The Role of PNAS-2 in Apoptotic Pathway.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4092-4092
Author(s):  
Hai-Rong Wang ◽  
Hua Zhong ◽  
Ji-Hua Zhong ◽  
Fei Xiao ◽  
Jian-Yi Zhu ◽  
...  
Keyword(s):  
Fas Ligand ◽  
Death Receptor ◽  
Multivesicular Body ◽  
Intermembrane Space ◽  
Intrinsic Apoptotic Pathway ◽  
Lysosomal Degradation ◽  
Antibody Microarray ◽  
Apoptotic Pathway ◽  
Endonuclease G ◽  
No Activation

Abstract We recently demonstrated PNAS-2 serving as both an anti-apoptotic gene and an oncogene which is specifically up-regulated in acute leukemic patients (Hai-Rong WANG, et al. Oncology, Accepted). However, its role in apoptotic pathway remains elusive. Antibody microarray was applied to study the apoptosis-associated proteins’ changes to elucidate its role in apoptotic pathway. After PNAS-2 was inhibited by RNA interference, caspase3 was up-regulated confirming that PNAS-2 had an anti-apoptotic function. The activation of extrinsic, receptor-mediated apoptotic pathway was characterized by binding a death ligand to the corresponding death receptor. In this study, there were no changes in TNFα, CD95/Fas, TNFR, FADD, TRADD and caspase8 though Fas ligand decreased, while the apoptotic inhibitors such as DcR1 and FLIP were up-regulated, indicating there was no activation in the extrinsic apoptotic pathway. As to the apoptogenic proteins of classical intrinsic apoptotic pathway, there were no changes in Bax and caspase 9 though Bim was down-regulated, while the apoptotic inhibitors such as Bcl-2, Bcl-XL were up-regulated, indicating there was no activation in the intrinsic apoptotic pathway as well. Apart from the two classical apoptotic pathways (intrinsic and extrinsic), Granzyme B (GraB)-perforin apoptotic pathway is one well defined extrinsic accessory way of triggering apoptosis. In our data, GraB and Perforin were up-regulated, suggesting the GraB-Perforin apoptotic pathway was also activated. The apoptosis-induced factor (AIF) and Cathepsin D (CatD) were also up-regulated. AIF could induce apoptosis via a caspase-independent pathway, and Cat D could selectively escape AIF from mitochondria when translocated into the cytosol from lysosome, while other apoptogenic proteins resided in the intermembrane space of mitochondria such as cytochrome c and endonuclease G remained in the mitochondria (Nicolas Bidere, et al. JBC, 2003). Our data indicated that the AIF-induced-caspase-independent pathway was activated when PNAS-2 was inhibited. The changes in protein levels of CatD, AIF and GraB were confirmed by Western Blot. Mannose-6-Phosphate receptor (M6Pr) is the target receptor for GraB and CatD, mediating uptake of the proteases to form vesicles via endocytosis. This receptor also serves to transport newly synthesized GraB and CatD into the vesicles. The vesicles would form a structure named multivesicular body (MVB), which served to sort vesicles’ content destined for degradation or routing to the lysosome. CHMP5 (the alias of PNAS-2) located in MVB and lysosome, and the loss of CHMP5/PNAS-2 could inhibit lysosomal degradation (Jae-Hyuck Shim, et al. JCB, 2006). As GraB and CatD could trigger apoptosis when they were effluxed from the lysosome, we hypothesize that the overexpression of PNAS-2 in leukemic patients might execute its anti-apoptotic function by increasing the lysosomal degradation of apoptogenic factors in lysosome such as CatD and GraB. Figure Figure

