Epigallocatechin gallate reduces hypoxia-induced apoptosis in human hepatoma cells

Cell detachment from extracellular matrix is closely related to induction of apoptosis. Epigallocatechin gallate (EGCG) has been shown to have antioxidant effect and to protect hypoxia-induced damage. We investigated whether EGCG reduced hypoxia-induced apoptosis and cell detachment in HepG2 cells. EGCG prevented cell death by hypoxia (0.5% O2) in a dose-dependent manner (hypoxic cell viability, 54.67%). RT-PCR and caspase3 activity assay showed that the hypoxia-induced cell death was caused by apoptosis increasing mRNA level of BAX, CASP3, and caspase3 activity. EGCG reduced increase of these mRNA and caspase3 activity. Western blot analysis and immunocytochemistry showed that EGCG increased cell adhesion proteins including E-cadherin (CDH1), tumor-associated calcium signal transducer 1 (TACSTD1), and protein tyrosine kinase 2 (PTK2) decreased by hypoxia. Hypoxia-induced apoptosis in HepG2 cells, and EGCG contributed to the HepG2 cell survival by attenuating the apoptosis.     

Hypoxia-induced cell death of HepG2 cells involves a necrotic cell death mediated by calpain

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of μ-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially μ-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.     

NOXA, a sensor of proteasome integrity, is degraded by 26S proteasomes by an ubiquitin-independent pathway that is blocked by MCL-1

Ubiquitin (Ub)-mediated proteasome-dependent proteolysis is critical in regulating multiple biological processes including apoptosis. We show that the unstructured BH3-only protein, NOXA, is degraded by an Ub-independent mechanism requiring 19S regulatory particle (RP) subunits of the 26S proteasome, highlighting the possibility that other unstructured proteins reported to be degraded by 20S proteasomes in vitro may be bona fide 26S proteasome substrates in vivo. A lysine-less NOXA (NOXA-LL) mutant, which is not ubiquitinated, is degraded at a similar rate to wild-type NOXA. Myeloid cell leukemia 1, but not other anti-apoptotic BCL-2 family proteins, stabilizes NOXA by interaction with the NOXA BH3 domain. Depletion of 19S RP subunits, but not alternate proteasome activator REG subunits, increases NOXA half-life in vivo. A NOXA-LL mutant, which is not ubiquitinated, also requires an intact 26S proteasome for degradation. Depletion of the 19S non-ATPase subunit, PSMD1 induces NOXA-dependent apoptosis. Thus, disruption of 26S proteasome function by various mechanisms triggers the rapid accumulation of NOXA and subsequent cell death strongly implicating NOXA as a sensor of 26S proteasome integrity.     

BH3分析技术研究进展及其应用

许多化疗药物发挥作用的重要方式是通过诱导线粒体介导的细胞凋亡.细胞凋亡在维持正常机体的内环境稳态中有重要作用,而在肿瘤细胞中,细胞凋亡的失调成为肿瘤细胞逃避化疗药物杀灭细胞作用的一道屏障. BCL-2家族蛋白在调节线粒体诱导的凋亡中处于中心地位,因此一项基于BCL-2家族蛋白的检测技术,BH3分析技术应运而生.该项技术的提出或许能为肿瘤的个性化治疗提供新的思路.本文重点综述BH3分析技术的原理,以及在肿瘤化疗药物选择和新型化疗药物开发中的应用.     

Platinum resistant cancer cells conserve sensitivity to BH3 domains and obatoclax induced mitochondrial apoptosis

Resistance to cisplatin chemotherapy remains a major hurdle preventing effective treatment of many solid cancers. BAX and BAK are pivotal regulators of the mitochondrial apoptosis pathway, however little is known regarding their regulation in cisplatin resistant cells. Cisplatin induces DNA damage in both sensitive and resistant cells, however the latter exhibits a failure to initiate N-terminal exposure of mitochondrial BAK or mitochondrial SMAC release. Both phenotypes are highly sensitive to mitochondrial permeabilisation induced by exogenous BH3 domain peptides derived from BID, BIM, NOXA (which targets MCL-1 and A1), and there is no significant change in their prosurvival BCL2 protein expression profiles. Obatoclax, a small molecule inhibitor of pro-survival BCL-2 family proteins including MCL-1, decreases cell viability irrespective of platinum resistance status across a panel of cell lines selected for oxaliplatin resistance. In summary, selection for platinum resistance is associated with a block of mitochondrial death signalling upstream of BAX/BAK activation. Conservation of sensitivity to BH3 domain induced apoptosis can be exploited by agents such as obatoclax, which directly target the mitochondria and BCL-2 family.     

