Apaf-1 and caspase-9 are required for cytokine withdrawal-induced apoptosis of mast cells but dispensable for their functional and clonogenic death

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 1872-1877 ◽  
Author(s):  
Vanessa S. Marsden ◽  
Thomas Kaufmann ◽  
Lorraine A. O'Reilly ◽  
Jerry M. Adams ◽  
Andreas Strasser
Keyword(s):  
Mast Cells ◽  
Fetal Liver ◽  
Wild Type ◽  
Caspase 9 ◽  
Receptor Stimulation ◽  
Apoptotic Pathway ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
Cytokine Deprivation

Cytokines promote survival of mast cells by inhibiting apoptotic pathways regulated by the Bcl-2 protein family. We previously showed that lymphocyte apoptosis can proceed via a Bcl-2-inhibitable pathway independent of the canonical initiator caspase, caspase-9, and its adaptor, Apaf-1. Here we report that mast cells lacking caspase-9 or Apaf-1 are refractory to apoptosis after cytotoxic insults but still lose effector function and ability to proliferate. In response to cytokine deprivation or DNA damage, fetal liver-derived mast cells lacking Apaf-1 or caspase-9 failed to undergo apoptosis. Nevertheless, the cytokine-starved cells were not functionally alive, because, unlike those overexpressing Bcl-2, they could not degranulate on Fcϵ receptor stimulation or resume proliferation on re-addition of cytokine. Furthermore, mast cells lacking Apaf-1 or caspase-9 had no survival advantage over wild-type counterparts in vivo. These results indicate that the Apaf-1/caspase-9-independent apoptotic pathway observed in lymphocytes is ineffective in cytokine-deprived mast cells. However, although Apaf-1 and caspase-9 are essential for mast cell apoptosis, neither is required for the functional or clonogenic death of the cells, which may be due to mitochondrial dysfunction.

scholarly journals The BH3-only protein Puma plays an essential role in cytokine deprivation–induced apoptosis of mast cells

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3209-3217 ◽  
Author(s):  
Maria Ekoff ◽  
Thomas Kaufmann ◽  
Maria Engström ◽  
Noboru Motoyama ◽  
Andreas Villunger ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Essential Role ◽  
Wild Type ◽  
Cell Numbers ◽  
Growth Factor Deprivation ◽  
Induced Apoptosis ◽  
Cytokine Deprivation

Abstract Mast cells play critical roles in the regulation of inflammation. One characteristic feature of mast cells is their relatively long lifespan in vivo. Members of the Bcl-2 protein family are regulators of cell survival and apoptosis, where the BH3-only proteins are critical proapoptotic proteins. In this study we investigated the role of the BH3-only proteins Noxa, Bad, Bim, Bmf, Bid, and Puma in apoptosis of mucosal-like mast cells (MLMCs) and connective tissue–like mast cells (CTLMCs). We demonstrate that Puma is critical for the induction of mast-cell death following cytokine deprivation and treatment with the DNA-damaging agent etoposide in MLMCs and CTLMCs. Using p53−/− mast cells, we found that cytokine deprivation–induced apoptosis, in contrast to that elicited by etoposide, is p53-independent. Interestingly, mast cells deficient in FOXO3a, previously proposed as a transcription factor for Puma induction in response to growth factor deprivation, were markedly resistant to cytokine withdrawal compared with wild-type cells. Moreover, overexpression of phosphorylation-deficient, constitutively active FOXO3a caused an up-regulation of Puma. In conclusion, our data demonstrate a pivotal role for Puma in the regulation of cytokine deprivation–induced mast-cell apoptosis and suggest a plausible role for Puma in the regulation of mast cell numbers in vivo.

scholarly journals Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo

2007 ◽  
Vol 408 (2) ◽  
pp. 181-191 ◽  
Author(s):  
Potchanapond Graidist ◽  
Michio Yazawa ◽  
Moltira Tonganunt ◽  
Akiko Nakatomi ◽  
Curtis Chun-Jen Lin ◽  
...  
Keyword(s):  
Biological Significance ◽  
Wild Type ◽  
Apoptotic Activity ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
The Impact ◽  
Overlay Assay ◽  
Dissociation Constant Kd ◽  
Flow Dialysis

Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1–72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 μM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.

scholarly journals Anti-Proliferative and Pro-Apoptotic Activities of Hederacolchiside A1 On MCF-7 Cells Via ROS Regulation and JAK2/STAT3 Inactivation

2021 ◽  
Author(s):  
Aijun Chen ◽  
Shushu Zhang ◽  
Dandan Zhang ◽  
Xingjiang Hu ◽  
Nana Xu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Ros Generation ◽  
Caspase 9 ◽  
Apoptotic Pathway ◽  
Hct 116 ◽  
Hct 116 Cells ◽  
Induced Apoptosis ◽  
Mcf 7

Abstract Many studies have shown that hederacolchiside A1 (HA1) is an important anticancer saponin, although its mechanism of action and in vivo investigations are still lacking. Our previous results revealed that HA1 may have the potential to treat breast cancer. Therefore, we attempted to verify the potential anti-breast cancer effect of HA1 in vitro and in vivo. MTT, flow cytometry, DCFH-DA fluorescence microscopy, and western blotting were used to evaluate the activities and mechanisms of action of HA1. Athymic nude mice were used to demonstrate the antitumor activity of HA1 in vivo. HA1 exhibited significant cytotoxic effects on HepG2, MCF-7, MDA-MB-231, SKBr-3, HT-29, and HCT-116 cells, especially MCF-7 cells. HA1 blocked the sub-G1 and G0/G1 phases, induced apoptosis, promoted reactive oxygen species (ROS) generation, and decreased the mitochondrial membrane potential of MCF-7 cells. HA1 upregulated Bax and downregulated Bcl-2 levels and activated caspase-9 and caspase-3 in MCF-7 cells Meanwhile, HA1 inhibited the phosphorylation of JAK2/STAT3 in MCF-7 cells. In addition, 50 and 100 mg/kg HA1 significantly inhibited the growth of transplanted tumors with inhibition rates of 46.95 ± 26.72% and 48.45 ± 22.36%, respectively. This preliminary study demonstrated that HA1 could inhibit proliferation and induce the apoptosis of MCF-7 cells via ROS-mediated activation of the mitochondrial apoptotic pathway and JAK2/STAT3 inactivation. HA1 may therefore be developed as a novel agent for breast cancer therapy.

Analysis of mouse LMIR5/CLM-7 as an activating receptor: differential regulation of LMIR5/CLM-7 in mouse versus human cells

Blood ◽  
2008 ◽  
Vol 111 (2) ◽  
pp. 688-698 ◽  
Author(s):  
Yoshinori Yamanishi ◽  
Jiro Kitaura ◽  
Kumi Izawa ◽  
Takayuki Matsuoka ◽  
Toshihiko Oki ◽  
...  
Keyword(s):  
Mast Cells ◽  
Cytokine Production ◽  
Fetal Liver ◽  
Human Cells ◽  
Cross Linking ◽  
Wild Type ◽  
Predominant Role ◽  
Innate Immunity Cells ◽  
Production Cell ◽  
Activating Receptor

We have analyzed leukocyte mono-Ig–like receptor 5 (LMIR5) as an activating receptor among paired LMIRs. Mouse LMIR5 (mLMIR5) is expressed in myeloid cells such as mast cells, granulocytes, macrophages, and dendritic cells. Cross-linking of transduced mLMIR5 in bone marrow–derived mast cells (BMMCs) caused activation events, including cytokine production, cell survival, degranulation, and adhesion to the extracellular matrix. mLMIR5 associated with DAP12 and to a lesser extent with DAP10, and mLMIR5-mediated functions of BMMCs were strongly inhibited by DAP12 deficiency. Importantly, cross-linking of endogenous mLMIR5 induced Syk-dependent activation of fetal liver–derived mast cells. Unlike mLMIR5, cross-linking of human LMIR5 (hLMIR5) induced cytokine production of BMMCs even in the absence of both DAP12 and DAP10, suggesting the existence of unidentified adaptors. Interestingly, hLMIR5 possessed a tyrosine residue (Y188) in the cytoplasmic region. Signaling via Y188 phosphorylation played a predominant role in hLMIR5-mediated cytokine production in DAP12-deficient, but not wild-type BMMCs. In addition, experiments using DAP10/DAP12 double-deficient BMMCs suggested the existence of Y188 phoshorylation-dependent and -independent signals from unidentified adaptors. Collectively, although both mouse and human LMIR5 play activatory roles in innate immunity cells, the functions of LMIR5 were differentially regulated in mouse versus human cells.