Acquired Resistance to Tumor Necrosis Factor-Related Apoptosis- Inducing Ligand (TRAIL): Histone Deacetylase Inhibitors Resensitize TRAIL-Resistant Jurkat Acute Lymphocytic Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5032-5032
Author(s):  
Pavel Klener ◽  
Jan Molinsky ◽  
Tereza Simonova ◽  
Emanuel Necas ◽  
Ladislav Andera ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Fas Ligand ◽  
Death Receptor ◽  
Lymphocytic Leukemia ◽  
Jurkat Cells ◽  
Tnf Alpha ◽  
Apoptotic Pathway ◽  
Induced Apoptosis ◽  
Disc Formation

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a death-ligand from the TNF family. TRAIL induces programmed cell death by the cell-extrinsic p53-independent apoptotic pathway. A potential of TRAIL as cancer-specific therapeutic agent has been proposed and is preclinically and clinically tested. Development of TRAIL-resistant clones in the TRAIL-sensitive tumor cells may be a serious complication of TRAIL based cancer therapy. Jurkat acute lymphocytic leukemia cells are sensitive to TRAIL-induced apoptosis, as well as other apoptosis inducing ligands from TNF family, Fas and TNF-alpha. Jurkat cells express only one of the four receptors for TRAIL, death receptor 5 (DR5). Prolonged exposure of TRAIL-sensitive Jurkat cells to recombinant soluble TRAIL (1000 ng/mL) resulted in the establishment of three TRAIL-resistant (TR) Jurkat cell subclones, Jurkat TR1, TR2, and TR3. The Jurkat TR subclones were also resistant to TNF-alpha and Fas ligand, suggesting disruption of the extrinsic apoptotic pathway. TRAIL-resistant subclone TR1, but not TR2 and TR3, demonstrated decreased susceptibility to undergo apoptosis in response to histone-deacetylase inhibitors, valproic acid (VA), sodium butyrate (SB) and suberoylanilide hydroxamic acid (SAHA) and was resistant to fludarabine. Flow cytometry analysis showed Jurkat TR subclones had unchanged expression of cell surface death receptor DR5, Fas, and receptors for TNF-alpha, TNF-R1 and TNF-R2, compared to TRAIL-sensitive Jurkat cells. Analysis of death-inducing signaling complex (DISC) formation by immunoprecipitation (anti-TRAIL, anti-DR5) and subsequent western blotting (anti-caspase 8, anti-FADD) clearly demonstrated that the DISC formation in response to TRAIL binding to DR5 was significantly decreased in subclones TR2 and TR3, but remained unchanged in subclone TR1 compared to TRAIL-sensitive Jurkat cells. To gain further insight into potential molecular aletarations associated with acquired TRAIL resistance of Jurkat subclones, we measured gene expression of several key apoptotic regulators, including receptors for TRAIL, cFLIP, BCL2 family, IAP family, HSP family members in TRAIL-resistant and TRAIL-sensitive Jurkat cells and did not detect any significant (>2-fold) change. These results suggest acquired TRAIL resistance of Jurkat cells might be mediated by changes on the protein rather than mRNA level. We analyzed whether the TRAIL-resistant Jurkat cells could be resensitized to TRAIL-induced apoptosis by pretreatment with diverse inhibitors of important prosurvival pathways, including inhibitors of proteosynthesis (cycloheximid), inhibitors of transcription (actinomycin D), NFkB inhibitors (bortezomib, SN-50), PI3K-Akt-mTOR inhibitors (rapamycine, LY294002, Hsp90 inhibitor (17-AAG), cyclin-dependent kinase inhibitors (roscovitine), casein kinase II inhibitors (DRB), or histone deacetylase inhibitors (HDACi: SAHA, VA, SB). Pretreatment with HDAC inhibitors for 12 hour was able to resensitize all three TRAIL-resistant Jurkat subclones to TRAIL-induced apoptosis. The percentage of apoptotic cells of HDACi-pretreated subclones was 70–95% 24 h after the exposure to TRAIL compared to 5–15% apoptosis for HDACi-untreated TRAIL-exposed controls, and to 10–15% apoptosis for HDACi-treated TRAIL unexposed controls. We established TRAIL-resistant subclones from the original TRAIL-sensitive Jurkat cells. Acquired resistance to TRAIL was not mediated by downregulation of TRAIL death receptor DR5 and was associated with (cross)resistance to TNFa and Fas ligand, suggesting disruption of cell-extrinsic apoptotic pathway. We assume diverse molecular mechanisms were involved in the development of TRAIL-resistant subclones upon exposure to TRAIL, as exemplified by disrupted formation of DISC in case of subclones TR2 and TR3 and normal DISC formation and fludarabine resistance in subclone TR1, suggesting deregulated apoptotic pathway downstream of DISC. Finally, we observed that HDACi resensitized the TRAIL-resistant subclones to TRAIL. The results provide substantiation for combinatorial approaches in the potential TRAIL-based therapies of hematological malignancies.

