Bax-mediated Pathway Research Articles

Sesamol Induces Apoptosis by Altering Expression of Bcl-2 and Bax Proteins and Modifies Skin Tumor Development in Balb/c Mice.

Chemoprevention using natural agents has emerged as a new and promising strategy for reducing cancer burden. Sesamol, a water soluble lignin, is a potent antioxidant with potential anticancer activities. Its small size (molecular weight: 138.34g) coupled with easy permeability (log P: 1.29) results in its excessive systemic loss therefore, compromising local bioavailability. Furthermore, irritant nature of sesamol limits its application on skin per se. Present study aims to evaluate chemopreventive efficacy of free and encapsulated (SLNs) sesamol, at gross and molecular level, in DMBA induced skin cancer animal model. Evaluation is done in terms of tumor burden quantification, histological evaluation of skin, determination of oxidative stress, and quantification of apoptotic proteins, bcl-2 and bax, using both western blot analysis and immunofluorescence studies. Sesamol administration (both in free and encapsulated form) significantly decreased the tumor burden and lipid peroxidation level and increased anti-oxidant levels, thereby hampering the development and promotion of skin tumors. Further, downregulation of bcl-2 and stimulation of bax protein expression on treatment with both free and encapsulated sesamol was responsible for the induction of apoptosis in tumor cells. Encapsulating sesamol into SLNs not only reduced its irritant nature which limits its direct topical application but also improved its local targeting to skin. Both free and encapsulated sesamol demonstrated the inhibition of tumor progression by inducing skin cell apoptosis via bcl-2/bax mediated pathway.

Pandemic Influenza A (H1N1) Virus Infection Increases Apoptosis and HIV-1 Replication in HIV-1 Infected Jurkat Cells.

Influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. It is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (HIV)-1 replication in HIV-1-infected patients. Using a lymphoma cell line, Jurkat, we examined the in vitro effects of pandemic influenza A (H1N1) virus (pH1N1) infection on cell death and HIV-1 RNA production in infected cells. We found that pH1N1 infection increased apoptotic cell death through Fas and Bax-mediated pathways in HIV-1-infected Jurkat cells. Infection with pH1N1 virus could promote HIV-1 RNA production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), nuclear factor of activated T-cells (NFAT) and activator protein 1 (AP-1) through mitogen-activated protein kinases (MAPK) pathways and T-cell antigen receptor (TCR)-related pathways. The replication of HIV-1 latent infection could be reactivated by pH1N1 infection through TCR and apoptotic pathways. These data indicate that HIV-1 replication can be activated by pH1N1 virus in HIV-1-infected cells resulting in induction of cell death through apoptotic pathways.

Presenilin 2 overexpression is associated with apoptosis in Neuro2a cells.

Presenilin 1 (PS1) and PS2 are evolutionarily conserved transmembrane proteins of the aspartyl protease family. Initially, they were reported to be associated with the early onset of familial, early-onset Alzheimer’s disease. PS1 has been implicated in several crucial brain functions including developmental processes, synaptic plasticity, and processing of various molecules, while PS2 has been poorly studied and is considered to be a compensatory partner of PS1. Certain controversial reports have suggested that PS2 has a role in apoptosis, though the underlying mechanism is not clear. To ascertain the role of PS2 in apoptosis, mouse neuroblastoma cells (Neuro2a) were transfected with a cDNA construct encoding full length mouse PS2 and analyzed for viability, expression of PS1, PS2, Bax and p53, Bax protein, and status of chromatin condensation. Our results showed reduced viability, condensed chromatin and higher expression of Bax at mRNA and protein levels, but no change in the expression of p53 and PS1 in PS2-overexpressing Neuro2a cells. Thus, it is evident that PS2, independent of PS1, is associated with apoptosis via a Bax-mediated pathway. These findings might help in the understanding of the involvement of PS2 in apoptosis and its associated brain disorders.