scholarly journals MDM2 Suppresses p73 Function without Promoting p73 Degradation

1999 ◽  
Vol 19 (5) ◽  
pp. 3257-3266 ◽  
Author(s):  
Xiaoya Zeng ◽  
Lihong Chen ◽  
Christine A. Jost ◽  
Ruth Maya ◽  
David Keller ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Feedback Loop ◽  
Human Lung ◽  
Activation Function ◽  
Wild Type ◽  
Mdm2 Protein ◽  
Induced Apoptosis ◽  
P53 Inactivation

ABSTRACT The newly identified p53 homolog p73 can mimic the transcriptional activation function of p53. We investigated whether p73, like p53, participates in an autoregulatory feedback loop with MDM2. p73 bound to MDM2 both in vivo and in vitro. Wild-type but not mutant MDM2, expressed in human p53 null osteosarcoma Saos-2 cells, inhibited p73- and p53-dependent transcription driven by the MDM2 promoter-derived p53RE motif as measured in transient-transfection and chloramphenicol acetyltransferase assays and also inhibited p73-induced apoptosis in p53-null human lung adenocarcinoma H1299 cells. MDM2 did not promote the degradation of p73 but instead disrupted the interaction of p73, but not of p53, with p300/CBP by competing with p73 for binding to the p300/CBP N terminus. Both p73α and p73β stimulated the expression of the endogenous MDM2 protein. Hence, MDM2 is transcriptionally activated by p73 and, in turn, negatively regulates the function of this activator through a mechanism distinct from that used for p53 inactivation.

Distinct Apoptotic Responses Imparted by c-myc and max

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 1003-1010 ◽  
Author(s):  
Chadd E. Nesbit ◽  
Saijun Fan ◽  
Hong Zhang ◽  
Edward V. Prochownik
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Ectopic Expression ◽  
Drug Induced ◽  
Enhanced Sensitivity ◽  
Contrast Enhanced ◽  
Apoptotic Pathways ◽  
American Society ◽  
Induced Apoptosis ◽  
Cytokine Deprivation

Abstract The c-myc oncoprotein accelerates programmed cell death (apoptosis) after growth factor deprivation or pharmacological insult in many cell lines. We have shown that max, the obligate c-myc heterodimeric partner protein, also promotes apoptosis after serum withdrawal in NIH3T3 fibroblasts or cytokine deprivation in interleukin-3 (IL-3)-dependent 32D murine myeloid cells. We now show that c-myc– and max-overexpressing 32D cells differ in the nature of their apoptotic responses after IL-3 removal or treatment with chemotherapeutic compounds. In the presence of IL-3, c-myc overexpression enhances the sensitivity of 32D cells to Etoposide (Sigma, St Louis, MO), Adriamycin (Pharmacia, Columbus, OH), and Camptothecin (Sigma), whereas max overexpression increases sensitivity only to Camptothecin. Drug treatment of c-myc–overexpressing cells in the absence of IL-3 did not alter the spectrum of drug sensitivity other than to additively accelerate cell death. In contrast, enhanced sensitivity to Adriamycin, Etoposide, and Taxol (Bristol-Meyers Squibb, Princeton, NJ) was revealed in max-overexpressing cells concurrently deprived of IL-3. Differential rates of apoptosis were not strictly correlated with the ability of the drugs to promote G1 or G2/M arrest. Ectopic expression of Bcl-2 or Bcl-XL blocked drug-induced apoptosis in both cell lines. In contrast, whereas Bcl-2 blocked apoptosis in both cell lines in response to IL-3 withdrawal, Bcl-XL blocked apoptosis in max-overexpressing cells but not in c-myc–overexpressing cells. These results provide mechanistic underpinnings for the idea that c-myc and max modulate distinct apoptotic pathways. © 1998 by The American Society of Hematology.