scholarly journals Caspase-8 inactivation in T cells increases necroptosis and suppresses autoimmunity in Bim−/− mice

2011 ◽  
Vol 195 (2) ◽  
pp. 277-291 ◽  
Author(s):  
Toshiyuki Bohgaki ◽  
Julien Mozo ◽  
Leonardo Salmena ◽  
Elzbieta Matysiak-Zablocki ◽  
Miyuki Bohgaki ◽  
...  
Keyword(s):  
T Cells ◽  
Death Receptor ◽  
Death Process ◽  
Caspase 8 ◽  
Intrinsic Apoptotic Pathway ◽  
Immune Disorders ◽  
Loss Of Function ◽  
T Cell Homeostasis ◽  
Apoptotic Pathway ◽  
Cell Death Process

Dysregulation of either the extrinsic or intrinsic apoptotic pathway can lead to various diseases including immune disorders and cancer. In addition to its role in the extrinsic apoptotic pathway, caspase-8 plays nonapoptotic functions and is essential for T cell homeostasis. The pro-apoptotic BH3-only Bcl-2 family member Bim is important for the intrinsic apoptotic pathway and its inactivation leads to autoimmunity that is further exacerbated by loss of function of the death receptor Fas. We report that inactivation of caspase-8 in T cells of Bim−/− mice restrained their autoimmunity and extended their life span. We show that, similar to caspase-8−/− T cells, Bim−/− T cells that also lack caspase-8 displayed elevated levels of necroptosis and that inhibition of this cell death process fully rescued the survival and proliferation of these cells. Collectively, our data demonstrate that inactivation of caspase-8 suppresses the survival and proliferative capacity of Bim−/− T cells and restrains autoimmunity in Bim−/− mice.

scholarly journals Cerebral Cortex Apoptosis in Early Aged Hypertension: Effects of Epigallocatechin-3-Gallate

2021 ◽  
Vol 13 ◽  
Author(s):  
Min-Huang Hsieh ◽  
Zhen-Yang Cui ◽  
Ai-Lun Yang ◽  
Nguyen Thanh Nhu ◽  
Shih-Ying Ting ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Green Tea ◽  
Fas Ligand ◽  
Therapeutic Effects ◽  
Apoptotic Pathway ◽  
Egcg Treatment ◽  
Endonuclease G ◽  
Survival Pathway ◽  
Wistar Kyoto ◽  
Apoptotic Pathways

