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This study aimed to assess the potential anti-angiogenic mechanism of Phyllanthus urinaria (P. urinaria) and characterize the major compound in P. urinaria that exerts anti-angiogenic effect. The water extract of P. urinaria and Ellagic Acid were used to evaluate the anti-angiogenic effect in chorioallantoic membrane (CAM) in chicken embryo and human vascular endothelial cells (HUVECs). The matrix metalloproteinase-2 (MMP-2) activity was determined by gelatin zymography. The mRNA expressions of MMP-2, MMP-14 and tissue inhibitor of metalloproteinase-2 (TIMP-2) were analyzed by reverse transcription polymerase chain reaction (RT-PCR). Level of MMP-2 proteins in conditioned medium or cytosol was determined by western blot analysis. We confirmed that P. urinaria's in vivo anti-angiogenic effect was associated with a reduction in MMP-2 activity. Ellagic acid, one of the major polyphenolic components as identified in P. urinaria by high performance liquid chromatography mass spectrometry (HPLC/MS), exhibited the same anti-angiogenic effect in vivo. Both P. urinaria and Ellagic Acid inhibited MMP-2 activity in HUVECs with unchanged mRNA level. The mRNA expression levels of MMP-14 and TIMP-2 were not altered either. Results from comparing the change of MMP-2 protein levels in conditioned medium and cytosol of HUVECs after the P. urinaria or Ellagic Acid treatment revealed an inhibitory effect on the secretion of MMP-2 protein. This study concluded that Ellagic Acid is the active compound in P. urinaria to exhibit anti-angiogenic activity and to inhibit the secretion of MMP-2 protein from HUVECs.

Background:

Phyllanthus (Euphorbiaceae) species have long been used in folk medicine to treat various pathological conditions including liver diseases. Some species of Phyllanthus were found to exhibit hepatoprotective activity against drugs or toxins and this property was majorly attributed to phyllanthin and hypophyllanthin. In this study, we examined the hepatoprotective activity of five different species of Phyllanthus, namely, Phyllanthus amarus, Phyllanthus fraternus, Phyllanthus maderaspatensis, Phyllanthus urinaria, and Phyllanthus Rotundifolius. The extracts were also evaluated for the presence of key phytoconstituents, phyllanthin and hypophyllanthin.

Materials and Methods:

The extracts were evaluated for hepatoprotective activity against tert-butyl hydroxide (t-BH)-induced cytotoxicity using human hepatocarcinoma cells (HepG2 cell line).

Results:

Only P. urinaria and P. maderaspatensis exhibited significant hepatoprotective activity as evident from increased cell viability. The HPLC profile revealed that except P. amarus, the other extracts did not contain phyllanthin and hypophyllanthin.

Conclusion:

P. urinaria and P. maderaspatensis demonstrated dose-dependent hepatoprotective activity and hence, can provide promising therapeutic interventions against chemical–induced liver damage.

Increased apoptotic cell death is believed to play a pathological role in septic patients and experimental animals. Apoptosis can be induced by either a cell death receptor (extrinsic) or mitochondrial (intrinsic) pathway. Bid, a pro-apoptotic member of the Bcl-2 family, is thought to mediate cross talk between the extrinsic and intrinsic pathways of apoptosis; however, little is known about the action of Bid in the development of apoptosis and organ specific tissue damage/cell death as seen in polymicrobial sepsis. Our results show that following the onset of sepsis, tBid (the active form of Bid) is significantly increased in mitochondrial fractions of the thymus, spleen, Peyer’s patches and liver and that Fas or FasL deficiency blocks Bid activation in various tissues after septic challenge. Increased Bid activation is correlated with increased active caspase-3, -9 and apoptosis during sepsis. Bid deficient mice exhibit significantly reduced apoptosis in the thymus, spleen and Peyer’s patches compared with background mice after sepsis. Furthermore, Bid deficient mice had significantly reduced systemic and local inflammatory cytokine levels and improved survival after sepsis. These data support not only the contribution of Bid to sepsis-induced apoptosis and the onset of septic morbidity/mortality, but also the existence of a bridge between extrinsic apoptotic signals, e.g., FasL:Fas, TNF:TNFR, etc., and the intrinsic mitochondrial pathway via Bid-tBid activation during sepsis.