scholarly journals Apigenin Suppresses Angiogenesis by Inhibiting Tube Formation and Inducing Apoptosis

2016 ◽  
Vol 11 (10) ◽  
pp. 1934578X1601101
Author(s):  
Hyun Ju Kim ◽  
Mok-Ryeon Ahn
Keyword(s):  
Umbilical Vein ◽  
Chorioallantoic Membrane ◽  
Tube Formation ◽  
Anticancer Activities ◽  
Apoptotic Pathway ◽  
Inhibition Of Angiogenesis ◽  
Induced Apoptosis ◽  
Umbilical Vein Endothelial Cells

Apigenin has been reported to exert angiogenic and anticancer activities in vitro. The mechanism of inhibition of angiogenesis by apigenin, however, has not been well-established. In this study, we investigated whether apigenin not only inhibited tube formation but also induced apoptosis in human umbilical vein endothelial cells (HUVECs). Furthermore, strong antiangiogenic activity of apigenin was observed in the in vivo assay using chick embryo chorioallantoic membrane (CAM). We also analyzed changes in survival signals and the apoptotic pathway through Western blotting. The results indicate that apigenin exerts its antiangiogenic effects through induction of endothelial apoptosis.

scholarly journals L-securinine inhibits cell growth and metastasis of human androgen-independent prostate cancer DU145 cells via regulating mitochondrial and AGTR1/MEK/ERK/STAT3/PAX2 apoptotic pathways

2019 ◽  
Vol 39 (5) ◽  
Author(s):  
Dongxu Zhang ◽  
Houxian Liu ◽  
Binbin Yang ◽  
Jiasheng Hu ◽  
Yue Cheng
Keyword(s):  
Prostate Cancer ◽  
Protein Expression ◽  
Annexin V ◽  
Inhibitory Effect ◽  
Apoptotic Pathway ◽  
Growth Inhibitory ◽  
Du145 Cells ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
Androgen Independent

Abstract The present study aims to evaluate the anticancer effect of L-securinine on androgen-independent prostate cancer (AIPC) DU145 cells. L-securinine (2.5, 5, and 10 μM) treatment for 24, 48 and 72 h displayed strong growth inhibitory effect on DU145 cells in a concentration and time-dependent fashion but has less toxicity toward normal androgen-dependent LNCaP cells. Hoechst 332582 staining of DU145 cells and Annexin V-FITC/ PI dual-labeling followed by flow cytometry assay identified that this growth inhibition by L-securinine would be due to the induction of apoptosis. Moreover Transwell assay revealed that L-securinine significantly inhibited the cell migration/invasion ability of DU145 cells. Furthermore, results of western blotting showed that the involvement of mitochondrial apoptotic pathway in the L-securinine-induced apoptosis of DU145 cell, as evidenced by an increase in the protein expression of Bax, cleaved caspase-9, cleaved caspase-3, cytosolic cytochrome c, and cleaved PARP, together with a unchanged cleaved caspase-8 and decreased Bcl-2 protein expression. Also, L-securinine-induced antimetastatic activity in DU145 cells was associated with decreased protein expression of MMP-2 and MMP-9 and concurrent reduction of VEGF. In addition, further studies revealed that L-securinine may inhibit the protein expression of AGTR1, p-MEK1/2, p-ERK1/2, p-STAT3, PAX2, and p-PAX2, while the expression of ERK1/2, MEK1/2, and STAT3 protein retains intact. These findings suggest that L-securinine may be a promising chemopreventive agent against AIPC.

