 activation, neither did the addition of F(ab’)2 fragments. However, in 5 of 20 serum-free cell cultures, all of had unmutated VH, active caspase-3 was clearly detectable after alemtuzumab treatment, and caspase-3 activity was further up-regulated when F(ab’)2 fragments were also added. Summary: in CLL cells mechanism and rate of cell death dramatically differed depending on in-vitro treatment with fludarabine, etoposide and alemtuzumab, and differed between genetic subgroups. CLL cells from high-risk patients were more capable of caspase-3 activation when treated with fludarabine or alemtuzumab. Alemtuzumab killed CLL cells effectively and independently of serum as a source of complement, but the mechanism of response was different when serum was added. In serum-free CLL cultures, alemtuzumab induced apoptosis with activation of caspase-3, and addition of cross-linking F(ab’)2 fragments increased the rate apoptosis, whereas in the presence of serum treatment with alemtuzumab induced no typical features of apoptosis, even in B-cell enriched cultures. These findings favor CDC rather than apoptosis or ADCC as the major cell kill mechanism activated by in vivo alemtuzumab. mean % of cells AnnexinV+/7AAD+ caspase-3 activation etoposide (48 hrs) fludarabine (48 hrs) fludarabine (48 hrs) IgVH unmutated 33% 24% 32% IgVH mutated 74% 38% 20% del 11q/del 17p 39% 25% 37% del 13q/normal karyotype 61% 32% 21%

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Abstract 380: Aging Enhances Endoplasmic Reticulum Stress-Induced Apoptosis in Murine Macrophages

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a380 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Daniel R Goldstein ◽

Yang Song

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Stress Proteins ◽

Fluorescent Microscopy ◽

Annexin V ◽

Peritoneal Macrophages ◽

Downstream Effector ◽

Induced Apoptosis ◽

Dependent Interaction

Introduction and hypothesis Aging enhances atherosclerosis for unclear reasons. As macrophage apoptosis and endoplasmic reticulum (ER) stress contribute to atherosclerosis, we examined if aging sensitizes these cells to apoptosis during ER stress. Methods and Results Peritoneal macrophages were isolated from young (aged 2-4 months) and aged (aged 16-18 months) mice, exposed to the ER stress inducer tunicamycin (TM) in vitro, and apoptosis was measured by Annexin V staining via fluorescent microscopy. We found that aged macrophages exhibited significantly more apoptosis than young macrophages (see Figure). We next measured key ER stress proteins in macrophages by Western blot to determine the underlying molecular pathways impacted by aging. With aging, we found reduced activation of inositol-requiring enzyme-1 (IRE1α), a key ER stress transducer. We next examined if augmenting activated IRE1α levels in aged macrophages reduced apoptosis during ER stress. We employed siRNA to knock down x-box binding protein 1 (XBP1), a downstream effector of IRE1α, which has been shown to induce feedback activation of IRE1α in hepatocytes. siRNA to XBP1 significantly reduced tunicamycin-induced cell apoptosis in aged macrophages from 26.1±0.408% to 5.48±1.38% (p<0.05) but not in young macrophages. Conclusions Our study has uncovered a novel, age-dependent interaction by which macrophages undergo apoptosis upon ER stress, and suggests that enhancing IRE1α activation will alleviate aging-augmented ER stress and subsequent apoptosis. This novel interaction may have important implications for the pathogenesis of atherosclerosis with aging.

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Xanthohumol Induces Apoptosis in B-CLL In Vitro by Sustained Endoplasmic Reticulum Stress and Inhibition of NFκ-B.

Blood ◽

10.1182/blood.v108.11.4979.4979 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4979-4979

Author(s):

Sofie Lust ◽

Barbara Vanhoecke ◽

Mireille Van Gele ◽

Mary Kaileh ◽

Jerina Boelens ◽

...

Keyword(s):

