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1.
Tumour Biol ; 39(10): 1010428317715039, 2017 Oct.
Article in English | MEDLINE | ID: mdl-29065794

ABSTRACT

Nitric oxide, a signaling molecule, inhibits mitochondrial respiration by binding with cytochrome c oxidase, resulting in elevated production of reactive superoxide species (reactive oxygen and nitrogen) in the mitochondria and increased susceptibility to cell death. Generation of mitochondrial superoxide species can be suppressed by natural compounds such as resveratrol, a dietary polyphenol found in the skin of red fruits. In various cancer cells, resveratrol shows anti-oxidant and cancer preventive properties. Since, the effect of resveratrol on reactive superoxide species-independent apoptosis in prostate cancer cells is not well illustrated; therefore, we investigated this phenomenon in TRAMP murine prostate cancer cells. To accomplish this, TRAMP cells were incubated with resveratrol, resveratrol + DETA-NONOate, DETA-NONOate (nitric oxide donor), resveratrol + L-NMMA, or L-NMMA (nitric oxide inhibitor) for 48 h, and reactive superoxide species in the mitochondria and culture supernatant were measured. In addition, the mitochondrial membrane potential, cell viability, expression of apoptotic markers (Bax and Bcl2), γ-H2A.x, p53, and caspase-3 was determined. We found that resveratrol suppressed reactive superoxide species such as reactive oxygen species in the mitochondria and nitric oxide in culture supernatant when compared to the DETA-NONOate treatment and disrupted the mitochondrial membrane potential. Resveratrol also reduced cell viability, altered the expression of apoptotic markers (Bax and Bcl2), and increased expression of γ-H2A.x (indicative marker of DNA fragmentation) and p53 (a critical DNA damage response protein). However, there was no appreciable modulation of the caspase-3. Therefore, our data suggest that resveratrol induces superoxide species-independent apoptosis and may act as a therapeutic agent against prostate cancer.


Subject(s)
Antioxidants/pharmacology , Apoptosis/drug effects , Prostatic Neoplasms/pathology , Stilbenes/pharmacology , Animals , Cell Line, Tumor , Disease Models, Animal , Male , Membrane Potential, Mitochondrial/drug effects , Mice , Reactive Oxygen Species/metabolism , Resveratrol
2.
Oncotarget ; 8(13): 20895-20908, 2017 Mar 28.
Article in English | MEDLINE | ID: mdl-28157696

ABSTRACT

Found in the skins of red fruits, including grapes, resveratrol (RES) is a polyphenolic compound with cancer chemopreventive activity. Because of this activity, it has gained interest for scientific investigations. RES inhibits tumor growth and progression by targeting mitochondria-dependent or -independent pathways. However, further investigations are needed to explore the underlying mechanisms.The present study is focused on examining the role of RES-induced, mitochondria-mediated, caspase-independent apoptosis of prostate cancer cells, namely transgenic adenocarcinoma of mouse prostate (TRAMP) cells. These cells were exposed to RES for various times, and cell killing, cell morphology, mitochondrial membrane potential (Δψm), expression of Bax and Bcl2 proteins, the role of caspase-3, and DNA fragmentation were analyzed.TRAMP cells exposed to RES showed decreased cell viability, altered cell morphology, and disrupted Δψm, which led to aberrant expression of Bax and Bcl2 proteins. Furthermore, since the caspase-3 inhibitor, z-VAD-fmk (benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethyl ketone), had no appreciable impact on RES-induced cell killing, the killing was evidently caspase-independent. In addition, RES treatment of TRAMP-C1, TRAMP-C2, and TRAMP-C3 cells caused an appreciable breakage of genomic DNA into low-molecular-weight fragments.These findings show that, in inhibition of proliferation of TRAMP cells, RES induces mitochondria-mediated, caspase-independent apoptosis. Therefore, RES may be utilized as a therapeutic agent to control the proliferation and growth of cancer cells.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Caspases/metabolism , Mitochondria/drug effects , Prostatic Neoplasms/pathology , Stilbenes/pharmacology , Animals , Caspase Inhibitors/pharmacology , Caspases/chemistry , Cell Cycle Proteins/metabolism , Cell Proliferation/drug effects , Humans , Male , Membrane Potential, Mitochondrial/drug effects , Mice , Mitochondria/metabolism , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Resveratrol , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
3.
Cancer Lett ; 374(1): 156-166, 2016 Apr 28.
Article in English | MEDLINE | ID: mdl-26898980