Selective induction of apoptosis in various cancer cells irrespective of drug sensitivity through a copper chelate, copper N-(2 hydroxy acetophenone) glycinate: crucial involvement of glutathione

Drug induced toxicity and drug resistance are the major impediments to successful application of cancer chemotherapy. Therefore, selective targeting of the key biochemical events of the malignant cells may have a great therapeutic potential in specifically kill the cancer cells. We have evaluated in vitro the cytotoxic efficacy of a previously reported copper complex viz. copper N-(2-hydroxy acetophenone) glycinate (CuNG) on different drug sensitive and resistant cancer cell lines by MTT, annexin V positivity and caspase 3 activation assays. We have also investigated the underlying signalling events in CuNG mediated apoptosis of cancer cells by Western blotting technique. We have found that CuNG preferentially induces apoptosis to malignant cells irrespective of drug sensitivity and spares the normal cells. Our studies disclose that CuNG causes cellular redox imbalance in cancer cells through depletion of intracellular GSH level. CuNG mediated depletion of intracellular GSH level induces mitochondrial superoxide generation, which detaches cyto C from mitochondrial membrane through lipid peroxidation. The detached cyto C then release into the extra mitochondrial milieu in Bax mediated pathway where CuNG facilitates the binding of Bax through dissociation of hexokinase II from mitochondrial membrane. The present study opens the possibility of developing effective chemotherapeutic drugs by synthesizing numerous chemical compounds capable of targeting cellular redox environment and thus specifically kills cancer cells of broad spectrum.

Astragaloside IV prevents MPP+-induced SH-SY5Y cell death via the inhibition of Bax-mediated pathways and ROS production

Parkinson's disease (PD) is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra. Oxidative stress and neural degeneration are suggested to be involved in the pathogenesis of PD. Previous studies have revealed that Astragaloside IV (AS-IV) can reduce inflammation and oxidation, making it a potential therapeutic agent for neurodegenerative disease. In this study, we investigated whether AS-IV protect against 1-methyl-4-phenylpyridnium ion (MPP(+))-induced dopaminergic neurotoxicity in SH-SY5Y cells and determined the mechanism of AS-IV neuroprotection. We found that pretreatment with AS-IV significantly reversed the loss of cell viability, nuclear condensation, the generation of intracellular reactive oxygen species (ROS), and the increase in Bax/Bcl-2 ratio and the activity of caspase-3 induced by MPP(+). Our study suggests that the neuroprotective effect of AS-IV is related to mechanisms including ROS production and the inhibition of Bax-mediated pathway. The present study supports the notion that AS-IV may be a promising neuroprotective agent for the treatment of neurodegenerative disorders such as PD.

Fasudil mesylate protects PC12 cells from oxidative stress injury via the Bax-mediated pathway.

We previously reported that fasudil mesylate (FM) improves neurological deficit and neuronal damage in rats with ischemia following middle cerebral artery occlusion and reperfusion in vivo. In this study, the properties of FM on hydrogen peroxide (H(2)O(2))-induced oxidative stress insult in cultured PC12 cells as well as the underlying mechanisms were investigated in vitro. Pretreatment with FM (5, 10 μM) prior to H(2)O(2) exposure significantly elevated cell viability, reduced cell apoptosis by MTT assay, LDH assay, Hoechst 33258 dye staining, and FM also decreased the accumulation of reactive oxygen species (ROS) by DCFH-DA staining and NBT test. Furthermore, FM also reversed the upregulation of Bax/Bcl-2 ratio, the downstream cascade following ROS. FM protected PC12 cells from oxidative stress insult via downregulating the Bax/Bcl-2 ratio. These findings indicate that a direct effect of fasudil mesylate on PC12 cells may be partly responsible for its protective effect against oxidative stress injury.

Thai Acanthamoeba isolate (T4) induced apoptotic death in neuroblastoma cells via the Bax-mediated pathway

A Thai Acanthamoeba isolate named AS recovered from a corneal scraping of a keratitis patient was genotypically determined as T4. AS trophozoites were used for studying Acanthamoeba-induced apoptosis in mouse neuroblastoma NA cells during in vitro co-cultivation. The Acanthamoeba-exposed NA cells showed signs of apoptosis including cell shrinkage, nuclear condensation and DNA fragmentation. The effect was confirmed by DNA laddering electrophoresis. Involvement of caspase enzymes and mitochondrial pro- and anti-apoptotic proteins (Bax and Bcl-2) in AS-induced apoptosis was determined. The use of Z-VAD-FMK, a pan-caspase inhibitor, significantly reduced the apoptotic effect, while Bax/Bcl-2 ratio analysis showed a significant increase in the expression of apoptotic proteins in AS-exposed NA cells. These results strongly indicated that apoptosis induced by AS trophozoites is caspase-dependent and is mediated by over-expression of pro-apoptotic proteins in the mitochondrial pathway. This is the first report on the role of Bax in mediating apoptosis induced by Acanthamoeba.

