Siyaram Pandey

Role of mitochondria in neuronal cell death induced by oxidative stress; neuroprotection by Coenzyme Q10.

Neuronal cells depend on mitochondrial oxidative phosphorylation for most of their energy needs and therefore are at a particular risk for oxidative stress. Mitochondria play an important role in energy production and oxidative stress-induced apoptosis. In the present study, we have demonstrated that external oxidative stress induces mitochondrial dysfunction leading to increased ROS generation and ultimately apoptotic cell death in neuronal cells. Furthermore, we have investigated the role of Coenzyme Q10 as a neuroprotective agent. Coenzyme Q10 is a component of the mitochondrial respiratory chain and a potent anti-oxidant. Our results indicate that total cellular ROS generation was inhibited by Coenzyme Q10. Further, pre-treatment with Coenzyme Q10 maintained mitochondrial membrane potential during oxidative stress and reduced the amount of mitochondrial ROS generation. Our study suggests that water-soluble Coenzyme Q10 acts by stabilizing the mitochondrial membrane when neuronal cells are subjected to oxidative stress. Therefore, Coenzyme Q10 has the potential to be used as a therapeutic intervention for neurodegenerative diseases.

Pancratistatin: A natural anti-cancer compound that targets mitochondria specifically in cancer cells to induce apoptosis

The major hurdle in the fight against cancer is the non-specific nature of current treatments. The search for specific drugs that are non-cytotoxic to normal cells and can effectively target cancer cells has lead some researchers to investigate the potential anti-cancer activity of natural compounds. Some natural compounds, such as Taxol, have been shown to posses some anti-cancer potential. Pancratistatin (PST) is a natural compound that was isolated from the spider lily Pancratium littorale and shown to exhibit antineoplastic activity. The specificity of PST to cancer cells and the mechanism of PST’s action remain unknown. This study provides a detailed look at the effect of PST treatment on cancerous and normal cells. Our results indicate that PST induced apoptosis selectively in cancer cells and that the mitochondria may be the site of action of PST in cancer cells. A biochemical target available specifically in cancer cells may lead to the development of new and more effective cancer fighting agents.

Pancratistatin causes early activation of caspase-3 and the flipping of phosphatidyl serine followed by rapid apoptosis specifically in human lymphoma cells.

Recently a major research effort has been focused on the development of anticancer drugs by targeting the components of a biochemical pathway to induce apoptosis in cancerous cells. Some of the natural products (e.g. paclitaxel) have been proven to be useful in inducing apoptosis in cancer cells with limited specificity. Pancratistatin, a natural product isolated and characterized over a decade ago, has been shown to be cytostatic and antineoplastic. We investigated the specificity and biochemical mechanism of action of pancratistatin. Pancratistatin seemed to show more specificity than VP-16 or paclitaxel as an efficient inducer of apoptosis in human lymphoma (Jurkat) cells, with minimal effect on normal nucleated blood cells. Caspase-3 activation and exposure of phosphatidyl serine on the outer leaflet of the plasma membrane were earlier events than the generation of ROS and DNA fragmentation observed following pancratistatin treatment. This indicates a possible involvement of caspase-3 and plasma membrane proteins in the induction phase of apoptosis. Our results indicate that pancratistatin does not cause DNA double-strand breaks or DNA damage prior to the execution phase of apoptosis in cancer cells. Parallel experimentation with VP-16, a currently used medication for cancer treatment, indicated that VP-16 causes substantial DNA damage in normal non-cancerous blood cells, while pancratistatin does not cause any DNA double-strand breaks or DNA damage in non-cancerous cells. Taken together, our finding that pancratistatin induces apoptosis in cancer cells using non-genomic targets, and more importantly does not seem to have any affect non-cancerous cells, presents a significant platform to develop non-toxic anticancer therapies.