Caspase-8 stably inserts into the mitochondrial outer membrane during extrinsic apoptosis. Inhibition of caspase-8 enrichment on the mitochondria impairs caspase-8 activation and prevents apoptosis. However, the function of active caspase-8 on the mitochondrial membrane remains unknown. In this study, we have identified a native complex containing caspase-8 and BID on the mitochondrial membrane, and showed that death receptor activation by Fas or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced the cleavage of BID (tBID formation) within this complex. tBID then shifted to separate mitochondria-associated complexes that contained other BCL-2 family members, such as BAK and BCL-XL. We report that cells stabilize active caspase-8 on the mitochondria in order to specifically target mitochondria-associated BID, and that BID cleavage on the mitochondria is essential for caspase-8-induced cytochrome c release. Our findings indicate that during extrinsic apoptosis, caspase-8 can specifically target BID where it is mostly needed, on the surface of mitochondria.

Background

Recently there has been a remarkable surge of interest about natural products and their applications in the cosmetic industry. Topical delivery of antioxidants from natural sources is one of the approaches used to reverse signs of skin aging. The aim of this research was to develop a nanoemulsion cream for topical delivery of 30% ethanolic extract derived from local Phyllanthus urinaria (P. urinaria) for skin antiaging.

Methods

Palm kernel oil esters (PKOEs)-based nanoemulsions were loaded with P. urinaria extract using a spontaneous method and characterized with respect to particle size, zeta potential, and rheological properties. The release profile of the extract was evaluated using in vitro Franz diffusion cells from an artificial membrane and the antioxidant activity of the extract released was evaluated using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method.

Results

Formulation F12 consisted of wt/wt, 0.05% P. urinaria extract, 1% cetyl alcohol, 0.5% glyceryl monostearate, 12% PKOEs, and 27% Tween® 80/Span® 80 (9/1) with a hydrophilic lipophilic balance of 13.9, and a 59.5% phosphate buffer system at pH 7.4. Formulation F36 was comprised of 0.05% P. urinaria extract, 1% cetyl alcohol, 1% glyceryl monostearate, 14% PKOEs, 28% Tween® 80/Span® 80 (9/1) with a hydrophilic lipophilic balance of 13.9, and 56% phosphate buffer system at pH 7.4 with shear thinning and thixotropy. The droplet size of F12 and F36 was 30.74 nm and 35.71 nm, respectively, and their nanosizes were confirmed by transmission electron microscopy images. Thereafter, 51.30% and 51.02% of the loaded extract was released from F12 and F36 through an artificial cellulose membrane, scavenging 29.89% and 30.05% of DPPH radical activity, respectively.

Conclusion

The P. urinaria extract was successfully incorporated into a PKOEs-based nanoemulsion delivery system. In vitro release of the extract from the formulations showed DPPH radical scavenging activity. These formulations can neutralize reactive oxygen species and counteract oxidative injury induced by ultraviolet radiation and thereby ameliorate skin aging.

Bcl-2 and other closely related members of the Bcl-2 family of proteins inhibit the death of neurons and many other cells in response to a wide variety of pathogenic stimuli. Bcl-2 inhibition of apoptosis is mediated by its binding to pro-apoptotic proteins, e.g., Bax and tBid, inhibition of their oligomerization, and thus inhibition of mitochondrial outer membrane pore formation, through which other pro-apoptotic proteins, e.g., cytochrome c, are released to the cytosol. Bcl-2 also exhibits an indirect antioxidant activity caused by a sub-toxic elevation of mitochondrial production of reactive oxygen species and a compensatory increase in expression of antioxidant gene products. While classic approaches to cytoprotection based on Bcl-2 family gene delivery have significant limitations, cellular protein transduction represents a new and exciting approach utilizing peptides and proteins as drugs with intracellular targets. The mechanism by which proteins with transduction domains are taken up by cells and delivered to their targets is controversial but usually involves endocytosis. The effectiveness of transduced proteins may therefore be limited by their release from endosomes into the cytosol.