This study aimed to investigate cerebral cortex apoptosis on the early aged hypertension and the effects of green tea flavonoid epigallocatechin-3-gallate (EGCG). Twenty-four rats were divided into three groups: a control Wistar-Kyoto group (WKY, n = 8), a spontaneously early aged hypertensive group (SHR, n = 8), and an early aged hypertension with EGCG treatment group (SHR-EGCG, n = 8; daily oral EGCG 200 mg/kg—94%, 12 weeks). At 48 weeks old, blood pressures (BPs) were evaluated and cerebral cortexes were isolated for TUNEL assay and Western blotting. Systolic, diastolic, and mean blood pressure levels in the SHR-EGCG were reduced compared to the SHR. The percentage of neural cell deaths, the levels of cytosolic Endonuclease G, cytosolic AIF (Caspase-independent apoptotic pathway), Fas, Fas Ligand, FADD, Caspase-8 (Fas-mediated apoptotic pathway), t-Bid, Bax/Bcl-2, Bak/Bcl-xL, cytosolic Cytochrome C, Apaf-1, Caspase-9 (Mitochondrial-mediated apoptotic pathway), and Caspase-3 (Fas-mediated and Mitochondria-mediated apoptotic pathways) were increased in the SHR relative to WKY and reduced in SHR-EGCG relative to SHR. In contrast, the levels of Bcl-2, Bcl-xL, p-Bad, 14-3-3, Bcl-2/Bax, Bcl-xL/Bak, and p-Bad/Bad (Bcl-2 family-related pro-survival pathway), as well as Sirt1, p-PI3K/PI3K and p-AKT/AKT (Sirt1/PI3K/AKT-related pro-survival pathway), were reduced in SHR relative WKY and enhanced in SHR-EGCG relative to SHR. In conclusion, green tea flavonoid epigallocatechin-3-gallate (EGCG) might prevent neural apoptotic pathways and activate neural survival pathways, providing therapeutic effects on early aged hypertension-induced neural apoptosis.

Involvement of Fas Signalling in 7β-Hydroxycholesterol-and Cholesterol-5β,6β-Epoxide-Induced Apoptosis

2008 ◽  
Vol 27 (3) ◽  
pp. 279-285 ◽  
Author(s):  
Sinéad Lordan ◽  
John J. Mackrill ◽  
Nora M. O’Brien
Keyword(s):  
Arterial Wall ◽  
Caspase 3 ◽  
Fas Ligand ◽  
Specific Interaction ◽  
Death Receptor ◽  
Critical Event ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

The induction of apoptosis in cells of the arterial wall is a critical event in the development of atheroma. 7 β-Hydroxycholesterol (7 β-OH) and cholesterol-5 β,6 β-epoxide ( β-epoxide) are components of oxLDL and have previously been shown to be potent inducers of apoptosis. However, the exact mechanisms through which these oxysterols induce apoptosis remains to be fully elucidated. The specific interaction of the Fas death receptor with Fas ligand (FasL) initiates a caspase cascade culminating in apoptosis. The purpose of the present study was to determine the involvement of Fas signalling in 7 β-OH- and β-epoxide-induced apoptosis. To this end we employed the Fas/FasL antagonist, Kp7-6, and examined the effect of Fas inhibition on oxysterol-induced cell death in U937 cells. Fas levels were increased following 24 h exposure to 30 μM 7 β-OH while treatment with 30 μM β-epoxide had no effect. Kp7-6 reduced the Fas content of 7 β-OH-treated cells to control levels and partially protected against 7 β-OH-induced apoptosis. This coincided with a decrease in cytochrome c release along with a reduction in caspase-3 and caspase-8 activity. Our data implicate Fas signalling in the apoptotic pathway induced by 7 β-OH and also highlight differences between apoptosis induced by 7 β-OH and β-epoxide.

PNAS-2’s Subcullar Location in Leukemic Cell Is Abnormal and Its Relation with Leukemogenesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4299-4299
Author(s):  
Hai-Rong Wang ◽  
Fang-Yuan Chen ◽  
Hua Zhong ◽  
Ji-Hua Zhong ◽  
Fei Xiao ◽  
...  
Keyword(s):  
Structure Prediction ◽  
De Novo ◽  
Secondary Structure Prediction ◽  
Granzyme B ◽  
Leukemic Cell ◽  
Multivesicular Body ◽  
Leukemic Cells ◽  
Antibody Microarray ◽  
Apoptotic Pathway ◽  
Apoptotic Gene