Oxidative stress-induced apoptosis in dividing fibroblasts involves activation of p38 MAP kinase and over-expression of Bax: Resistance of quiescent cells to oxidative stress

Oxidative stress has been postulated to be involved in aging and age-related degenerative diseases. Cell death as a result of oxidative stress plays an important role in the age related diseases. Using human diploid fibroblasts (HDF) as model to study the mechanism of cell death induced by oxidative stress, a condition was standardized to induce apoptosis in the early passage sub-confluent HDFs by a brief exposure of cells to 250 μM hydrogen peroxide. It was observed that p38 MAP kinase (MAPK) was activated soon after the treatment followed by over-expression of Bax protein in cells undergoing apoptosis. An interesting finding of the present study is that the confluent, quiescent HDFs were resistant to cell death under identical condition of oxidative stress. The contact-inhibited quiescent HDFs exhibited increased glutathione level following H2O2-treatment, did not activate p38 MAP kinase, or over-express Bax, and were resistant to cell death. These findings indicated that there was a correlation between the cell cycle and sensitivity to oxidative stress. This is the first report to our knowledge that describes a relationship between the quiescence state and anti-oxidative defense. Furthermore, our results also suggest that the p38MAPK activation-Bax expression pathway might be involved in apoptosis induced by oxidative stress.

Oxidative toxicity in neurodegenerative diseases: role of mitochondrial dysfunction and therapeutic strategies.

Besides fluorine, oxygen is the most electronegative element with the highest reduction potential in biological systems. Metabolic pathways in mammalian cells utilize oxygen as the ultimate oxidizing agent to harvest free energy. They are very efficient, but not without risk of generating various oxygen radicals. These cells have good antioxidative defense mechanisms to neutralize these radicals and prevent oxidative stress. However, increased oxidative stress results in oxidative modifications in lipid, protein, and nucleic acids, leading to mitochondrial dysfunction and cell death. Oxidative stress and mitochondrial dysfunction have been implicated in many neurodegenerative disorders including Alzheimers disease, Parkinsons disease, and stroke-related brain damage. Research has indicated mitochondria play a central role in cell suicide. An increase in oxidative stress causes mitochondrial dysfunction, leading to more production of reactive oxygen species and eventually mitochondrial membrane permeabilization. Once the mitochondria are destabilized, cells are destined to commit suicide. Therefore, antioxidative agents alone are not sufficient to protect neuronal loss in many neurodegenerative diseases. Combinatorial treatment with antioxidative agents could stabilize mitochondria and may be the most suitable strategy to prevent neuronal loss. This review discusses recent work related to oxidative toxicity in the central nervous system and strategies to treat neurodegenerative diseases.

Structure-activity studies on the lycorine pharmacophore: a potent inducer of apoptosis in human leukemia cells.

Abstract The direct chemoselective differential functionalization of the ring-C hydroxyl groups present in the Amaryllidaceae alkaloid lycorine is described allowing for selective manipulation of the 1,2-hydroxyl groups. A mini-library comprised of synthetic and natural lycorane alkaloids was prepared and their apoptosis-inducing activity investigated in human leukemia (Jurkat) cells. Further insights into the nature of this interesting apoptosis-inducing pharmacophore are described, including the requirement of both free hydroxyl groups in ring-C.

Paraquat induces oxidative stress, neuronal loss in substantia nigra region and Parkinsonism in adult rats: Neuroprotection and amelioration of symptoms by water-soluble formulation of Coenzyme Q10

BackgroundParkinsons disease, for which currently there is no cure, develops as a result of progressive loss of dopamine neurons in the brain; thus, identification of any potential therapeutic intervention for disease management is of a great importance.ResultsHere we report that prophylactic application of water-soluble formulation of coenzyme Q10 could effectively offset the effects of environmental neurotoxin paraquat, believed to be a contributing factor in the development of familial PD. In this study we utilized a model of paraquat-induced dopaminergic neurodegeneration in adult rats that received three weekly intra-peritoneal injections of the herbicide paraquat. Histological and biochemical analyses of rat brains revealed increased levels of oxidative stress markers and a loss of approximately 65% of dopamine neurons in the substantia nigra region. The paraquat-exposed rats also displayed impaired balancing skills on a slowly rotating drum (rotorod) evidenced by their reduced spontaneity in gait performance. In contrast, paraquat exposed rats receiving a water-soluble formulation of coenzyme Q10 in their drinking water prior to and during the paraquat treatment neither developed neurodegeneration nor reduced rotorod performance and were indistinguishable from the control paraquat-untreated rats.ConclusionOur data confirmed that paraquat-induced neurotoxicity represents a convenient rat model of Parkinsonian neurodegeneration suitable for mechanistic and neuroprotective studies. This is the first preclinical evaluation of a water-soluble coenzyme Q10 formulation showing the evidence of prophylactic neuroprotection at clinically relevant doses.