IL-9 and IL-13 Induce Mucous Cell Metaplasia That Is Reduced by IFN-γ in a Bax-Mediated Pathway

One of the major aspects of airway remodeling in asthma is the development of mucous cell metaplasia (MCM). The role of cytokines in the generation and resolution of MCM has been studied in mice and in isolated airway epithelial cells in culture. However, studies using organ cultures that keep the tubular structure of the airways intact and allow studies in the absence of inflammatory cells have not been reported. We established an organ culture system that replicates the allergen-induced MCM in mice and analyzed the role of Bax in the IFN-gamma-induced resolution of MCM. IL-9 or IL-13 induced MCM independently, but a combined IL-9/IL-13 treatment enhanced MCM synergistically. Addition of IFN-gamma at 0.1 ng/ml concentration further increased MCM to levels observed in allergen-exposed mice in vivo. However, MCM was reduced when explants were treated with 50 ng/ml IFN-gamma after MCM was established. While IL-9/IL-13 induced MCM in bronchioles microdissected from bax(+/+) and bax(-/-) mice to a similar extent, IFN-gamma treatment reduced MCM only in bronchioles from bax(+/+) but not in bax(-/-) bronchioles. Restoration of Bax expression in bax(-/-) bronchioles using an adenoviral expression system reduced IL-9/IL-13-induced MCM while MCM was similar in noninfected or adenoviral green fluorescent protein-infected bax(-/-) bronchioles. Furthermore, expressing Bax using an adenoviral expression system reduced allergen-induced MCM in mice. These studies show that allergen-induced MCM is a response to a combination of various cytokines at defined concentrations and that IFN-gamma requires Bax for the resolution of MCM.

Transforming Growth Factor (TGF)-β1 Stimulates Pulmonary Fibrosis and Inflammation via a Bax-dependent, Bid-activated Pathway That Involves Matrix Metalloproteinase-12

Fibrosis, apoptosis, and the exaggerated production of transforming growth factor (TGF)-beta(1) are juxtaposed in a variety of pulmonary diseases including the interstitial lung diseases and asthma. In these disorders, the relationships between these responses are not well defined. In addition, the apoptosis pathways that contribute to these responses and the mechanism(s) of their contribution have not been described. We hypothesized that BH3 domain-only protein-induced apoptosis plays an important role in the pathogenesis of TGF-beta(1)-induced pulmonary responses. To test this hypothesis, we characterized the effects of transgenic TGF-beta(1) in mice with wild type (WT) and null Bax loci. To investigate the mechanisms of Bax activation and its effector functions, we also compared the effects of TGF-beta(1) in mice with WT and null Bid and matrix metalloproteinase (MMP)-12 loci, respectively. These studies demonstrate that TGF-beta(1) is a potent stimulator of Bax, Bid, and MMP-12. The studies also demonstrate that Bax and Bid play key roles in the pathogenesis of TGF-beta(1)-induced inflammation, fibrosis, and apoptosis; that TGF-beta(1) stimulates MMP-12, TIMP-1, and cathepsins and inhibits MMP-9 and p21 via Bax- and Bid-dependent mechanisms; and that TGF-beta(1)-stimulated pulmonary fibrosis is ameliorated in MMP-12-deficient animals. Finally, they demonstrate that Bax, Bid, and MMP-12 play similar roles in bleomycin-induced fibrosis, thereby highlighting the importance of this Bid-activated, Bax-mediated pathway and downstream MMP-12 in a variety of fibrogenic settings.

Enhanced Killing of Melanoma Cells by Simultaneously Targeting Mcl-1 and NOXA

By deciphering the dysregulation of apoptosis in melanoma cells, new treatment approaches exploiting aberrant control mechanisms regulating cell death can be envisioned. Among the Bcl-2 family, a BH3-only member, NOXA, functions in a specific mitochondrial-based cell death pathway when melanoma cells are exposed to a proteasome inhibitor (e.g., bortezomib). Some therapeutic agents, such as bortezomib, not only induce proapoptotic Bcl-2 family members and active conformational changes in Bak and Bax but also are associated with undesirable effects, including accumulation of antiapoptotic proteins, such as Mcl-1. To enhance the bortezomib-mediated killing of melanoma cells, the apoptotic pathway involving NOXA was further investigated, leading to identification of an important target (i.e., the labile Bcl-2 homologue Mcl-1 but not other survival proteins). To reduce Mcl-1 levels, melanoma cells were pretreated with several different agents, including Mcl-1 small interfering RNA (siRNA), UV light, or the purine nucleoside analogue fludarabine. By simultaneously triggering production of NOXA (using bortezomib) as well as reducing Mcl-1 levels (using siRNA, UV light, or fludarabine), significantly enhanced killing of melanoma cells was achieved. These results show binding interactions between distinct Bcl-2 family members, such as NOXA and Mcl-1, in melanoma cells, paving the way for novel and rational therapeutic combination strategies, which target guardians of the proapoptotic Bak- and Bax-mediated pathways, against this highly aggressive and often fatal malignancy.