scholarly journals Topical 2′-Hydroxyflavanone for Cutaneous Melanoma

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1556 ◽  
Author(s):  
Bose ◽  
Singh ◽  
Igid ◽  
Green ◽  
Singhal ◽  
...  
Keyword(s):  
Cell Lines ◽  
Topical Therapy ◽  
Systemic Absorption ◽  
Caspase 9 ◽  
Epidermal Growth ◽  
Dietary Flavonoid ◽  
Parp1 Inhibitors ◽  
Induced Apoptosis ◽  
Immunocompetent Mice

2′-hydroxyflavanone (2HF) is a dietary flavonoid with anticancer activity towardsmultiple cancers. Here, we report that topically applied 2HF inhibits the growth of intradermalimplants of melanoma in immunocompetent mice. 2HF induced apoptosis and inhibited the growthof the human SK-MEL-24 as well as murine B16-F0 and B16-F10 melanoma cell lines in vitro.Apoptosis was associated with depletion of caspase-3, caspase-9, and PARP1 in B16-F0 and SKMEL-24 cells. Caspase-9 and MEKK-15 were undetected even in untreated B16-F10 cells. Signalingproteins TNFα, and phospho-PDGFR-β were depleted in all three cell lines; MEKK-15 was depletedby 2HF in SK-MEL-24 cells. 2HF enhanced sunitinib (an MEK and PDGFR-β inhibitor) and AZD2461 (a PARP1 inhibitor) cytotoxicity. 2HF also depleted the Ral-regulated, stress-responsive,antiapoptotic endocytic protein RLIP76 (RALBP1), the inhibition of which has previously beenshown to inhibit B16-F0 melanoma growth in vivo. Functional inhibition of RLIP76 was evidentfrom inhibition of epidermal growth factor (EGF) endocytosis by 2HF. We found that topicallyapplied 2HF–Pluronic Lecithin Organogel (PLO) gel inhibited B16-F0 and B16-F10 tumorsimplanted in mice and caused no overt toxicity despite significant systemic absorption. 2HFtreatment reduced phospho-AKT, vimentin, fibronectin, CDK4, cyclinB1, and BCL2, whereas itincreased BIM and phospho-AMPK in excised tumors. Several cancer signals are controlled byendocytosis, a process strongly inhibited by RLIP76 depletion. We conclude that 2HF–PLO gel maybe useful for topical therapy of cutaneous metastases of melanoma and could enhance theantineoplastic effects of sunitinib and PARP1 inhibitors. The mechanism of action of 2HF inmelanoma overlaps with RLI76 inhibitors.

Identification of apoptotic genes mediating TGF-β/Smad3-induced cell death in intestinal epithelial cells using a genomic approach

2007 ◽  
Vol 292 (1) ◽  
pp. G28-G38 ◽  
Author(s):  
Yanna Cao ◽  
Lu Chen ◽  
Weili Zhang ◽  
Yan Liu ◽  
Harry T. Papaconstantinou ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Target Genes ◽  
Transforming Growth Factor ◽  
Intestinal Epithelial ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Genomic Approach ◽  
Apoptotic Genes ◽  
Induced Apoptosis

Transforming growth factor (TGF)-β-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. Previously, we have shown that TGF-β inhibits the growth of rat intestinal epithelial (RIE)-1 cells. However, RIE-1 cells are relatively resistant to TGF-β-induced apoptosis due to a low endogenous Smad3-to-Akt ratio. Overexpression of Smad3 sensitizes RIE-1 cells (RIE-1/Smad3) to TGF-β-induced apoptosis by altering the Smad3-to-Akt ratio in favor of apoptosis. In this study, we utilized a genomic approach to identify potential downstream target genes that are regulated by TGF-β/Smad3. Total RNA samples were analyzed using Affymetrix oligonucleotide microarrays. We found that TGF-β regulated 518 probe sets corresponding to its target genes. Interestingly, among the known apoptotic genes included in the microarray analyses, only caspase-3 was induced, which was confirmed by real-time RT-PCR. Furthermore, TGF-β activated caspase-3 through protein cleavage. Upstream of caspase-3, TGF-β induced mitochondrial depolarization, cytochrome c release, and cleavage of caspase-9, which suggests that the intrinsic apoptotic pathway mediates TGF-β-induced apoptosis in RIE-1/Smad3 cells.

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