Subhasree Roy Choudhury

Synergistic efficacy of sorafenib and genistein in growth inhibition by down regulating angiogenic and survival factors and increasing apoptosis through upregulation of p53 and p21 in malignant neuroblastoma cells having N-Myc amplification or non-amplification

SummaryNeuroblastoma is an extracranial, solid, and heterogeneous malignancy in children. The conventional therapeutic modalities are mostly ineffective and thus new therapeutic strategies for malignant neuroblastoma are urgently warranted. We examined the synergistic efficacy of combination of sorafenib (SF) and genistein (GST) in human malignant neuroblastoma SK-N-DZ (N-Myc amplified) and SH-SY5Y (N-Myc non-amplified) cell lines. MTT assay showed dose-dependent decrease in cell viability and the combination therapy more prominently inhibited the cell proliferation in both cell lines than either treatment alone. Apoptosis was confirmed morphologically by Wright staining. Flow cytometric analysis of cell cycle phase distribution and Annexin V-FITC/PI staining showed increase in subG1 DNA content and early apoptosis, respectively, after treatment with the combination of drugs. Apoptosis was further confirmed by scanning electron microscopy. Combination therapy showed activation of caspase-8, cleavage of Bid to tBid, increase in p53 and p21 expression, down regulation of anti-apoptotic Mcl-1, and increase in Bax:Bcl-2 ratio to trigger apoptosis. Down regulation of MDR, hTERT, N-Myc, VEGF, FGF-2, NF-κB, p-Akt, and c-IAP2 indicated suppression of angiogenic and survival pathways. Mitochondrial release of cytochrome c and Smac into cytosol indicated involvement of mitochondia in apoptosis. Increases in proteolytic activities of calpain and caspase-3 were also confirmed. Our results suggested that combination of SF and GST inhibited angiogenic and survival factors and increased apoptosis via receptor and mitochondria mediated pathways in both neuroblastoma SK-N-DZ and SH-SY5Y cell lines. Thus, this combination of drugs could be a potential therapeutic strategy against human malignant neuroblastoma cells having N-Myc amplification or non-amplification.

Targeting Angiogenesis for Controlling Neuroblastoma

Neuroblastoma, a progressive solid tumor in childhood, continues to be a clinical challenge. It is highly vascular, heterogeneous, and extracranial tumor that originates from neural crest. Angiogenesis, genetic abnormalities, and oncogene amplification are mainly responsible for malignant phenotype of this tumor. Survivability of malignant neuroblastoma patients remains poor despite the use of traditional therapeutic strategies. Angiogenesis is a very common and necessary pre-requisite for tumor progression and metastasis. Angiogenesis is also a major factor in making malignant neuroblastoma. Thus, prevention of angiogenesis can be a highly significant strategy in the treatment of malignant neuroblastoma. Here, we summarize our current understanding of angiogenesis in malignant neuroblstoma and describe the use of experimental anti-angiogenic agents either alone or in combination therapy. This review will clearly indicate the importance of angiogenesis in the pathogenesis of malignant neuroblastoma, its prevention as a promising therapy in preclinical models of malignant neuroblastoma, and prospective clinical trials.

Bcl-2 inhibitor and apigenin worked synergistically in human malignant neuroblastoma cell lines and increased apoptosis with activation of extrinsic and intrinsic pathways

Neuroblastoma is the most common extracranial solid tumor in infants and young children. Current treatments are not always effective and new therapies are needed. We examined efficacy of combination of the small molecule Bcl-2 inhibitor HA14-1 (HA) and the dietary isoflavonoid apigenin (APG) in human malignant neuroblastoma cells. Dose-response studies indicated that treatment with HA and APG for 24 h synergistically reduced cell viability in human malignant neuroblastoma SK-N-DZ, SH-SY5Y, and IMR32 cells. For further studies, we selected SK-N-DZ cells that showed the highest sensitivity following treatment with 2.5 microM HA, 100 microM APG, or combination (2.5 microM HA+100 microM APG). Wright staining showed increase in morphological features of apoptosis. Cell cycle distribution and Annexin V assay showed that combination therapy caused more apoptosis than either treatment alone. Western blotting revealed that combination therapy downregulated angiogenic factors and also induced extrinsic pathway of apoptosis with activation of caspase-8 for Bid cleavage to tBid. Alterations in Bax and Bcl-2 levels resulted in an increase in Bax:Bcl-2 ratio to activate intrinsic pathway of apoptosis with mitochondrial release of cytochrome c into the cytosol and activation of proteases. Increases in calpain and caspase-3 activities generated 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Results showed that combination of HA and APG could be used for downregulation of angiogenic factors and activation of extrinsic and intrinsic pathways of apoptosis in malignant neuroblastoma cells.

Valproic Acid Induced Differentiation and Potentiated Efficacy of Taxol and Nanotaxol for Controlling Growth of Human Glioblastoma LN18 and T98G Cells

Glioblastoma shows poor response to current therapies and warrants new therapeutic strategies. We examined the efficacy of combination of valproic acid (VPA) and taxol (TX) or nanotaxol (NTX) in human glioblastoma LN18 and T98G cell lines. Cell differentiation was manifested in changes in morphological features and biochemical markers. Cell growth was controlled with down regulation of vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), nuclear factor-kappa B (NF-κB), phospho-Akt (p-Akt), and multi-drug resistance (MDR) marker, indicating suppression of angiogenic, survival, and multi-drug resistance pathways. Cell cycle analysis showed that combination therapy (VPA and TX or NTX) increased the apoptotic sub G1 population and apoptosis was further confirmed by Annexin V-FITC/PI binding assay and scanning electron microscopy. Combination therapy caused activation of caspase-8 and cleavage of Bid to tBid and increased Bax:Bcl-2 ratio and mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF). Upregulation of calpain and caspases (caspase-9 and caspase-3) and substrate degradation were also detected in course of apoptosis. The combination of VPA and NTX most effectively controlled the growth of LN18 and T98G cells. Therefore, this combination of drugs can be used as an effective treatment for controlling growth of human glioblastoma cells.