Abstract We used functional analyses of PNAS-2 by RNAi and RNA overexpression and demonstrated PNAS-2 as an anti-apoptotic gene. PNAS-2 expression was significantly increased in de novo and relapsed acute leukemia patients, but in patients in complete remission, the expression of PNAS-2 decreased to the levels comparable to those found in normal controls. In carcinomas, PNAS-2 expression was not up-regulated, indicating that PNAS-2 overexpression was specific for leukemia. So we hypothesized that PNAS-2 could act as an oncogene and anti-apoptotic gene which probably participates leukemogenesis (Hai-Rong WANG, et al. Oncology, Accepted). To study the subcellular localization of PNAS protein, we constructed PNAS-2-GFP expression plasmids and transfected them into U937 leukemic cell line. We found that PNAS-2-GFP fusion protein not only located in vesicles near the nucleus but also distributed throughout the cytosol instead of Diane’s report that CHMP5/PNAS-2 was found in vesicles near the nucleus in Cos-7 cells. This phenomenon indicates the abnormal location of PNAS-2 protein either in U937 leukemia cells or in Cos-7 cells. For the protein classification and secondary structure prediction system: SOSUI showed that PNAS-2 wasn’t a membranal protein and the function of CHMP5/PNAS-2 protein was involved in the lysosome and MVB (multivesicular body), so PNAS-2 should be a protein in the interior of the MVB and lysosome, thus abnormal location of PNAS-2 protein might occur in U937 leukemic cells. From our data and related articles, PNAS-2 served as an anti-apoptotic gene and located in MVB and lysosome. We suggested that PNAS-2 might execute its anti-apoptotic function via following ways: it might be a protease which could degrade apoptogenic factors such as CatD, GraB and perforin in MVB and lysosome; or it could return CatD, GraB and perforin to the lysosome; or it could stabilize the membrane of MVB and lysosome. When PNAS-2 was set free into the cytosol from MVB structure in leukemic cells, it would enhance its anti-apoptotic function via enhanced above-mentioned ways. Figure Figure Now compelling evidences showed that insufficient apoptosis could result in tumor genesis, thus we thought abnormal subcellular location of PNAS-2 in leukemic cells might participate in leukemogenesis. Antibody microarray was used to study the protein changes after inhibition of PNAS-2, we found cancer suppressor proteins such as p53, Rb increased after PNAS-2 was suppressed, while the rest cancer suppressor proteins did not change, indicating the overexpression of PNAS-2 might participate in tumorigeness by suppressing tumor suppressor proteins. Of notice, Granzyme B-perforin apoptotic pathway was inhibited when PNAS-2 was up-regulated. Granzyme-mediated apoptosis was part of the immune surveillance mechanism where malignantly transformed cells were targeted by CTL and NK cells before cancer could ensue. In conclusion, the abnormal location of PNAS-2 in leukemic cells could enhance its anti-apoptotic function. PNAS-2 might participate in leukemogenesis via its inhibition of apoptosis, cancer suppressor proteins and Granzyme B-perforin pathway.

Activation of the intrinsic-apoptotic pathway in LNCaP prostate cancer cells by genistein- topotecan combination treatments

2013 ◽  
Vol 3 (3) ◽  
pp. 66 ◽  
Author(s):  
Vanessa Hörmann ◽  
Sivanesan Dhandayuthapani ◽  
James Kumi-Diaka ◽  
Appu Rathinavelu
Keyword(s):  
Prostate Cancer ◽  
Cell Death ◽  
Cancer Cells ◽  
Apoptotic Cell ◽  
Treatment Options ◽  
Attractive Alternative ◽  
Intrinsic Apoptotic Pathway ◽  
Prostate Cancer Cells ◽  
Apoptotic Pathway ◽  
Combination Treatments

