Mitochondrial Apoptotic Pathway Participates In Apoptosis Induced By Bufalin In Human Leukemia HL-60 Cells

ObjectiveApoptosis is a cell - autonomous programmed cell death mechanism that is utilized extensively during development, tissue homeostasis and maintenance of the immune system in adult organisms. Defective apoptosis (programmed cell death) represents a major causative factor in the development and progression of cancer. The ability of tumor cells to evade engagement of apoptosis can play a significant role in their resistance to conventional therapeutic regimens. Mitochondria are central to the apoptosis activation pathway in many physiological and pathological conditions. the mitochondrial inner transmembrane potential ( Δψm) collapses in apoptosis, indicating the opening of a large conductance channel known as the mitochondrial PT pore( PTP). Opening of this nonselective channel in the inner membrane allows for an equilibration of ions within the matrix and intermembrane space of mitochondria, thus dissipating the H + gradient across the inner membrane and uncoupling the respiratory chain. Inhibitors of PTP opening, including cyclosporins (which bind cyclophilin D) appear to block apoptosis in some systems, thus providing support for the idea that the PT is central to apoptotic processes. The Bcl - 2 family of proteins, comprising both antiapoptotic ( Bcl - 2 and Bcl - x/L) and proapoptotic ( Bax, Bak, Bid, NOXA, and PUMA) members, integrate diverse cell death signals and regulate the integrity of mitochondrial membrane. The BCL -2 family of pro - and antiapoptotic proteins are known to focus much of their response to the mitochondria level,upstream the irreversible cellular damage,involving dimerization,phospho-rylation, proteolytic cleavage and mitochondrial translocation.Perhaps more importantly, PT pore opening results in a volume dysregula-tion of mitochondria due to the hyperosmolality of the matrix, which causes the matrix space to expand. Because the inner membrane with its folded cristae possesses a larger surface area than the outer membrane, this matrix volume expansion can eventually cause outer membrane rupture, releasing caspase - activating proteins,such as cyt -c,Smac/DIABLO and AIF, located within the inter-membrane space into the cytosol. Smac/DIABLO is a mitochondrial protein that binds and inhibits one or more members of the IAP family of apoptosis inhibitory proteins(e. g. survivin) and, by doing so, releases active caspases that are then able to mediate apoptosis.Bufalin, a component of the traditional Chinese medicine chansu, has been shown to induce leukemia cell differentiation and apoptosis under certain experimental conditions. In an attempt to characterize the mechanisms of the induction of apoptosis — inducing effect of bufalin on human leukemia HL - 60 cells, we detected the MTP and the expression of mitochondria - mediated apoptotic pathway related genes. To further investigate the role of mitochondrial FTP in apoptosis induced by bufalin, we also detected the effect of CsA pretreatment on apoptosis induced by bufalin.Materials and Methods1. Cell viability was determined by trypan blue dye exclusion and apoptosis by morphology and flow cytometry.2. Expression of Bel -2 ^Bax^ Surviving Smac/DIABLO and caspase -3 were analyzed by Western blot.3. Expression of Survivin mRNA were determined by RT - PCR.4. The protein concentrations were determined by the Lowry method.5. Mitochondrial transmembrane potential were determined by monitoring the uptake by cells of fluorescent dye Rhodamine 123 and PI or DiOC 6(3).6. Results were statistically analyzed with software SPSS10.0.Results1. Proliferation of HL - 60 cells was inhibited by bufalin and the IC50 at24, 48 and 72 h were 25. 8,8.0 and 2.1 nmol/L,respectively. Apoptosis was induced when the cells were treated with bufalin at concentrations of 50nmol/L and higher.2. Compared with control,treatment with bufalin at 50nmol/L for 6,12,24 and 48h resulted in decrease of Bel -2 protein expression to 88. 6% ,53. 3% , 19.2% and 9.5%, Bcf-2/Bax ratio from 2.0 to 1.7,1.1,0.4 andO.2,Sur-vivin protein expression to 75. 2% ,54. 8% ,37. 5% and 20. 3% ,and Survivin mRNA to 85.7% ,39.4% , 12.5%and 0% .respectively.3. Compared with control, expression of Smac/DIABLO protein in mitochon-drial fraction were downregulated to 77. 5% ( 12h) ,21. 2% (24h) and 15. 3% (48h) ,and upregulated to 1.4(12h) ,2. 0(24h) and 3. 