Bcl-2 homology domain 3 (BH3)-only protein Bid is posttranslationally cleaved by caspase-8 into its truncated form (tBid) and couples with stress signals to the mitochondrial cell death pathway. However, the physiological relevance of Bid is not clearly understood. Hepatocyte-specific knockout (KO) of Bcl-xL leads to naturally-occurring apoptosis despite co-expression of Mcl-1, which shares a similar anti-apoptotic function. We generated Bcl-xL KO, Bcl-xL/Bid double KO, Bcl-xL/Bak double KO, Bcl-xL/Bax double KO, and Bcl-xL/Bak/Bax triple KO mice and found that hepatocyte apoptosis caused by Bcl-xL deficiency was completely dependent on Bak and Bax, and surprisingly on Bid. This indicated that, in the absence of Bid, Bcl-xL is not required for the integrity of differentiated hepatocytes, suggesting a complicated interaction between core Bcl-2 family proteins and BH3-only proteins even in a physiological setting. Indeed, a small but significant level of tBid was present in wild-type liver under physiological conditions. tBid was capable of binding to Bcl-xL and displacing Bak and Bax from Bcl-xL, leading to release of cytochrome c from wild-type mitochondria. Bcl-xL–deficient mitochondria were more susceptible to tBid-induced cytochrome c release. Finally, administration of ABT-737, a pharmacological inhibitor of Bcl-2/Bcl-xL, caused Bak/Bax-dependent liver injury, but this was clearly ameliorated with a Bid KO background.

Conclusion

Bid, originally considered to be a sensor for apoptotic stimuli, is constitutively active in healthy liver cells and is involved in the Bak/Bax-dependent mitochondrial cell death pathway. Healthy liver cells are addicted to a single Bcl-2–like molecule because of BH3 stresses, and therefore special caution may be required for the use of the Bcl-2 inhibitor for cancer therapy.

Background

Phyllanthus is a traditional medicinal plant that has been used in the treatment of many diseases including hepatitis and diabetes. The main aim of the present work was to investigate the potential cytotoxic effects of aqueous and methanolic extracts of four Phyllanthus species (P.amarus, P.niruri, P.urinaria and P.watsonii) against skin melanoma and prostate cancer cells.

Methodology/Principal Findings

Phyllanthus plant appears to possess cytotoxic properties with half-maximal inhibitory concentration (IC50) values of 150–300 µg/ml for aqueous extract and 50–150 µg/ml for methanolic extract that were determined using the MTS reduction assay. In comparison, the plant extracts did not show any significant cytotoxicity on normal human skin (CCD-1127Sk) and prostate (RWPE-1) cells. The extracts appeared to act by causing the formation of a clear “ladder” fragmentation of apoptotic DNA on agarose gel, displayed TUNEL-positive cells with an elevation of caspase-3 and -7 activities. The Lactate Dehydrogenase (LDH) level was lower than 15% in Phyllanthus treated-cancer cells. These indicate that Phyllanthus extracts have the ability to induce apoptosis with minimal necrotic effects. Furthermore, cell cycle analysis revealed that Phyllanthus induced a Go/G1-phase arrest on PC-3 cells and a S-phase arrest on MeWo cells and these were accompanied by accumulation of cells in the Sub-G1 (apoptosis) phase. The cytotoxic properties may be due to the presence of polyphenol compounds such as ellagitannins, gallotannins, flavonoids and phenolic acids found both in the water and methanol extract of the plants.

Conclusions/Significance

Phyllanthus plant exerts its growth inhibition effect in a selective manner towards cancer cells through the modulation of cell cycle and induction of apoptosis via caspases activation in melanoma and prostate cancer cells. Hence, Phyllanthus may be sourced for the development of a potent apoptosis-inducing anticancer agent.