Background: Prostate cancer is the second most common cancer in American men. The development of alternative preventative and/or treatment options utilizing a combination of phytochemicals and chemotherapeutic drugs could be an attractive alternative compared to conventional carcinoma treatments. Genistein isoflavone is the primary dietary phytochemical found in soy and has demonstrated anti-tumor activities in LNCaP prostate cancer cells. Topotecan Hydrochloride (Hycamtin) is an FDA-approved chemotherapy for secondary treatment of lung, ovarian and cervical cancers. The purpose of this study was to detail the potential activation of the intrinsic apoptotic pathway in LNCaP prostate cancer cells through genistein-topotecan combination treatments. Methods: LNCaP cells were cultured in complete RPMI medium in a monolayer (70-80% confluency) at 37ºC and 5% CO2. Treatment consisted of single and combination groups of genistein and topotecan for 24 hours. The treated cells were assayed for i) growth inhibition through trypan blue exclusion assay and microphotography, ii) classification of cellular death through acridine/ ethidium bromide fluorescent staining, and iii) activation of the intrinsic apoptotic pathway through Jc-1: mitochondrial membrane potential assay, cytochrome c release and Bcl-2 protein expression.Results: The overall data indicated that genistein-topotecan combination was significantly more efficacious in reducing the prostate carcinoma’s viability compared to the single treatment options. In all treatment groups, cell death occurred primarily through the activation of the intrinsic apoptotic pathway.Conclusion: The combination of topotecan and genistein has the potential to lead to treatment options with equal therapeutic efficiency as traditional chemo- and radiation therapies, but lower cell cytotoxicity and fewer side effects in patients. Key words: topotecan; genistein; intrinsic apoptotic cell death

scholarly journals sFasL—The Key to a Riddle: Immune Responses in Aging Lung and Disease

2021 ◽  
Vol 22 (4) ◽  
pp. 2177
Author(s):  
Shulamit B. Wallach-Dayan ◽  
Dmytro Petukhov ◽  
Ronit Ahdut-HaCohen ◽  
Mark Richter-Dayan ◽  
Raphael Breuer
Keyword(s):  
Cell Death ◽  
Lung Disease ◽  
Fas Ligand ◽  
Death Receptor ◽  
Mesenchymal Cells ◽  
Soluble Form ◽  
Cell Accumulation ◽  
Key Factor ◽  
Aged Subjects

By dint of the aging population and further deepened with the Covid-19 pandemic, lung disease has turned out to be a major cause of worldwide morbidity and mortality. The condition is exacerbated when the immune system further attacks the healthy, rather than the diseased, tissue within the lung. Governed by unremittingly proliferating mesenchymal cells and increased collagen deposition, if inflammation persists, as frequently occurs in aging lungs, the tissue develops tumors and/or turns into scars (fibrosis), with limited regenerative capacity and organ failure. Fas ligand (FasL, a ligand of the Fas cell death receptor) is a key factor in the regulation of these processes. FasL is primarily found in two forms: full length (membrane, or mFasL) and cleaved (soluble, or sFasL). We and others found that T-cells expressing the mFasL retain autoimmune surveillance that controls mesenchymal, as well as tumor cell accumulation following an inflammatory response. However, mesenchymal cells from fibrotic lungs, tumor cells, or cells from immune-privileged sites, resist FasL+ T-cell-induced cell death. The mechanisms involved are a counterattack of immune cells by FasL, by releasing a soluble form of FasL that competes with the membrane version, and inhibits their cell death, promoting cell survival. This review focuses on understanding the previously unrecognized role of FasL, and in particular its soluble form, sFasL, in the serum of aged subjects, and its association with the evolution of lung disease, paving the way to new methods of diagnosis and treatment.

Role of testosterone in the intrinsic apoptotic pathway of skeletal muscle

Bone ◽  
2011 ◽  
Vol 48 (6) ◽  
pp. S288
Author(s):  
L. Pronsato ◽  
R. Boland ◽  
L. Milanesi
Keyword(s):  
Skeletal Muscle ◽  
Intrinsic Apoptotic Pathway ◽  
Apoptotic Pathway
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