IEX-1-induced cell death requires BIM and is modulated by MCL-1

MCL-1 (myeloid cell leukemia-1) is a distinguished and pivotal member of the pro-survival BCL-2 family of proteins, and we isolated IEX-1 (immediate early response gene X-1) as a MCL-1-interacting protein using the yeast two-hybrid system and confirmed their endogenous association in human cells. The underlying mechanisms by which IEX-1 affects cell survival and death are largely unknown. Ectopic expression of IEX-1-induced caspase-dependent apoptosis in 293T cells, and the response was significantly modulated by changes in the MCL-1 expression level in cells. Forced expression of IEX-1 was unable to induce cell death or to perturb mitochondrial membrane potential in BIM-depleted cells. Additionally, knockouts of NOXA or PUMA did not affect the activities of IEX-1, indicating that the pro-death action of IEX-1 specifically requires BIM. Our findings provide insight into a new regulatory circuit that controls cell death and survival by the coordinated action of MCL-1, IEX-1, and BIM.     

Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies

Although the BCL-2 family constitutes a crucial checkpoint in apoptosis, the intricate interplay between these family members remains elusive. Here, we demonstrate that BIM and PUMA, similar to truncated BID (tBID), directly activate BAX-BAK to release cytochrome c. Conversely, anti-apoptotic BCL-2-BCL-X(L)-MCL-1 sequesters these 'activator' BH3-only molecules into stable complexes, thus preventing the activation of BAX-BAK. Extensive mutagenesis of BAX-BAK indicates that their activity is not kept in check by BCL-2-BCL-X(L)-MCL-1. Anti-apoptotic BCL-2 members are differentially inactivated by the remaining 'inactivator' BH3-only molecules including BAD, NOXA, BMF, BIK/BLK and HRK/DP5. BAD displaces tBID, BIM or PUMA from BCL-2-BCL-X(L) to activate BAX-BAK, whereas NOXA specifically antagonizes MCL-1. Coexpression of BAD and NOXA killed wild-type but not Bax, Bak doubly deficient cells or Puma deficient cells with Bim knockdown, indicating that activator BH3-only molecules function downstream of inactivator BH3-only molecules to activate BAX-BAK. Our data establish a hierarchical regulation of mitochondrion-dependent apoptosis by various BCL-2 subfamilies.     

Multiple Functions of BCL-2 Family Proteins

BCL-2 family proteins are the regulators of apoptosis, but also have other functions. This family of interacting partners includes inhibitors and inducers of cell death. Together they regulate and mediate the process by which mitochondria contribute to cell death known as the intrinsic apoptosis pathway. This pathway is required for normal embryonic development and for preventing cancer. However, before apoptosis is induced, BCL-2 proteins have critical roles in normal cell physiology related to neuronal activity, autophagy, calcium handling, mitochondrial dynamics and energetics, and other processes of normal healthy cells. The relative importance of these physiological functions compared to their apoptosis functions in overall organismal physiology is difficult to decipher. Apoptotic and noncanonical functions of these proteins may be intertwined to link cell growth to cell death. Disentanglement of these functions may require delineation of biochemical activities inherent to the characteristic three-dimensional shape shared by distantly related viral and cellular BCL-2 family members.     

NOXA as critical mediator for drug combinations in polychemotherapy

During polychemotherapy, cytotoxic drugs are given in combinations to enhance their anti-tumor effectiveness. For most drug combinations, underlying signaling mechanisms responsible for positive drug–drug interactions remain elusive. Here, we prove a decisive role for the Bcl-2 family member NOXA to mediate cell death by certain drug combinations, even if drugs were combined which acted independently from NOXA, when given alone. In proof-of-principle studies, betulinic acid, doxorubicin and vincristine induced cell death in a p53- and NOXA-independent pathway involving mitochondrial pore formation, release of cytochrome c and caspase activation. In contrast, when betulinic acid was combined with either doxorubicine or vincristine, cell death signaling changed considerably; the drug combinations clearly depended on both p53 and NOXA. Similarly and of high clinical relevance, in patient-derived childhood acute leukemia samples the drug combinations, but not the single drugs depended on p53 and NOXA, as shown by RNA interference studies in patient-derived cells. Our data emphasize that NOXA represents an important target molecule for combinations of drugs that alone do not target NOXA. NOXA might have a special role in regulating apoptosis sensitivity in the complex interplay of polychemotherapy. Deciphering the differences in signaling of single drugs and drug combinations might enable designing highly effective novel polychemotherapy regimens.     