Flow Cytometry ◽

Endoplasmic Reticulum ◽

Cell Death ◽

Er Stress ◽

Caspase 3 ◽

Protein Translation ◽

Mrna Levels ◽

Induced Apoptosis ◽

Sustained Phosphorylation

Abstract Introduction Correct folding of new proteins is supervised in the endoplasic reticulum (ER) unfolded protein response (UPR). Misfolded proteins recruit the chaperone Grp78 that is thereby released from the transcription factors ATF6, IRE-1 leading to compensatory increase in Grp78, and PERK, leading to phosphorylation of eIF2α and block of further protein translation. UPR overload leads to ER stress and cell death. Targeting the endoplasmic reticulum (ER) is a new strategy explored in B-CLL. The hop-derived chalcone Xanthohumol (X) has been characterized as a ‘broad-spectrum’ cancer chemopreventive agent. Recently, we demonstrated that X induces dose- and time-dependent cell death of MCF7/6 breast cancer cells accompanied by ER stress. X induces apoptosis and cleavage of poly(ADP)-ribose-polymerase (PARP) in B-CLL in vitro. The present study investigates the branches of the UPR in relation to X induced apoptosis of B-CLL cells. Materials and methods. Lymphocytes were isolated by Lymphoprep from 15 patients with B-CLL after informed consent. CD19 positive cells were selected by EasySep positive selection kit. Apoptosis was assessed by flow-cytometry (AnnexinV-PI). Western Blotting was used for Grp78, ATF6, XBP1, phospho-eIF2a, eIF2a, ATF4, CHOP, phospho-IKK, IKK, PARP, caspase-9, -8, -7, -4, cleaved caspase-3, mcl-1, bcl-xL, bax, bak, and bid. NF-kB activity was assessed by EMSA. Quantitative RT-PCR was performed to analyze Grp78 mRNA levels. Bcl-2 protein level was detected by flow cytometry and reactive oxygen species (ROS) by fluorescence microscopy. Results and conclusion X induced an upregulation of Grp78 mRNA levels which was not translated in an increase in protein. X treatment stimulated a rapid and sustained phosphorylation of eIF2a, suggesting the involvement of PERK. In contrast, the ER-stress transducers ATF6 and IRE1 were not activated. X-induced ER stress was associated with strong induction of the pro-apoptotic protein CHOP and inhibition of the NF-kB pathway. Furthermore, the pro-apoptotic effect of X was accompanied by an accumulation of ROS, a downregulation of the anti-apoptotic proteins mcl-1, bcl-xL, bcl-2 and processing of caspase-3, -7 and -9.In conclusion, the chalcone X is capable of inducing cell death with down-regulation of bcl-2, mcl-1, bcl-xL, and activation of the caspase cascade. This is accompanied by ER-stress as evidenced by the upregulation of Grp78 mRNA levels, induction of a rapid and sustained phosphorylation of eIF2a, upregulation of CHOP, and inhibition of the NF-kB signaling.

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Xao tam phan (Paramignya trimera) methanol extract induced apoptosis in hepatocellular carcinoma HepG2 cell line in vitro

Science and Technology Development Journal ◽

10.32508/stdj.v23i1.2013 ◽

2020 ◽

Vol 23 (1) ◽

pp. 484-489

Author(s):

Sinh Truong Nguyen ◽

Nghia Minh Do ◽

Phuc Hong Vo ◽

Trinh Thi – Tu Nguyen ◽

Kiet Dinh Truong ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Flow Cytometry ◽

Hepg2 Cells ◽

Annexin V ◽

Ic50 Values ◽

Induced Apoptosis ◽

Index Value ◽

Nuclear Degradation ◽

Alamarblue Assay

Introduction: Xao Tam Phan (Paramignya trimera) has long been used in Viet Nam as an herbal medicine for the treatment of Hepatitis, hepatocellular carcinoma, and diabetes. This study aimed to determine the anti-proliferation effect of Paramignya trimera extract (P. trimera extract) on HepG2 hepatocellular carcinoma cells. Methods: AlamarBlue assay was used to determine the IC50 values of P. trimera extract on HepG2 cells. Adipose-derived stem cells (ADSCs) was used as normal cell control. For apoptosis examination, P. trimera extract-treated HepG2 cells were incubated with Annexin V/Propidium iodide (PI). Then they have been analyzed their expression of Annexin-V and PI by flow cytometry. The cell nuclear degradation also was evaluated by PI/Hoechst 33342 staining assay. Results: Doxorubicin and P. trimera extract IC50 values on HepG2 cells were 55.13 +/- 2.028 ng/ml and 582.533 +/- 16.521 mg/ml, respectively. Those on ADSCs were 5.96 +/- 0.56 ng/ml and 268.976 +/- 19.325 mg/ml, respectively. Side effect index value (SEI) of P. trimera extract was 2.175 +/- 0.12, and the SEI of doxorubicin was 8.71 +/- 0.36. Flow cytometry analysis indicated significant apoptosis on P. trimera extract-treated HepG2 cells at a dose of 500 mg/ml (32.39 +/- 2.28% apoptotic cells, and 14.63 +/- 1.59% necrotic cells). Nuclear aggregation and degradation was seen on 500 mg/ml P. trimera treated HepG2 cells. Conclusion: P. trimera extract could inhibit HepG2 hepatocellular carcinoma cell proliferation by inducing apoptosis.

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Action of inhibitors Heat shock proteins 90 and 27 on dexamethasone-induced apoptosis of tumor cells

Bulletin of Siberian Medicine ◽

10.20538/1682-0363-2010-3-68-71 ◽

2010 ◽

Vol 9 (3) ◽

pp. 68-71 ◽

Cited By ~ 1

Author(s):

Ye. V. Kaigorodova ◽

N. V. Ryazantseva ◽

V. V. Novitsky ◽

A. N. Maroshkina ◽

M. V. Belkina ◽

...