Methoxychlor Directly Affects Ovarian Antral Follicle Growth and Atresia through Bcl-2- and Bax-Mediated Pathways

Methoxychlor (MXC) is an organochlorine pesticide and reproductive toxicant. While in vivo studies indicate that MXC exposure increases antral follicle atresia, in part by altering apoptotic regulators (Bcl-2 and Bax), they do not distinguish whether MXC does so via direct or indirect mechanisms. Therefore, we utilized an in vitro follicle culture system to test the hypothesis that MXC is directly toxic to antral follicles, and that overexpression of anti-apoptotic Bcl-2, or deletion of pro-apoptotic Bax, protects antral follicles from MXC-induced toxicity. Antral follicles were isolated from wild-type (WT), Bcl-2 overexpressing (Bcl-2 OE), or Bax deficient (BaxKO) mice, and exposed to dimethylsulfoxide (control) or MXC (1-100 microg/ml) for 96 h. Follicle diameters were measured every 24 h to assess growth. After 96 h, follicles were histologically evaluated for atresia or collected for quantitative PCR analysis of Bcl-2 and Bax mRNA levels. MXC (10-100 microg/ml) significantly inhibited antral follicle growth at 72 and 96 h, and increased atresia (100 microg/ml) compared to controls at 96 h. Furthermore, MXC increased Bax mRNA levels between 48-96 h and decreased Bcl-2 mRNA levels at 96 h. While MXC inhibited growth of WT antral follicles beginning at 72 h, it did not inhibit growth of Bcl-2 OE or BaxKO follicles until 96 h. MXC also increased atresia of small and large WT and BaxKO antral follicles over controls, but it did not increase atresia of large Bcl-2 OE antral follicles over controls. These data suggest that MXC directly inhibits follicle growth partly by Bcl-2 and Bax pathways, and increases atresia partly through Bcl-2 pathways.

Docosahexaenoic Acid Induces Apoptosisviathe Bax-Independent Pathway in HL-60 Cells

We attempted to determine whether docosahexaenoic acid (DHA)-induced apoptosis is mediated via the Bax-mediated pathway in human myeloid leukemia HL-60 cells. DHA-induced apoptosis was confirmed by morphological analysis and caspase-3 activation. But, cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition (MPT), did not inhibit DHA-induced Bax translocation to mitochondria or caspase-3 activation. These data suggest that DHA can induce apoptosis via the Bax-independent pathway.

Neoplastic cell apoptosis in nude mice transplants with nasopharyngeal carcinoma cell lines

Objective: To observe the morphological features of neoplastic cell apoptosis developed in nude mice transplants with nasopharyngeal carcinoma (NPC) cell lines, CNE-1 and CNE-2, and to investigate the roles of p53, bcl-2 and bax playing in the process of apoptosis. Methods: CNE-1 and CNE-2 cell lines were inoculated and passed in nude mice for 3 generations. The cell apoptosis was detected on H & E and TUNEL staining slides. The expression of p53, bcl-2 and bax were detected by using immunohistochemistry. p53 gene alteration was assayed in cell lines and transplants by PCR-SSCP. Results: A considerable number of neoplastic cells underwent apoptosis in CNE-1 and CNE-2 transplant tissues. The “shrinkage necrosis” and apoptotic bodies were the main appearances of apoptosis. The p53 alteration was detected in exon 8 by PCR-SSCP and p53 protein accumulation observed in the cell smears and nude mice transplant tissue sections. All the transplant tissue sections of 3 passages showed bcl-2 negativity and bax overexpression. Conclusion: The neoplastic cells of CNE-1 and CNE-2 transplants underwent death mainly taking the way of apoptosis. The “shrinkage necrosis” and apoptotic bodies were the main morphological features of apoptosis seen in those transplants. The apoptosis in CNE-1 or CNE-2 nude mice transplant is highly probable through a p53-independent and bax-mediated pathway.