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

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

PURPOSE: This study demonstrated that apoptosis induced by mycophenolic acid (MPA) is mediated by mitochondrial membrane potential transition (MPT) changes in Jurkat cells. METHODS: Cell viability and MPT changes were measured by flow cytometry. Western blotting was performed to evaluate the expression of Bcl-2 family proteins, Bid, truncated Bid (tBid), cytochrome c, voltage dependent anion channel (VDAC), poly ADP-ribose polymerase (PARP), and protein kinase C-delta (PKC-delta). The catalytic activity of caspase-9 and -3 was also measured. RESULTS: Cell viability was decreased in time- and dose-dependent manners. Bcl-2 protein expression was decreased, but Bax protein expression was identified. A decreased Bcl-XL /Bcl-XS ratio was also noted. The expression of tBid protein also increased in a time-dependent manner in Jurkat cells treated with MPA. While normal MPT appeared as orange fluorescence, abnormal MPT corresponded to green fluorescence. Green fluorescence increased as orange decreased in the MPA-treated cells. Significantly increased concentrations of MPA induced the release of cytosolic cytochrome c. MPA also augmented the catalytic activity of caspase-9 and caspase-3 in Jurkat cells. Our findings demonstrated that MPA-induced apoptosis is mediated by MPT changes accompanied by decreased Bcl-XL expression and the appearance of tBid protein. The release of cytosolic cytochrome c from mitochondria and increased catalytic activity of caspase-9 and caspase-3 were observed in MPA-treated Jurkat cells. CONCLUSION: These results suggest that mitochondrial dysfunction caused by MPA induces human T lymphocyte apoptosis.
Adenosine Diphosphate Ribose ; Apoptosis ; bcl-2-Associated X Protein ; BH3 Interacting Domain Death Agonist Protein ; Blotting, Western ; Caspase 3 ; Caspase 9 ; Cell Survival ; Citrus sinensis ; Cytochromes c ; Cytosol ; Flow Cytometry ; Fluorescence ; Humans ; Jurkat Cells ; Lymphocytes ; Membrane Potential, Mitochondrial ; Mitochondria ; Mitochondrial Membranes ; Mycophenolic Acid ; Protein Kinase C-delta ; Proteins

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

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

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

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

PURPOSE: A multi-subunit transcription factor NF-kappaB mediates the antiapoptotic signals in several cancer cell lines and it is activated in a broad range of human tumors. In this study, we investigated whether the expression levels of the NF-kappaB and the apoptosis inducing genes were related to the pathogenesis and clinical properties of human bladder tumor. MATERIALS AND METHODS: The expressions of NF-kappaB, BCL2-associated X protein (BAX), BCL2-associated death protein (BAD) and BH3-interacting domain death agonist protein (BID) were investigated by performing immunohistochemical staining on 133 archival bladder tissue paraffin blocks; these blocks included 122 transitional cell carcinomas of the urinary bladder and 11 normal bladder mucosae. RESULTS: The expression levels of NF-kappaB were significantly higher in the bladder tumors than those of the normal bladder mucosae (p=0.001). The expression levels of BAX in the superficial and low-grade (grade 1 and 2) bladder tumors were significantly enhanced more than those of the high-grade and invasive cases (p=0.042 and p=0.045, respectively), while the expression levels of BAD in the tumor tissues and low-grade tumors were significantly elevated compared with those of the normal mucosae and high grade tumor (p=0.007 and p=0.048, respectively). But the expressions of BID were not correlated with any pathologic and clinical properties. CONCLUSIONS: The expressions of the NF-kappaB and apoptosis inducing genes such as BAX and BAD are strongly associated with the pathogenesis and clinical properties of bladder tumor. (Korean J Urol 2007;48:483-488)
Apoptosis* ; bcl-2-Associated X Protein ; BH3 Interacting Domain Death Agonist Protein ; Carcinoma, Transitional Cell ; Cell Line ; Humans ; Mucous Membrane ; NF-kappa B* ; Paraffin ; Transcription Factors ; Urinary Bladder Neoplasms* ; Urinary Bladder*