ABSTRACT

In all organisms, heat-shock proteins (HSPs) provide an ancient defense system. These proteins act as molecular chaperones by assisting proper folding and refolding of misfolded proteins and aid in the elimination of old and damaged cells. HSPs include Hsp100, Hsp90, Hsp70, Hsp40, and small HSPs. Through its substrate-binding domains, Hsp70 interacts with wide spectrum of molecules, ranging from unfolded to natively folded and aggregated proteins, and provides cytoprotective role against various cellular stresses. Under pathophysiological conditions, the high expression of Hsp70 allows cells to survive with lethal injuries. Increased Hsp70, by interacting at several points on apoptotic signaling pathways, leads to inhibition of apoptosis. Elevated expression of Hsp70 in cancer cells may be responsible for tumorigenesis and for tumor progression by providing resistance to chemotherapy. In contrast, inhibition or knockdown of Hsp70 reduces the size of tumors and can cause their complete regression. Moreover, extracellular Hsp70 acts as an immunogen that participates in cross presentation of MHC-I molecules. The goals of this review are to examine the roles of Hsp70 in cancer and to present strategies targeting Hsp70 in the development of cancer therapeutics.


Subject(s)
HSP70 Heat-Shock Proteins/antagonists & inhibitors , HSP70 Heat-Shock Proteins/immunology , Neoplasms/therapy , Animals , Gene Knockdown Techniques , HSP70 Heat-Shock Proteins/biosynthesis , HSP70 Heat-Shock Proteins/genetics , Humans , Molecular Targeted Therapy , Neoplasms/genetics , Neoplasms/immunology
4.
Avian Dis ; 53(3): 376-81, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19848075

ABSTRACT

Transgenic lines of Arabidopsis thaliana producing recombinant TC protein were developed. The S1 gene encoding sigmaC protein of an avian reovirus (ARV) was amplified by reverse-transcription PCR (RT-PCR). The amplified product was cloned into a plant-expression vector, pE1857, with a strong promoter for expression. The resulting construct with the BAR gene cassette for bialaphos selection was designated rpE-sigmaC and was introduced into Agrobacterium tumefaciens by electroporation. Agrobacterium containing the rpE-sigmaC constructs that transform A. thaliana and transgenic plants were selected using bialaphos selection. The presence of S1 transcript in plants and their relative level of expression were determined by real time RT-PCR. Western blot analysis further confirmed the presence of sigmaC protein in the plants. Transgenic lines with high levels of sigmaC protein were selected for immunization experiments using specific-pathogen free chickens. Recombinant sigmaC protein produced in plants induced a variety of immunoglobulin G (IgG) antibody responses in chickens. Recombinant protein administered either subcutaneously or orally in birds showed significant protection against challenge. Results suggested that the recombinant sigmaC protein produced in plants has the potential for large-scale vaccination against ARV in commercial poultry.


Subject(s)
Capsid Proteins/immunology , Capsid Proteins/metabolism , Chickens , Poultry Diseases/prevention & control , Reoviridae Infections/veterinary , Viral Vaccines/immunology , Aging , Animals , Antibodies, Viral/blood , Arabidopsis , Capsid Proteins/genetics , Enzyme-Linked Immunosorbent Assay/veterinary , Immunoglobulin G/blood , Plants, Genetically Modified , Poultry Diseases/virology , Reoviridae/immunology , Reoviridae Infections/prevention & control , Specific Pathogen-Free Organisms , Viral Vaccines/administration & dosage
5.
Biotechnol Lett ; 31(10): 1511-7, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19543855

ABSTRACT

An open reading frame representing cDNA from a hemagglutinin (HA) encoding gene of a low pathogenic avian influenza virus (AIV) subtype H10N7 was cloned in the pNMT1-TOPO vector under the control of thiamine response promoter. This construct was designated as pNMT1-HA. The pNMT1-HA construct was transformed into Schizosaccharomyces pombe for expression of HA antigen. The correct expression of recombinant HA protein was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The level of expression of recombinant HA protein was approximately 0.2% of total soluble protein. Purified yeast-derived recombinant HA protein showed hemagglutination activity. The 2-D and 3-D scanning images of recombinant HA protein were observed with an atomic force microscope (AFM). The structural integrity of the HA protein under AFM and hemagglutination activity provided support that the recombinant HA protein may be suitable for development of AIV subunit vaccine for mass administration to poultry.