The BH3-only BCL-2 family protein BID is activated by caspase-8 cleavage upon engagement of cell surface death receptors. The resulting 15 kDa C-terminal fragment, tBID, translocates to mitochondria triggering the release of cytotoxic molecules and cell death. The pro-apoptotic activity of tBID is regulated by its interactions with pro-survival BCL-XL and pro-death BAX, both in the cytosol and at the mitochondrial membrane. In this study we characterize the molecular interactions between full-length tBID and BCL-XL using NMR spectroscopy and isothermal titration calorimetry (ITC). In aqueous solution, tBID adopts an α-helical but dynamically disordered conformation, however, the three-dimensional conformation is stabilized when tBID engages its BH3 domain in the BH3-binding hydrophobic groove of BCL-XL to form a stable heterodimeric complex. Characterization of the binding thermodynamics by ITC reveals that the interaction between tBID and BCL-XL is driven by enthalpy, but disfavored by the entropy associated with the conformational order induced in tBID upon binding BCL-XL.

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-δ (PKC-δ). 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.

To search for novel strategies to enhance the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis pathways in glioblastoma, we used the B-cell lymphoma 2/Bcl2-like 2-inhibitor ABT-737. Here we report that ABT-737 and TRAIL cooperate to induce apoptosis in several glioblastoma cell lines in a highly synergistic manner (combination index <0.1). Interestingly, the concerted action of ABT-737 and TRAIL to trigger the accumulation of truncated Bid (tBid) at mitochondrial membranes is identified as a key underlying mechanism. ABT-737 and TRAIL cooperate to cleave BH3-interacting domain death agonist (Bid) into its active fragment tBid, leading to increased accumulation of tBid at mitochondrial membranes. Coinciding with tBid accumulation, the activation of Bcl2-associated X protein (Bax), loss of mitochondrial membrane potential, release of cytochrome-c and second mitochondria-derived activator of caspase (Smac) into the cytosol and caspase activation are strongly increased in cotreated cells. Of note, knockdown of Bid significantly decreases ABT-737- and TRAIL-mediated Bax activation and apoptosis. Also, caspase-3 silencing reduces ABT-737- and TRAIL-induced Bid cleavage and apoptosis, indicating that a caspase-3-driven, mitochondrial feedback loop contributes to Bid processing. Importantly, ABT-737 profoundly enhances TRAIL-triggered apoptosis in primary cultured glioblastoma cells derived from tumor material, underlining the clinical relevance. Also, ABT-737 acts in concert with TRAIL to suppress tumor growth in an in vivo glioblastoma model. In conclusion, the rational combination of ABT-737 and TRAIL cooperates to trigger tBid mitochondrial accumulation and apoptosis. This approach presents a promising strategy for targeting the apoptosis pathways in glioblastoma, which warrants further investigation.

Background

The pro-apoptotic effector Bid induces mitochondrial apoptosis in synergy with Bax and Bak. In response to death receptors activation, Bid is cleaved by caspase-8 into its active form, tBid (truncated Bid), which then translocates to the mitochondria to trigger cytochrome c release and subsequent apoptosis. Accumulating evidence now indicate that the binding of tBid initiates an ordered sequences of events that prime mitochondria from the action of Bax and Bak: (1) tBid interacts with mitochondria via a specific binding to cardiolipin (CL) and immediately disturbs mitochondrial structure and function idependently of its BH3 domain; (2) Then, tBid activates through its BH3 domain Bax and/or Bak and induces their subsequent oligomerization in mitochondrial membranes. To date, the underlying mechanism responsible for targeting tBid to mitochondria and disrupting mitochondrial bioenergetics has yet be elucidated.

Principal Findings

The present study investigates the mechanism by which tBid interacts with mitochondria issued from mouse hepatocytes and perturbs mitochondrial function. We show here that the helix αH6 is responsible for targeting tBid to mitochondrial CL and disrupting mitochondrial bioenergetics. In particular, αH6 interacts with mitochondria through electrostatic interactions involving the lysines 157 and 158 and induces an inhibition of state-3 respiration and an uncoupling of state-4 respiration. These changes may represent a key event that primes mitochondria for the action of Bax and Bak. In addition, we also demonstrate that tBid required its helix αH6 to efficiently induce cytochrome c release and apoptosis.

