Caspase-2, a highly conserved member of the caspase family, is considered an initiator caspase that triggers apoptosis in response to some cellular stresses. Previous studies suggest that an intracellular multi-protein complex PIDDosome, induced by genotoxic stress, serves as a platform for caspase-2 activation. Due to caspase-2's inability to process effector caspases, however, the mechanism underlying caspase-2-mediated cell death upon PIDDosome activation remains unclear. Here, we conducted an unbiased genome-wide genetic screen and identified that the Bcl2 family protein BID is required for PIDDosome-induced, caspase-2-mediated apoptosis. PIDDosome-activated caspase-2 directly and functionally processes BID to signal the mitochondrial pathway for apoptosis induction. In addition, a designed chemical screen identified a compound, HUHS015, which specifically activates caspase-2-mediated apoptosis. HUHS015-stimulated apoptosis also requires BID but is independent of the PIDDosome. Through extensive structure-activity relationship efforts, we identified a derivative with a potency of ~60 nmol/L in activating caspase-2-mediated apoptosis. The HUHS015-series of compounds act as efficient agonists that directly target the interdomain linker in caspase-2, representing a new mode of initiator caspase activation. Human and mouse caspase-2 differ in two crucial residues in the linker, rendering a selectivity of the agonists for human caspase-2. The caspase-2 agonists are valuable tools to explore the physiological roles of caspase-2-mediated cell death and a base for developing small-molecule drugs for relevant diseases.
Caspase 2/genetics* ; Humans ; BH3 Interacting Domain Death Agonist Protein/metabolism* ; Apoptosis/drug effects* ; Death Domain Receptor Signaling Adaptor Proteins/metabolism* ; Animals ; Mice ; Cysteine Endopeptidases

Adult ; Altitude Sickness ; metabolism ; pathology ; Apoptosis ; BH3 Interacting Domain Death Agonist Protein ; metabolism ; Erythroblasts ; cytology ; pathology ; Humans ; Male ; Middle Aged ; RNA, Messenger ; genetics ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism

OBJECTIVETo explore the effects of oridonin (Ori) on human multiple myeloma (MM) cell line LP-1 apoptosis and its mechanisms.

METHODSThe human MM LP-1 cells were incubated in vitro by different concentrations of Ori. The proliferation activities of LP-1 cells were detected using MTT assay. The apoptosis rate of LP-1 cells was detected using Annexin V/PI double staining method. The ultrastructural changes of LP-1 cells were observed under transmission electron microscope (TEM) after they were treated with Ori. The mRNA expression of apoptosis correlated genes were detected using Real-time PCR.

RESULTSOri inhibited the proliferation of LP-1 cells in a dose- and time-dependent way. Results of Annexin V/PI double staining method showed that, along with increased drug concentration and prolonged drug action time, the apoptosis rate of LP-1 cells significantly increased. Under TEM, chromatin margination and mitochondrial swelling could be seen in LP-1 cells after they were treated by Ori. The mRNA expressions of PDCD5 and Bid were up-regulated, and those of Bcl-2 and NK-kappaB were down-regulated after action of Ori.

CONCLUSIONSOri induced cell apoptosis by up-regulating the mRNA expression of Bid and down-regulating the mRNA expression of Bcl-2 to decrease the mitochondrial membrane potential, trigger mitochondrial apoptosis way of LP-1 cells. Ori, also as the inhibitor of NF-kappaB activities, blocked the NF-kappaB activation, induced cell apoptosis, and inhibited the cell proliferation. Of them, it is necessary to further study the role of PDCD5 as an apoptosis promoter.

Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; BH3 Interacting Domain Death Agonist Protein ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Diterpenes, Kaurane ; pharmacology ; Humans ; Multiple Myeloma ; pathology ; NF-kappa B p50 Subunit ; metabolism ; Neoplasm Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism

In the present study, the apoptotic mechanism and polyamine transporter recognition of WJH-6, a novel polyamine conjugate, were investigated in K562 and HL-60 cells. The cytotoxicity of WJH-6 was assessed by MTT assay; cell cycle distribution and apoptosis were measured by flow cytometry; the protein expression of Caspase-3, Caspase-8, Caspase-9, Bid and mitochondrial membrane potential (MMP) were evaluated by high content screening (HCS) analysis; the protein expression of cytochrome c was measured by Western blotting. The results showed that WJH-6 could be recognized and transported by polyamine transporter (PAT). Furthermore, WJH-6 was able to inhibit K562 and HL-60 cells proliferation and induce apoptosis. This apoptotic effect was relative to MMP loss, cytochrome c release from mitochondria to cytoplasm and the activation of Caspase-8, Caspase-9, Caspase-3 and Bid. These results suggested that WJH-6-induced K562 and HL-60 cells apoptosis was related with mitochondrial damage.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; BH3 Interacting Domain Death Agonist Protein ; metabolism ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cytochromes c ; metabolism ; Cytoplasm ; metabolism ; Enzyme Activation ; drug effects ; HL-60 Cells ; Humans ; K562 Cells ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; metabolism ; Polyamines ; pharmacology