MCP-1/CCL2 protects cardiac myocytes from hypoxia-induced apoptosis by a G(alphai)-independent pathway

Chemokines, in addition to their chemotactic properties, act upon resident cells within a tissue and mediate other cellular functions. In a previous study, we demonstrated that CCL2 protects cultured mouse neonatal cardiac myocytes from hypoxia-induced cell death. Leukocyte chemotaxis has been shown to contribute to ischemic injury. While the chemoattractant properties of CCL2 have been established, the protective effects of this chemokine suggest a novel role for CCL2 in myocardial ischemia/reperfusion injury. The present study examined the cellular signaling pathways that promote this protection. Treatment of cardiac myocyte cultures with CCL2 protected them from hypoxia-induced apoptosis. This protection was not mediated through the activation of G(alphai) signaling that mediates monocyte chemotaxis. Inhibition of the ERK1/2 signaling pathway abrogated CCL2 protection. Caspase 3 activation and JNK/SAPK phosphorylation were decreased in hypoxic myocytes co-treated with CCL2 as compared to hypoxia only-treated cultures. Expression of the Bcl-2 family proteins, Bcl-xL and Bag-1, was increased in CCL2-treated myocytes subjected to hypoxia. There was also downregulation of Bax protein levels as a result of CCL2 co-treatment. These data suggest that CCL2 cytoprotection and chemotaxis may occur through distinct signaling mechanisms.     

Mutation of BCL-2 Family Proteins in Cancer

Apoptosis is a genetically controlled cell death process that is required for normal development and tissue homeostasis. Suppression of apoptosis can confer a growth advantage to cells and contribute to cancer; many cancers are relatively resistant to apoptosis, including that induced by radiation or chemotherapeutics. Mutations which inactivate pro-apoptotic or activate anti-apoptotic proteins in cancer cells are therefore likely to be responsible for some of these differences. BCL-2 family proteins are key regulators of apoptosis and there is evidence supporting a role for mutation of BCL-2 family proteins in cancer. This includes well established events such as activation of BCL-2 via translocations in follicular lymphoma, as well as more recent observations implicating activation of Bcl-X_L expression and frameshift and missense mutations of BAX and BCL-2 in cancer.     

ROS and protein oxidation in early stages of cytotoxic drug induced apoptosis

Cytotoxic drugs induce cell death through induction of apoptosis. This can be due to activation of a number of cell death pathways. While the downstream events in drug induced cell death are well understood, the early events are less clear. We therefore used a proteomic approach to investigate the early events in apoptosis induced by a variety of drugs in HL60 cells. Using 2D-gel electrophoresis, we were able to identify a number of protein changes that were conserved between different drug treatments. Identification of post-translational modifications (PTM) responsible for these proteome changes revealed an increase in protein oxidation in drug treated cells, as well as changes in protein phosphorylation. We demonstrate an accumulation of oxidised proteins within the ER, which lead to ER stress and calcium release and may result in the induction of apoptosis. This study demonstrates the importance of ROS mediated protein modifications in the induction of the early stages of apoptosis in response to chemotherapeutic drug treatment.     

Human embryonic stem cells express elevated levels of multiple pro-apoptotic BCL-2 family members

Two of the greatest challenges in regenerative medicine today remain (1) the ability to culture human embryonic stem cells (hESCs) at a scale sufficient to satisfy clinical demand and (2) the ability to eliminate teratoma-forming cells from preparations of cells with clinically desirable phenotypes. Understanding the pathways governing apoptosis in hESCs may provide a means to address these issues. Limiting apoptosis could aid scaling efforts, whereas triggering selective apoptosis in hESCs could eliminate unwanted teratoma-forming cells. We focus here on the BCL-2 family of proteins, which regulate mitochondrial-dependent apoptosis. We used quantitative PCR to compare the steady-state expression profile of all human BCL-2 family members in hESCs with that of human primary cells from various origins and two cancer lines. Our findings indicate that hESCs express elevated levels of the pro-apoptotic BH3-only BCL-2 family members NOXA, BIK, BIM, BMF and PUMA when compared with differentiated cells and cancer cells. However, compensatory expression of pro-survival BCL-2 family members in hESCs was not observed, suggesting a possible explanation for the elevated rates of apoptosis observed in proliferating hESC cultures, as well as a mechanism that could be exploited to limit hESC-derived neoplasms.     

ROS and protein oxidation in early stages of cytotoxic drug induced apoptosis

Cytotoxic drugs induce cell death through induction of apoptosis. This can be due to activation of a number of cell death pathways. While the downstream events in drug induced cell death are well understood, the early events are less clear. We therefore used a proteomic approach to investigate the early events in apoptosis induced by a variety of drugs in HL60 cells. Using 2D-gel electrophoresis, we were able to identify a number of protein changes that were conserved between different drug treatments. Identification of post-translational modifications (PTM) responsible for these proteome changes revealed an increase in protein oxidation in drug treated cells, as well as changes in protein phosphorylation. We demonstrate an accumulation of oxidised proteins within the ER, which lead to ER stress and calcium release and may result in the induction of apoptosis. This study demonstrates the importance of ROS mediated protein modifications in the induction of the early stages of apoptosis in response to chemotherapeutic drug treatment.     