Conclusions

Our findings provide new insights into the mechanism of action of tBid, and particularly emphasize the importance of the interaction of the helix αH6 with CL for both mitochondrial targeting and pro-apoptotic activity of tBid. These support the notion that tBid acts as a bifunctional molecule: first, it binds to mitochondrial CL via its helix αH6 and destabilizes mitochondrial structure and function, and then it promotes through its BH3 domain the activation and oligomerization of Bax and/or Bak, leading to cytochrome c release and execution of apoptosis. Our findings also imply an active role of the membrane in modulating the interactions between Bcl-2 proteins that has so far been underestimated.

Phyllanthus urinaria Linnea (Euphorbiaceae) is one of the traditional medicinal plants widely used by oriental people to treat various diseases. We have previously demonstrated that the acetone extract of P. urinaria inhibits herpes simplex virus type 2 (HSV-2) but not HSV-1 infection. In a continuing effort to clarify the antiviral mechanisms of P. urinaria, we isolated the pure compound excoecarianin from the whole plant of P. urinaria through acetone extraction, and investigated its anti-HSV-1 and HSV-2 activities. Our results indicated that excoecarianin protected Vero cells from HSV-2 but not HSV-1 infection, and its 50% inhibitory concentration (IC50) was 1.4 ± 0.1 μM. The antiviral effective concentration of excoecarianin did not affect the viability or the morphology of Vero cells. Although excoecarianin inhibited HSV-2 infection, the inhibitory effect, however, was most prominent when excoecarianin was concurrently added with the virus. Pretreatment of Vero cells with excoecarianin with removal of the drug prior to infection did not yield any antiviral effects, and the same observation was made for post viral entry treatment. Subsequent studies revealed that excoecarianin inactivated HSV-2 virus particles to prevent viral infection. A synergistic antiviral effect against HSV-2 was also observed when Vero cells were treated with a combination of acyclovir (ACV) and excoecarianin. These results suggested that excoecarianin merits to be further explored as an entry inhibitor against HSV-2 and could potentially be investigated for combinatorial drug treatment with nucleoside analogues such as ACV in therapeutic management of HSV-2 infection.

During initiation of apoptosis, Bcl-2 family proteins regulate the permeability of mitochondrial outer membrane. BH3-only protein, tBid, activates pro-apoptotic Bax to release cytochrome c from mitochondria. tBid also activates anti-apoptotic Bcl-2 in the mitochondrial outer membrane, changing it from a single-spanning to a multi-spanning conformation that binds the active Bax and inhibits cytochrome c release. However, it is not known whether other mitochondrial proteins are required to elicit the tBid-induced Bcl-2 conformational alteration. To define the minimal components that are required for the functionally important Bcl-2 conformational alteration, we reconstituted the reaction using purified proteins and liposomes. We found that purified tBid was sufficient to induce a conformational alteration in the liposome-tethered, but not cytosolic Bcl-2, resulting in a multi-spanning form that is similar to the one found in the mitochondrial outer membrane of drug treated cells. Mutations that abolished tBid/Bcl-2 interaction also abolished the conformational alteration, demonstrating that a direct tBid/Bcl-2 interaction at the membrane is both required and sufficient to elicit the conformational alteration. Furthermore, active Bax also elicited the Bcl-2 conformational alteration. Bcl-2 mutants that displayed increased or decreased activity in the conformational alteration assay, showed corresponding activities in inhibiting pore formation by Bax in vitro, and in preventing apoptosis in vivo. Thus, there is a strong correlation between the direct interaction of membrane-bound Bcl-2 and tBid with activation of Bcl-2 in vitro and in vivo.

Abstract

Two principal pathways exist by which cells can undergo apoptotic death, known as the extrinsic and the intrinsic pathways. Binding of a ligand to a death receptor activates the extrinsic pathway. In the intrinsic pathway, an apoptotic stimulus, such as neurotrophin withdrawal or exposure to a toxin, causes a proapoptotic member of the Bcl-2 family of proteins, such as Bax, to permeabilize the outer mitochondrial membrane. This allows redistribution of cytochrome c from the mitochondrial intermembrane space into the cytoplasm, where it causes activation of caspase proteases and, subsequently, cell death. A dramatic increase occurs in mitochondria-derived reactive oxygen species (ROS) during the apoptotic death of sympathetic, cerebellar granule, and cortical neurons. These ROS lie downstream of Bax in each cell type. Here I review possible mechanisms by which Bax causes increased ROS during neuronal apoptosis. I also discuss evidence that these ROS are an important part of the apoptotic cascade in these cells. Finally, I discuss evidence that suggests that neurotrophins prevent release of cytochrome c in neurons through activation of an antioxidant pathway. Antioxid. Redox Signal. 14, 1437–1448.