Pore-formation and protein-protein interactions are considered to play critical roles in the regulation of apoptosis by Bcl-2 family proteins. During the initiation of apoptosis, the anti-apoptotic Bcl-2 and the pro-apoptotic Bax form different pores to regulate the permeability of mitochondrial outer membrane, playing their opposite functions. Overexpression of Bcl-2 has been found in various cancer cells, therefore it is gaining widespread interest to discover small molecules to compromise Bcl-2 function for anti-cancer treatment. Since Bax binds to Bcl-2's hydrophobic groove via its BH3 domain (composed of helices 2 and 3), by which their functions are inhibited each other, the H2-H3 peptide that contains the functional BH3 domain of Bax has been considered as a potential Bcl-2 antagonist. We recently reported that Bax peptide H2-H3 promotes cell death by inducing Bax-mediated cytochrome c release and by antagonizing Bcl-2's inhibitory effect on Bax. However, the mechanism of how H2-H3 inhibits the anti-apoptotic activity of Bcl-2 remains poorly understood. To address this question, we reconstituted the Bcl-2 or Bax pore-forming process in vitro. We found that H2-H3 inhibited Bcl-2's pore formation and neutralized Bcl-2's inhibitory effect on Bax pore formation in the membrane, whereas the mutant H2-H3 peptide that does not induce apoptosis in cells was shown to have no effect on Bcl-2's activities. Thus, inhibiting Bcl-2's pore-forming and anti-Bax activities in the membrane is strongly correlated with H2-H3's pro-apoptosis function in cells.
Apoptosis ; physiology ; BH3 Interacting Domain Death Agonist Protein ; chemistry ; Humans ; Membrane Proteins ; chemistry ; metabolism ; Mitochondrial Membrane Transport Proteins ; Mitochondrial Membranes ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; antagonists & inhibitors ; chemistry ; bcl-2 Homologous Antagonist-Killer Protein ; chemistry ; bcl-2-Associated X Protein ; chemistry

The permeability of mitochondrial outer membrane (MOM) is regulated by the proteins of the Bcl-2 family via their interactions at the membrane. While pro-apoptotic Bax protein promotes MOM permeabilization (MOMP) releasing cytochrome c after activation by BH3-only protein, anti-apoptotic Bcl-2 protein protects MOM. However both Bax and Bcl-2 can form pores in model membranes. Unlike Bax pore that has been extensively studied and reported to be directly linked to MOMP, Bcl-2 pore is much less known; thus we investigated the pore-forming property of recombinant Bcl-2 lacking the C-terminal transmembrane sequence (Bcl-2deltaTM) in liposomal membranes of MOM lipids. We found that: (1) Bcl-2 formed pores at acidic pH that induced the association of Bcl-2 with liposome; (2) Bcl-2 pore size was dependent on Bcl-2 concentration, suggesting that oligomerization is involved in Bcl-2 pore formation; (3) Unlike Bax pore that could release large molecules up to 2 mega-Da, Bcl-2 pore was smaller and could only release the molecules of a few kilo-Da. Therefore, Bcl-2 and Bax may form different size pores in MOM, and while the large pore formed by Bax may release cytochrome c during apoptosis, the small pore formed by Bcl-2 may maintain the normal MOM permeability.
BH3 Interacting Domain Death Agonist Protein ; metabolism ; Cell Membrane Permeability ; Cytochrome c Group ; metabolism ; Humans ; Hydrogen-Ion Concentration ; Liposomes ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Mitochondrial Membranes ; metabolism ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism

A vast portion of human disease results when the process of apoptosis is defective. Disorders resulting from inappropriate cell death range from autoimmune and neurodegenerative conditions to heart disease. Conversely, prevention of apoptosis is the hallmark of cancer and confounds the efficacy of cancer therapeutics. In the search for optimal targets that would enable the control of apoptosis, members of the BCL-2 family of anti- and pro-apoptotic factors have figured prominently. Development of BCL-2 antisense approaches, small molecules, and BH3 peptidomimetics has met with both success and failure. Success-because BCL-2 proteins play essential roles in apoptosis. Failure-because single targets for drug development have limited scope. By examining the activity of the BCL-2 proteins in relation to the mitochondrial landscape and drawing attention to the significant mitochondrial membrane alterations that ensue during apoptosis, we demonstrate the need for a broader based multi-disciplinary approach for the design of novel apoptosis-modulating compounds in the treatment of human disease.
Apoptosis/*drug effects/physiology ; BH3 Interacting Domain Death Agonist Protein/physiology ; Drug Design ; Genes, bcl-2 ; Humans ; Mitochondria/physiology/ultrastructure ; Mitochondrial Membranes/*metabolism/physiology ; Multigene Family ; Proto-Oncogene Proteins c-bcl-2/*antagonists & inhibitors ; Signal Transduction