Molecular mechanisms of anti-cancer action of garlic compounds in neuroblastoma.

The medicinal properties of garlic (Allium sativum) have been well known and widely used since historical times. Garlic compounds have received increasing attention during the last few years due to their cancer chemopreventive properties. The anti-cancer activity of garlic-derived organosulfur compounds (OSCs) are extensively reported in many cancers but only a few in the pediatric tumor neuroblastoma, which warrants exploration of new therapy for its management. There are some recent reports suggesting that garlic-derived OSCs cause cell cycle arrest, generate reactive oxygen species (ROS), activate stress kinases, and also stimulate the mitochondrial pathway for apoptosis in malignant neuroblastoma. The comprehensive mechanisms of anti-cancer action of OSCs still remain unclear and require more studies in neuroblastoma. This review is designed to highlight the molecular mechanisms of anti-cancer actions of garlic-derived OSCs in neuroblastoma and as well as in several other cancers. Further studies should be conducted to establish the clinical expediency of garlic-derived OSCs for treatment of malignant neuroblastoma in humans.

Activation of Multiple Molecular Mechanisms for Increasing Apoptosis in Human Glioblastoma T98G Xenograft.

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.

Bcl-2 inhibitor HA14-1 and genistein together adeptly down regulated survival factors and activated cysteine proteases for apoptosis in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells.

Neuroblastoma is a pediatric extracranial tumor and a major cause of death in children under age 2. Conventional therapy shows inefficacy in most cases and thus development of new therapeutic strategies is urgently needed. We explored the efficacy of combination of the small molecule Bcl-2 inhibitor HA14-1 (HA) and the isoflavonoid genistein (GST) in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells. Combination of 10 microM HA and 250 microM GST was optimal for SK-N-BE2 cells and combination of 5 microM HA and 100 microM GST was optimal for SH-SY5Y cells for induction of apoptosis. Phase-contrast microscopy and Wright staining showed morphological features of apoptosis. Cell cycle analysis and Annexin V-FITC/PI binding assay showed that combination of HA and GST was more effective in inducing apoptosis in both cell lines than either HA or GST alone. Western blotting showed that combination of HA and GST caused upregulation of Bax and down regulation of Bcl-2 resulting in increased Bax:Bcl-2 ratio and mitochondrial release of cytochrome c, Smac, and AIF. Down regulation of survival factors such as NF-kappaB, N-Myc, and survivin promoted apoptosis. Activation of caspase-8, calpain, and caspase-3 occurred in course of apoptosis. Increased calpain and caspase-3 activities were confirmed in the degradation of alpha-spectrin to 145 kD spectrin break down product (SBDP) and 120 kD SBDP, respectively. Thus, combination of HA and GST could serve as a promising therapeutic strategy for increasing apoptosis in different human malignant neuroblastoma cells.

Purification, crystallization and preliminary X-ray structural studies of a 7.2 kDa cytotoxin isolated from the venom of Daboia russelli russelli of the Viperidae family

A cytotoxin (MW 7.2 kDa) from Indian Russells viper (Daboia russelli russelli) venom possessing antiproliferative activity, cardiotoxicity, neurotoxicity and myotoxicity has been purified, characterized and crystallized. The crystals belong to the tetragonal space group P4(1), with unit-cell parameters a = b = 47.94, c = 50.2 A. Larger crystals, which diffracted to 1.5 A, were found to be twinned; diffraction data were therefore collected to 2.93 A resolution using a smaller crystal. Molecular-replacement calculations identified two molecules of the protein in the asymmetric unit, which is in accordance with the calculated VM value.

Toxic impact of nanomaterials on microbes, plants and animals

Nanotechnology has many potential applications in medical, agriculture, electronic and sports industries. Nonetheless, there is actually little knowledge on toxic effects of nanomaterial. Here we review nanotoxicity, action mechanisms and fate of nanomaterials. We discuss the impact of nanotoxicity on microbes, plant, animal and human health, and factors like size, shape, surface charge, composition, ionic concentration and physiological. We explain the detection of nanotoxicity at cell and genome levels. Toxicities of commercial nanomaterials, risk management, rules and regulations regarding marketed nanoproducts are also summarized.

Nanomaterial Toxicity in Microbes, Plants and Animals

Nanotechnology has gained public interest due to its extensive use in commercial products including industry, electronic components, agriculture, sports, sunscreens, medicine and biomedical field. As a consequence there is an unprecedented growth of research in medicine for nanotoxicity following inhalation, ingestion and skin contact. This review presents the toxicity, fate, behavior and mechanism of action of nanomaterials. It includes the effect of nanomaterials on microbes, plant, animal and human. Factors controlling toxicity are nanoparticle size, shape, surface charge, composition, ionic concentration and other physicochemical properties. We also list the market-available nanoproducts having toxic effects. We describe the tests for detection of cytotoxity and genotoxicity. Risk management, rules and regulations for marketed nanomaterials are also highlighted.