Oxidative stress and activation of proteasome protease during serum deprivation-induced apoptosis in rat hepatoma cells; inhibition of cell death by melatonin.

Growth factor deprivation-induced apoptosis has been shown in various cell systems and is recognized as one of the standard models for the study of programmed cell death. The mechanism of induction of apoptosis by serum deprivation is still not clear. The objective of the present study was to investigate if serum-deprivation causes oxidative stress, which then leads to apoptotic death. We have demonstrated that indeed, there was a significant increase in reactive oxygen species following serum deprivation of 5123tc hepatoma cells. Furthermore, treatment with anti-oxidants; melatonin or vitamin E, prevented cell death caused by serum-deprivation. We also demonstrated that there was activation of proteasome proteases and decrease in glutathione levels following serum deprivation. Interestingly, melatonin treatment blocked these changes and rescued the cells from apoptosis induced by serum-deprivation. These results indicated that oxidative stress may play a causal role in the induction of apoptosis induced by serum deprivation.

Identification of single-domain, Bax-specific intrabodies that confer resistance to mammalian cells against oxidative-stress-induced apoptosis

Bax is a proapoptotic protein implicated in cell death involved in several neurodegenerative diseases. Intracellularly expressed antibody (Ab) fragments (intrabodies) inhibiting Bax function would have potential for developing therapeutics for the aforementioned diseases and can serve as research tools. We report identification, cloning, and functional characterization of several Bax‐specific single‐domain antibodies (sdAbs). These minimal size Ab fragments, which were isolated from a llama VHH phage display library by panning, inhibited Bax function in in vitro assays. Importantly, as intrabodies, these sdAbs, which were stably expressed in mammalian cells, were nontoxic to their host cells and rendered them highly resistant to oxidative‐stress‐induced apoptosis. The intrabodies prevented mitochondrial membrane potential collapse and apoptosis after oxidative stress in the host cells. These anti‐Bax VHHs could be used as tools for studying the role of Bax in oxidative‐stress‐induced apoptosis and for developing novel therapeutics for the degenerative diseases involving oxidative stress.—Gueorguieva, D., Li, S., Walsh, N., Mukerji, A., Tanha, J., Pandey, S. Identification of single‐domain, Bax‐specific intrabodies that confer resistance to mammalian cells against oxidative‐stress‐induced apoptosis. FASEB J. 20, E2209–E2219 (2006)

Chemoenzymatic Synthesis of Amaryllidaceae Constituents and Biological Evaluation of their C-1 Analogues. The Next Generation Synthesis of 7-Deoxypancratistatin and trans-Dihydrolycoricidine†

An efficient synthesis of C-1 derivatives of 7-deoxypancratistatin is reported. The key steps include the following: selective opening of an epoxide with aluminum acetylide in the presence of an aziridine; solid-state silica-gel-catalyzed opening of an aziridine; and oxidative cleavage of a phenanthrene core and its recyclization to phenanthridone to provide the key C-1 aldehyde 22. The conversion of this aldehyde to C-1 acetoxymethyl and C-1 hydroxymethyl derivatives is described along with the evaluation of their biological activity against several cancer cell lines and in an apoptosis study. The C-1 acetoxymethyl derivative has shown promising activity comparable to that of the natural product. In addition, a total synthesis of trans-dihydrolycoricidine and a formal total synthesis of 7-deoxypancratistatin are reported from aldehyde 22. Detailed experimental and spectral data are provided for all new compounds.