BID, a proapoptotic BCL-2 family member, plays an essential role in the tumor necrosis factor alpha (TNF-α)/Fas death receptor pathway in vivo. Activation of the TNF-R1 receptor results in the cleavage of BID into truncated BID (tBID), which translocates to the mitochondria and induces the activation of BAX or BAK. In TNF-α-activated FL5.12 cells, tBID becomes part of a 45-kDa cross-linkable mitochondrial complex. Here we describe the biochemical purification of this complex and the identification of mitochondrial carrier homolog 2 (Mtch2) as part of this complex. Mtch2 is a conserved protein that is similar to members of the mitochondrial carrier protein family. Our studies with mouse liver mitochondria indicate that Mtch2 is an integral membrane protein exposed on the surface of mitochondria. Using blue-native gel electrophoresis we revealed that in viable FL5.12 cells Mtch2 resides in a protein complex of ca. 185 kDa and that the addition of TNF-α to these cells leads to the recruitment of tBID and BAX to this complex. Importantly, this recruitment was partially inhibited in FL5.12 cells stably expressing BCL-XL. These results implicate Mtch2 as a mitochondrial target of tBID and raise the possibility that the Mtch2-resident complex participates in the mitochondrial apoptotic program.

Ethanol intoxication stimulates the production of proinflammatory cytokines, increases the formation of reactive oxygen species, and induces mitochondrial impairment. However, information is limited as to the exact sequence and components involved in ethanol-induced hepatotoxicity. Acute ethanol exposure enhances mitochondrial superoxide (O2.−) production and impairs mitochondrial Ca2+ handling. In turn, O2.− facilitates cytochrome c release and mitochondrial membrane potential loss that is not dependent upon H2O2 and divalent cations and requires Bak in a Bax-independent fashion. Furthermore, triggering of Bak's proapoptotic activity requires the cytosolic presence of Bid, a BH3-only protein that is processed by the initiator caspase-2. Together, these studies identify an O2.−-driven, caspase-initiated apoptotic pathway that selectively involves the Bcl-2 family proteins Bid and Bak. This pathway manifests itself during chronic ethanol consumption in aged animals and identifies caspase-2, Bid, and Bak as essential mediators of O2.−-induced apoptosis that may prove effective targets for the development of therapeutics to treat alcoholic liver disease.

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was >95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. Bid cleavage was caspase-dependent (55–60%) and calcium-dependent (40–45%). Intracellular calcium as an intrinsic mechanism and extracellular calcium as an extrinsic mechanism were responsible for about 30 and 70% of calcium dependence for Bid cleavage, respectively. The results reveal electric field-mediated cell death induction and progression, activating pro-apoptotic-like mechanisms and affecting plasma membrane and intracellular functions, primarily through extrinsic-like pathways with smaller contributions from intrinsic-like pathways. Nanosecond second pulsed electric fields trigger heterogeneous cell death mechanisms in E4 SCC populations to delete them, with caspase-associated cell death as a predominant, but not an unaccompanied event.

Glioblastoma is the most malignant brain tumor of astroglial origin. It renders poor response or resistance to existing therapeutics. We used all-trans retinoic acid (ATRA) and interferon gamma (IFN-γ) alone and in combination for controlling human glioblastoma T98G xenografted in nude mice. Histopathological examination showed astrocytic differentiation in ATRA group, some apoptosis in IFN-γ group, and occurrence of differentiation and enhancement of apoptosis in ATRA plus IFN-γ group. ATRA plus IFN-γ induced extrinsic pathway of apoptosis by activation of caspase-8 and cleavage of Bid to tBid and also down regulation of hTERT, c-IAP2, and survivin and upregulation of Smac/Diablo to promote apoptosis. Mitochondrial release of apoptosis-inducing factor (AIF) induced caspase-independent pathway and also upregulation of calpain and caspase-dependent pathways ultimately activated caspase-3 for apoptosis. Increased activities of calpain and caspase-3 degraded 270 kD α-spectrin at the specific sites to generate 145 kD spectrin breakdown product (SBDP) and 120 kD SBDP, respectively. In situ TUNEL and double immunofluorescent labelings detected apoptosis with increased expression of calpain, caspase-12, caspase-3, and AIF in tumors after treatment with IFN-γ and most effectively with ATRA plus IFN-γ. Results indicated that ATRA plus IFN-γ activated multiple molecular mechanisms for increasing apoptosis in human glioblastoma in vivo.