Subject(s)
Hemagglutination , Hemagglutinins, Viral/chemistry , Hemagglutinins, Viral/metabolism , Influenza A virus/genetics , Amino Acid Sequence , Blotting, Western , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Gene Expression , Hemagglutinins, Viral/genetics , Microscopy, Atomic Force , Molecular Sequence Data , Protein Conformation , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Schizosaccharomyces/genetics
6.
Vaccine ; 24(8): 1213-24, 2006 Feb 20.
Article in English | MEDLINE | ID: mdl-16194585

ABSTRACT

Chlamydia trachomatis is a major human health pathogen due to its role in sexually transmitted diseases. Thus, there is a need to develop an effective vaccine at the mucosal surface against this pathogen. In an effort to develop a mucosal vaccine, a modified cholera toxin gene was genetically linked to the C. trachomatis MoPn NiggII MOMP gene to generate a recombinant protein with the mucosal adjuvant properties of the cholera toxin and immunological antigenicity of the chlamydial protein. The recombinant fusion protein (rMOMP) was expressed in E. coli, purified and analyzed by SDS-PAGE, immunoblot, and GM1-ELISA, and subsequently used to immunize BALB/c mice via intranasal (i.n.) and intravaginal (vag.) routes. The rMOMP protein administered via the i.n. route induced a higher concentration of anti-MOMP specific antibodies in both serum and vaginal washes as compared to mice immunized with Chlamydia or PBS. Antibody isotype analysis revealed that i.n. administration of rMOMP to mice induced higher concentrations of serum and vaginal wash IgA, IgG1, IgG2a, and IgG2b antibodies. Vaginal washes from all immunized mice following a chlamydial challenge infection were analyzed by indirect immunoflourescence to study the level of protection provided by various immunogens. Maximum protection against C. trachomatis as assessed by reduction in C. trachomatis inclusion forming units (IFU) was provided by i.n. immunization of mice with rMOMP. This is a first report using genetic fusion of cholera toxin and MOMP genes and provides a novel approach for the design and development of a mucosal vaccine against Chlamydia.


Subject(s)
Bacterial Vaccines/immunology , Chlamydia Infections/prevention & control , Chlamydia trachomatis/immunology , Cholera Toxin/immunology , Porins/immunology , Recombinant Fusion Proteins/immunology , Vaccines, Synthetic/immunology , Administration, Intranasal , Animals , Antibodies, Bacterial/blood , Cholera Toxin/genetics , Enzyme-Linked Immunosorbent Assay , Female , Immunity, Mucosal , Immunization , Mice , Mice, Inbred BALB C , Porins/genetics
7.
J Mol Biol ; 356(2): 288-99, 2006 Feb 17.
Article in English | MEDLINE | ID: mdl-16376379

ABSTRACT

Mitochondrial genomes in higher plants are much larger and more complex as compared to animal mitochondrial genomes. There is growing evidence that plant mitochondrial genomes exist predominantly as a collection of linear and highly branched DNA molecules and replicate by a recombination-dependent mechanism. However, biochemical evidence of mitochondrial DNA (mtDNA) recombination activity in plants has previously been lacking. We provide the first report of strand-invasion activity in plant mitochondria. Similar to bacterial RecA, this activity from soybean is dependent on the presence of ATP and Mg(2+). Western blot analysis using an antibody against the Arabidopsis mitochondrial RecA protein shows cross-reaction with a soybean protein of about 44 kDa, indicating conservation of this protein in at least these two plant species. mtDNA structure was analyzed by electron microscopy of total soybean mtDNA and molecules recovered after field-inversion gel electrophoresis (FIGE). While most molecules were found to be linear, some molecules contained highly branched DNA structures and a small but reproducible proportion consisted of circular molecules (many with tails) similar to recombination intermediates. The presence of recombination intermediates in plant mitochondria preparations is further supported by analysis of mtDNA molecules by 2-D agarose gel electrophoresis, which indicated the presence of complex recombination structures along with a considerable amount of single-stranded DNA. These data collectively provide convincing evidence for the occurrence of homologous DNA recombination in plant mitochondria.


Subject(s)
DNA/metabolism , Glycine max , Mitochondria/genetics , Recombination, Genetic , DNA/chemistry , DNA/ultrastructure , Electrophoresis/methods , Nucleic Acid Conformation , Glycine max/cytology , Glycine max/genetics
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