5 times (48h) in cy-tosolic fraction,respectively.4. The cleaved active subunits of caspase - 3 were observed following bufalin treatment from 8h to 48h.5. Furthermore, compared with control, incubation with bufalin at 50nmol/L elicited reduction of MTP to 34.8% (6h) N22.8% (12h) and 13.9% (24h).6. The loss of MTP and apoptosis - inducing effect of bufalin were partialy inhibited by a PTP inhibitor,CsA.DiscussionIn order to eliminate cells that are redundant, damaged, or infected, meta-zoan organisms have evolved the cell suicide mechanism termed apoptosis. This genetic program is vital for normal development, for maintenance of tissue home-ostasis, and for an effective immune system. Not surprisingly therefore, its disturbance is implicated in numerous pathological conditions, ranging from degenerative disorders to autoimmunity and cancer. Mitochondria are pivotal in controlling cell life and death. Reduced mitochondrial membrane potential ( Ai|jm) is considered as an initial and irreversible step towards apoptosis . The PTP is a multiprotein complex spanning the inner and outer mitochondrial membranes whose activation results in dissipation of the mitochondrial inner membrane potential , and eventual disruption of the outer mitochondrial membrane. The structure and composition of the PTP remains only partially defined, but its constitu-entsinclude both inner membrane proteins, such as the adenine nucleotidetrans-locator ( ANT) , and outer membrane proteins, such as porin ( voltage - dependent anion channel;VDAC) , which operate in concert, presumably at inner and outer membrane contact sites. Members of the Bel - 2 family of proteins are known to affect mitochondrial function and regulate the release of apoptosis - activating factors. In most cases, Bel - 2 family proteins are postulated to play a central role by controlling the permeabilization of mitochondrial membranes, either as pore forming sub - units or as regulators of intrinsic mitochondrial channels. Anti - apoptotic members of Bel - 2 family (e. g. Bel - 2 and Bel - xL) act primarily to preserve mitochondrial integrity by suppressing the release of cy-tochrome c. Mitochondrial efflux of Smac/DIABLO, in response to a variety of pro - apoptotic agents, was profoundly inhibited in Bel - 2 - overexpressing cells. Thus, in addition to modulating apoptosis -associated mitochondrial cyto-chrome c release, Bel — 2 also regulates Smac release. In contrast, pro - apoptotic members ( Bax, Bid, etc. ) induce the release of the apoptosis - activating factors and cause mitochondrial dysfunction. Bel -2 prevents Bax oligomerization by physically interfering with complete insertion of Bax into the membrane, a requirement for oligomerization. A pre - set of Bel - 2/Bax appeare to determine the survival or death of cells following an apoptotic stimulus.In cells stimulated to die in response to diverse pro - apoptotic agents, including death receptor ligation, cytotoxic.drugs and DNA -damaging agents, Smac/DIABLO accumulation within the cytosol was readily detected . This coincided with a concomitant loss of Smac/DIABLO from mitochondria. Smac has been shown to exit mitochondria and enter the cytosol during apoptosis. Through the interaction with IAPs (inhibitor of apoptosis protein) , Smac could hydrolyze caspase - 3 protein and enhance the catalytic activity of mature caspase - 3, finally promoting apoptosis. Survivin contains a baculovirus inhibitor of apoptosis repeat (BIR) protein domain that classifies it as a member of the LAP family. Survivin is a protein that inhibits apoptosis and regulates cell division. Survivin is expressed in embryonic tissues as well as in the majority of human cancers, but is not expressed in most normal adult tissues. The cancer - specific expression of survivin, coupled with its importance in inhibiting cell death and in regu-lating cell division, makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Survivin inhibits apoptosis, via its BIR domain, by either directly or indirectly interfering with the function of caspases. Smac/DLA-BLO interferes with the ability of survivin to prevent caspaseactivation.Our data indicates for the first time that bufalin induced apoptosis in HL -60 cells, and this coincided with a concomitant release of Smac/DIABLO from mitochondria to cytosol, downregulation of Bel - 2 and survivin expression, activation of caspase - 3 and loss of mitochondrial membrane permeability. Pretreat-ment of cells with cyclosporine A, prior to/concomitant with exposure to bufalin, effectively prevented the deleterious effects of bufalin on cellular integrity and viability. As mitochondrial permeability is known to be important in the regulation of Smac/DIABLO release, our observations indicate that mitochondria are the principal target of bufalin. More specifically, we propose that bufalin causes the mitochondrial membranes to lose their permeability, resulting in caspase - 3 activation and apoptosis.ConclusionApoptosis induced by bufalin in HL - 60 cells is associated with downregulation of expression of Bel -2 and Survivin,decrease of Bel -2/Bax ratio,mitochondrial release of Smac/DIABLO, activation of caspase - 3 and mitochondrial transmembrane potential collapses. Cyclosporin A , an inhibitor of PT pore could attenuate the apoptosis - inducing effect of bufalin and reverse the mitochondrial transmembrane potential. These results indicated that the mitochondria - mediated apoptotic pathway may be involved in the apoptosis induced by bufalin in HL - 60 cells.