Keyword(s):

Cell Death ◽

Heat Shock ◽

Programmed Cell Death ◽

Heat Shock Protein ◽

Tumor Cells ◽

Propidium Iodide ◽

Fluorescent Microscopy ◽

Annexin V ◽

Induced Apoptosis ◽

Shock Proteins

Programmed cell death of tumor cells of line Jurkat in conditions of cultivation with various concentration of dexamethasone, selective inhibitors of Hsp90 (Heat shock protein — Hsp) (17-AAG) and Hsp27 (KRIBB3) was investigated. An estimation of realisation apoptosis spent by method of fluorescent microscopy with use annexin V-FITC and propidium iodide. Inhibition of Hsp90 and Hsp27 leads to activation of tumor cells Jurkat apoptotic program and strengthening of dexamethasone-induced apoptosis. Hsp27 and Hsр90 play an antiapoptotic role in tumor cells of line Jurkat.

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Poly-L-arginine: Enhancing Cytotoxicity and Cellular Uptake of Doxorubicin and Necrotic Cell Death

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180412114750 ◽

2019 ◽

Vol 18 (10) ◽

pp. 1448-1456 ◽

Cited By ~ 3

Author(s):

Bahareh Movafegh ◽

Razieh Jalal ◽

Zobeideh Mohammadi ◽

Seyyede A. Aldaghi

Keyword(s):

Cell Death ◽

Cellular Uptake ◽

Combined Treatment ◽

Annexin V ◽

Cell Penetrating Peptides ◽

Short Exposure ◽

Dependent Manner ◽

Du145 Cells ◽

Induced Apoptosis ◽

Resistance To Doxorubicin

Objective: Cell resistance to doxorubicin and its toxicity to healthy tissue reduce its efficiency. The use of cell-penetrating peptides as drug delivery system along with doxorubicin is a strategy to reduce its side effects. In this study, the influence of poly-L-arginine on doxorubicin cytotoxicity, its cellular uptake and doxorubicin-induced apoptosis on human prostate cancer DU145 cells are assessed. Methods: The cytotoxicity of doxorubicin and poly-L-arginine, alone and in combination, in DU145 cells was evaluated at different exposure times using MTT assay. The influence of poly-L-arginine on doxorubicin delivery into cells was evaluated by fluorescence microscopy and ultraviolet spectroscopy. DAPI and ethidium bromide- acridine orange stainings, flow cytometry using annexin V/propidium iodide, western blot analysis with anti-p21 antibody and caspase-3 activity were used to examine the influence of poly-L-arginine on doxorubicininduced cell death. Results: Poly-L-arginine had no cytotoxicity at low concentrations and short exposure times. Poly-L-arginine increased the cytotoxic effect of doxorubicin in DU145 cells in a time-dependent manner. But no significant reduction was found in HFF cell viability. Poly-L-arginine seems to facilitate doxorubicin uptake and increase its intracellular concentration. 24h combined treatment of cells with doxorubicin (0.5 µM) and poly-L-arginine (1 µg ml-1) caused a small increase in doxorubicin-induced apoptosis and significantly elevated necrosis in DU145 cells as compared to each agent alone. Conclusion: Our results indicate that poly-L-arginine at lowest and highest concentrations act as proliferationinducing and antiproliferative agents, respectively. Between these concentrations, poly-L-arginine increases the cellular uptake of doxorubicin and its cytotoxicity through induction of necrosis.

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iPLA2β Contributes to ER Stress-Induced Apoptosis during Myocardial Ischemia/Reperfusion Injury

Cells ◽

10.3390/cells10061446 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1446

Author(s):

Tingting Jin ◽

Jun Lin ◽

Yingchao Gong ◽

Xukun Bi ◽

Shasha Hu ◽

...

Keyword(s):

Cell Death ◽

Heart Disease ◽

Myocardial Ischemia ◽

Ischemic Heart Disease ◽

Er Stress ◽

Ischemia Reperfusion ◽

Myocardial Ischemia Reperfusion ◽

Induced Apoptosis

Both calcium-independent phospholipase A2 beta (iPLA2β) and endoplasmic reticulum (ER) stress regulate important pathophysiological processes including inflammation, calcium homeostasis and apoptosis. However, their roles in ischemic heart disease are poorly understood. Here, we show that the expression of iPLA2β is increased during myocardial ischemia/reperfusion (I/R) injury, concomitant with the induction of ER stress and the upregulation of cell death. We further show that the levels of iPLA2β in serum collected from acute myocardial infarction (AMI) patients and in samples collected from both in vivo and in vitro I/R injury models are significantly elevated. Further, iPLA2β knockout mice and siRNA mediated iPLA2β knockdown are employed to evaluate the ER stress and cell apoptosis during I/R injury. Additionally, cell surface protein biotinylation and immunofluorescence assays are used to trace and locate iPLA2β. Our data demonstrate the increase of iPLA2β augments ER stress and enhances cardiomyocyte apoptosis during I/R injury in vitro and in vivo. Inhibition of iPLA2β ameliorates ER stress and decreases cell death. Mechanistically, iPLA2β promotes ER stress and apoptosis by translocating to ER upon myocardial I/R injury. Together, our study suggests iPLA2β contributes to ER stress-induced apoptosis during myocardial I/R injury, which may serve as a potential therapeutic target against ischemic heart disease.

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