Apoptosis/*drug effects/physiology ; BH3 Interacting Domain Death Agonist Protein/physiology ; Drug Design ; Genes, bcl-2 ; Humans ; Mitochondria/physiology/ultrastructure ; Mitochondrial Membranes/*metabolism/physiology ; Multigene Family ; Proto-Oncogene Proteins c-bcl-2/*antagonists & inhibitors ; Signal Transduction

OBJECTIVETo investigate the reversing effect of Bcl-XL small interfering RNA (siRNA) on the acquired resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human colon cancer.

METHODSHuman colon cancer cells DLD1-TRAIL/R, with acquired resistance to TRAIL, were firstly transfected with Bcl-XL siRNA for 24 h followed by the treatment of TRAIL protein. The survival rate of DLD1-TRAIL/R cells was assessed by FACS analysis and cell number counting, respectively, and activation of its apoptotic signaling was evaluated by Western blot.

RESULTSBcl-XL siRNA effectively downregulated the expression of Bcl-XL protein and reversed the acquired resistance to TRAIL in DLD1-TRAIL/R cells. After combination treatment of Bcl-XL siRNA and TRAIL protein, the apoptotic rate of DLD1-TRAIL/R cells was more than 50% and survival rate was less than 40%, whereas there was no effect on the survival of DLD1-TRAIL/R cells after treatment with control treatment or TRAIL protein treatment alone (P < 0.05). Western blot analysis demonstrated that caspase-8, caspase-9, Bid, caspase-3, and poly (ADP-ribose) polymerase (PARP) were obviously activated after combination treatment with Bcl-XL siRNA and TRAIL protein, and the release of cytochrome C was also significantly increased.

CONCLUSIONBcl-XL siRNA can effectively reverse the acquired resistance to TRAIL in human colon cancer cells, suggesting that it might be a new strategy for overcoming the resistance in cancer therapy.

Apoptosis ; BH3 Interacting Domain Death Agonist Protein ; metabolism ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Caspases ; metabolism ; Cell Line, Tumor ; Cell Survival ; Colonic Neoplasms ; metabolism ; pathology ; Cytochromes c ; metabolism ; Drug Resistance, Neoplasm ; Humans ; Poly(ADP-ribose) Polymerases ; metabolism ; RNA, Small Interfering ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Transfection ; bcl-X Protein ; genetics ; metabolism

OBJECTIVETo investigate the role of reactive oxygen species (ROS) and ROS-caused mitochondrial transmembrane potential loss in sodium selenite-induced apoptosis in NB4 cells.

METHODSROS production was measured by ROS-specific probe DCFH-DA. Sodium selenite mitochondrial transmembrane potential loss was evaluated by flow cytometry with Rh123 staining. Protein levels of cytochrome C, Bid, Bcl-xl, and Bax were measured by Western blot using protein-specific antibodies. NB4 cells were pre-incubated by MnTmPy or BSO before selenite treatment to further confirm the effects of ROS on NB4 cells.

RESULTS20 micromol/L sodium selenite induced ROS production and mitochondrial transmembrane potential loss in NB4 cells time-dependently. Cytochrome C accumulated in cytoplasm after selenite treatment. Sodium selenite also downregulated Bcl-xl and activated Bax and Bid at protein level. Pretreatment with antioxidant MnTmPy almost fully abrogated the proapoptotic effect of sodium selenite prevented the cleavage of Bid protein and in turn the mitochondrail transmembrane potential loss. On the contrary, pretreatment with BSO intensified the mitochondrail transmembrane potential loss induced by sodium selenite.

CONCLUSIONSSodium selenite may induce apoptosis by inducing ROS production in NB4 cells, which leads to the downregulation of Bcl-xl, upregulation of Bax, and cleavage and activation of Bid. Bax and tBid then agregate on mitochondrial membrane, which in turn causes a decrease of mitochondrial transmembrane potential and release of cytochrome C into cytoplasm.

Apoptosis ; BH3 Interacting Domain Death Agonist Protein ; biosynthesis ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Reactive Oxygen Species ; metabolism ; Sodium Selenite ; pharmacology ; bcl-2-Associated X Protein ; biosynthesis ; bcl-X Protein ; biosynthesis