Although Bcl-XL and Bax are structurally similar, activated Bax forms large oligomers that permeabilize the outer mitochondrial membrane, thereby committing cells to apoptosis, whereas Bcl-XL inhibits this process. Two different models of Bcl-XL function have been proposed. In one, Bcl-XL binds to an activator, thereby preventing Bax activation. In the other, Bcl-XL binds directly to activated Bax. It has been difficult to sort out which interaction is important in cells, as all three proteins are present simultaneously. We examined the mechanism of Bax activation by tBid and its inhibition by Bcl-XL using full-length recombinant proteins and measuring permeabilization of liposomes and mitochondria in vitro. Our results demonstrate that Bcl-XL and Bax are functionally similar. Neither protein bound to membranes alone. However, the addition of tBid recruited molar excesses of either protein to membranes, indicating that tBid activates both pro- and antiapoptotic members of the Bcl-2 family. Bcl-XL competes with Bax for the activation of soluble, monomeric Bax through interaction with membranes, tBid, or t-Bid-activated Bax, thereby inhibiting Bax binding to membranes, oligomerization, and membrane permeabilization. Experiments in which individual interactions were abolished by mutagenesis indicate that both Bcl-XL–tBid and Bcl-XL–Bax binding contribute to the antiapoptotic function of Bcl-XL. By out-competing Bax for the interactions leading to membrane permeabilization, Bcl-XL ties up both tBid and Bax in nonproductive interactions and inhibits Bax binding to membranes. We propose that because Bcl-XL does not oligomerize it functions like a dominant-negative Bax in the membrane permeabilization process.

Author Summary

During development and under stress, cells can become committed to die via programmed cell death (apoptosis). In most cases, the permeabilization of the outer mitochondrial membrane is a key component of this commitment. The membrane permeablization step is both positively and negatively regulated by members of the Bcl-2 family of proteins. One member of this protein family with only a BH3 region, such as tBid, activates another family member, Bax, causing it to form large complexes that generate membrane-spanning pores, hence making the membrane permeable. Antiapoptotic members of the Bcl-2 family, such as Bcl-XL, are structurally similar to Bax but inhibit the membrane permeabilization process by an unknown mechanism. Two mutually exclusive models have been proposed to explain how the Bcl-2 family is operating: one states that Bcl-XL binds to tBid, thereby preventing Bax activation, while the second suggests that Bcl-XL binds directly to activated Bax. It has been difficult to sort out which interaction is important in cells, where multiple members of all three protein families are present simultaneously. Here, we describe an in vitro system containing the three recombinant proteins and the use of mutagenesis to selectively remove individual interactions. We show that Bcl-XL inhibits Bax by competing with it for binding to membranes, tBid, and activated Bax. Because Bcl-XL does not form pores, it inhibits apoptosis by acting as if it is a dominant-negative version of Bax.

Bcl-XL and Bax are structurally similar members of the Bcl-2 family of cell-death-related proteins, and they compete for binding to membranes, as well as to Bcl-2 family member tBid and activated Bax. Unlike Bax, Bcl-XL is unable to oligomerize and form pores in membranes, so it inhibits membrane permeabilization--a key step during commitment to apoptosis--by functioning like a dominant-negative Bax.

Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a “BH3-domain–only” BCL-2 family member, triggers the homooligomerization of “multidomain” conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a “multidomain” proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.