B Cell Lymphoma-2 (BCL-2) Homology Domain 3 (BH3) Mimetics Demonstrate Differential Activities Dependent upon the Functional Repertoire of Pro- and Anti-apoptotic BCL-2 Family Proteins

The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these “BH3 mimetics” in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.     

BNIP3 and Nix: Atypical regulators of cell fate

Since their discovery nearly 25 years ago, the BCL-2 family members BNIP3 and BNIP3L (aka Nix) have been labelled ‘atypical’. Originally, this was because BNIP3 and Nix have divergent BH3 domains compared to other BCL-2 proteins. In addition, this atypical BH3 domain is dispensable for inducing cell death, which is also unusual for a ‘death gene’. Instead, BNIP3 and Nix utilize a transmembrane domain, which allows for dimerization and insertion into and through organelle membranes to elicit cell death. Much has been learned regarding the biological function of these two atypical death genes, including their role in metabolic stress, where BNIP3 is responsive to hypoxia, while Nix responds variably to hypoxia and is also down-stream of PKC signaling and lipotoxic stress. Interestingly, both BNIP3 and Nix respond to signals related to cell atrophy. In addition, our current view of regulated cell death has expanded to include forms of necrosis such as necroptosis, pyroptosis, ferroptosis, and permeability transition-mediated cell death where BNIP3 and Nix have been shown to play context- and cell-type specific roles. Perhaps the most intriguing discoveries in recent years are the results demonstrating roles for BNIP3 and Nix outside of the purview of death genes, such as regulation of proliferation, differentiation/maturation, mitochondrial dynamics, macro- and selective-autophagy. We provide a historical and unbiased overview of these ‘death genes’, including new information related to alternative splicing and post-translational modification. In addition, we propose to redefine these two atypical members of the BCL-2 family as versatile regulators of cell fate.     

Involvement of transforming growth factor-beta 1 signaling in hypoxia-induced tolerance to glucose starvation

Because survival and growth of human hepatoma cells are maintained by nutrient, especially glucose, glucose starvation induces acute cell death. The cell death is markedly suppressed by hypoxia, and we have reported involvement of AMP-activated protein kinase-alpha (AMPK-alpha), Akt, and ARK5 in hypoxia-induced tolerance. In the current study we investigated the mechanism of hypoxia-induced tolerance in human hepatoma cell line HepG2. ARK5 expression was induced in HepG2 cells when they were subjected to glucose starvation, and we found that glucose starvation transiently induced Akt and AMPK-alpha phosphorylation and that hypoxia prolonged phosphorylation of both protein kinases. We also found that hypoxia-induced tolerance was partially abrogated by blocking the Akt/ARK5 system or by suppressing AMPK-alpha expression and that suppression of both completely abolished the tolerance, suggesting that AMPK-alpha activation signaling and the Akt/ARK5 system play independent essential roles in hypoxia-induced tolerance. By using chemical compounds that specifically inhibit kinase activity of type I-transforming growth factor-beta (TGF-beta) receptor, we showed an involvement of TGF-beta in hypoxia-induced tolerance. TGF-beta1 mRNA expression was induced by hypoxia in an hypoxia-inducible factor-1alpha-independent manner, and addition of recombinant TGF-beta suppressed cell death during glucose starvation even under normoxic condition. AMPK-alpha, Akt, and ARK5 were activated by TGF-beta1, and Akt and AMPK-alpha phosphorylation, which was prolonged by hypoxia, was suppressed by an inhibitor of type I TGF-beta receptor. Based on these findings, we propose that hypoxia-induced tumor cell tolerance to glucose starvation is caused by hypoxia-induced TGF-beta1 through AMPK-alpha activation and the Akt/ARK5 system.     

BCL-2 in the crosshairs: tipping the balance of life and death

The discovery of B-cell lymphoma-2 (BCL-2) over 20 years ago revealed a new paradigm in cancer biology: the development and persistence of cancer can be driven by molecular roadblocks along the natural pathway to cell death. The subsequent identification of an expansive family of BCL-2 proteins provoked an intensive investigation of the interplay among these critical regulators of cell death. What emerged was a compelling tale of guardians and executioners, each participating in a molecular choreography that dictates cell fate. Ten years into the BCL-2 era, structural details defined how certain BCL-2 family proteins interact, and molecular targeting of the BCL-2 family has since become a pharmacological quest. Although many facets of BCL-2 family death signaling remain a mechanistic mystery, small molecules and peptides that effectively target BCL-2 are eliminating the roadblock to cell death, raising hopes for a medical breakthrough in cancer and other diseases of deregulated apoptosis.