The objective of this study was to elucidate the cytotoxic mechanism of Compound K, with respect to the involvement of reactive oxygen species (ROS) and the mitochondrial involved apoptosis, in HT-29 human colon cancer cells. Compound K exhibited a concentration of 50% growth inhibition (IC50) at 20 μg/mL and cytotoxicity in a time dependent manner. Compound K produced intracellular ROS in a time dependent fashion; however, N-acetylcysteine (NAC) pretreatment resulted in the inhibition of this effect and the recovery of cell viability. Compound K induced a mitochondria-dependent apoptotic pathway via the modulation of Bax and Bcl-2 expressions, resulting in the disruption of the mitochondrial membrane potential (Δψm). Loss of the Δψm was followed by cytochrome c release from the mitochondria, resulting in the activation of caspase-9, -3, and concomitant poly ADP-ribosyl polymerase (PARP) cleavage, which are the indicators of caspase-dependent apoptosis. The apoptotic effect of Compound K, exerted via the activation of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), was abrogated by specific MAPK inhibitors. This study demonstrated that Compound K-mediated generation of ROS led to apoptosis through the modulation of a mitochondria-dependent apoptotic pathway and MAPK pathway.

The death receptor apoptosis pathway is intimately connected with the mitochondrial apoptosis pathway. Bid is a BH3-only pro-death Bcl-2 family protein and is the major molecule linking the two pathways. Bid-mediated mitochondrial activation occurs early and is responsible for the prompt progress of tumor necrosis factor alpha (TNF-α)-induced apoptosis. However, in both cultured cells and animal models of TNF-α-induced injury, later-phase Bid-independent mitochondrial activation could be demonstrated. Consequently, bid-deficient mice are still susceptible to endotoxin-induced liver injury and mortality. Notably, embryonic hepatocyte apoptosis and lethality caused by TNF-α in the absence of p65relA cannot be rescued by the simultaneous deletion of bid. Further studies indicate that multiple mechanisms including reactive oxygen species, JNK, and permeability transition are critically involved in Bid-independent mitochondrial activation. Inhibition of these events suppresses TNF-α-induced mitochondrial activation and apoptosis in bid-deficient cells. These findings thus indicate that there are at least two sets of mechanisms of mitochondrial activation upon TNF-α stimulation. While the Bid-mediated mechanism is rapid and potent, the Bid-independent mechanism progresses gradually and involves multiple players. The critical involvement of Bid-independent mitochondrial activation in TNF-α-induced apoptosis demands the intervention of TNF-α-mediated tissue injury via multiple avenues.

Background

Bcl-2 homology domain (BH) 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid), which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid) from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM).

Results

Western blot experiments confirmed a translocation of FL-Bid to the mitochondria during excitotoxic apoptosis that was associated with the release of cytochrome-C from mitochondria. These results were confirmed by immunofluorescence analysis of Bid translocation during excitotoxic cell death using an antibody raised against the amino acids 1–58 of mouse Bid that is not able to detect tBid. Finally, inducible overexpression of FL-Bid or a Bid mutant that can not be cleaved by caspase-8 was sufficient to induce apoptosis in the hippocampal neuron cultures.

Conclusion

Our data suggest that translocation of FL-Bid is sufficient for the activation of mitochondrial cell death pathways in response to glutamate receptor overactivation.

The accumulation of misfolded proteins stresses the endoplasmic reticulum (ER) and triggers cell death through activation of the multidomain proapoptotic BCL-2 proteins BAX and BAK at the outer mitochondrial membrane. The signaling events that connect ER stress with the mitochondrial apoptotic machinery remain unclear, despite evidence that deregulation of this pathway contributes to cell loss in many human degenerative diseases. In order to “trap” and identify the apoptotic signals upstream of mitochondrial permeabilization, we challenged Bax−/− Bak−/− mouse embryonic fibroblasts with pharmacological inducers of ER stress. We found that ER stress induces proteolytic activation of the BH3-only protein BID as a critical apoptotic switch. Moreover, we identified caspase-2 as the premitochondrial protease that cleaves BID in response to ER stress and showed that resistance to ER stress-induced apoptosis can be conferred by inhibiting caspase-2 activity. Our work defines a novel signaling pathway that couples the ER and mitochondria and establishes a principal apoptotic effector downstream of ER stress.