| Survivin, an inhibitor of apoptosis protein, is highly expressed in most cancers and associated with chemotherapy resistance, increased tumor recurrence, and shorter patient survival, making anti-survivin therapy an attractive cancer treatment strategy. Survivin expression has been extensively evaluated in cancer; however, its expression and function in normal tissues are not well defined. Survivin has been shown to increase tumor resistance to various apoptotic stimuli, primarily through caspase-dependent mechanisms, although it can also block apoptosis in a caspase-independent fashion. Numerous studies have shown that loss of survivin expression or function causes spontaneous apoptosis or sensitizes cancer cells to apoptotic stimuli. Experimental work carried out in vitro and in transgenic animals has assigned a dual function to survivin: protection from apoptosis and regulation of cell division. Reduction or loss of survivin in mammalian cells has been associated with apoptosis and a panoply of cell division defects that include supernumerary centrosomes, aberrant spindle assembly, mislocalization of mitotic kinases, loss of mitotic checkpoint(s) , and cytokinesis failure with the appearance of multinucleated cells. Because of its dual role, its study in normal cells is important. The fact that targeting survivin for cancer therapeutics causes little toxicity in normal tissues/cells suggests different mechanisms for its regulation and function in normal vs. abnormal tissue. Characterization of these differences at the molecular level would certainly provide opportunities to enhance the therapeutic index of targeting survivin in cancer and possibly other diseases.In this study, we used RNA interference (RNAi) to reduce survivin expression in HeLa cells. RNAi is a novel technique used to knockdown target gene level and protein expression. RNAi represents an evolutionarily conserved cellular defense mechanism for controlling the expression of alien genes in almost all eukaryotes, including humans. It is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. In mammalian cells, long dsRNA similar to that used in C. elegans research, provokes a strong cytotoxic response. This non-specific effect can be circumvented by use of synthetic short [21- to 22-nucleotide (nt)] interfering RNAs (siRNAs), which can mediate strong and specific suppression of gene expression. In order to successfully interfere with mRNA levels in mammalian cells, two siRNA delivery technologies are used. One is the delivery of siRNAs to mammalian cells in culture, using siRNA transfected with chemical delivery agents, delivery of siRNA expression vectors, and viral methods for delivering siRNA expression cassettes. The other tool is in vivo delivery of siRNA and siRNA-expression constructs, utilizing hydrodynamic intravascular injection, synthetic delivery vehicles, and viral vectors. In this study, we use the pSUPER RNAi system, which provides a mammalian expression vector that directs intracellular synthesis of siRNA-like transcripts.In this study, we used RNA interference to knockdown the expression of surviving and then measured the extent of apoptosis in HeLa cells using flow cytometry analysis and caspase-3 activity. We were able to successfully knockdown the mRNA levels in the cells, which then showed an increase in apoptosis. To further investigate this effect, we also studied the p53 gene, which is the correlative gene of apoptosis. RT-PCR confirmed that after RNAi, the levels of p53 increased, indicating that survivin RNAi influences the apoptosis of HeLa cells.In almost every moment, air-living organisms are subjected to oxidative stress both external and internal sources. Consequently, all those organisms contain antioxidative enzymes that limit oxidative stress by detoxifying reactive oxygen species (ROS), including H2O2. When generation of ROS overwhelms these antioxidants capacity, the excessive ROS damages on most biological molecules and has been implicated in association with many diseases, from heart failure, to parkinson's disease, cancer, and aging. H2O2 is a main ROS form in some cell types and diffuses freely among the membrane systems of the cells, so that its reduction becomes a major challenge for the normal functions of these cells. Most biological sources of H2O2 involve the spontaneous or catalytic breakdown of superoxide anions (O2-), produced by the partial reduction of oxygen during aerobic respiration and following the exposure of cells to a variety of physical, chemical, and biological agents. In multicellular organisms, H2O2 can activate signaling pathways to stimulate cell proliferation, differentiation, migration, or apoptosis. There is considerable variation between cells in the concentration of exogenous H2O2 required to initiate a particular biological response. However, increased levels of H2O2 in cells induce nuclear DNA fragmentation and lipid peroxidation and lead to apoptotic cell death. Indeed, such damage is associated with the initiation and progression of many diseases, including neurodegenerative disorders, diabetes, atherosclerosis, and cancer.Consequently, there is great interest in developing antioxidants that can protect cells against oxidative stress. The micro-peroxidases (MP-8, MP-9, and MP-11), proteolytic products from cytochrome c, have been demonstrated to be effective anti-cataract agents in an in vitro cataract model. Although these micro-peroxidases have promising potential to become broader therapeutic agents, several problems have to be solved, such as difficulties in preparation and purification, short half-life in vivo, etc. To obtain readily available microperoxidase, we studied extensively the structure of ascorbate peroxidase (APX, EC1.11.1.11) that has iron-heme in its catalatic center. We designed and chemically synthesized a series of peroxidase mimics containing histidine and deuterohemin (DhHP), and analyzed their enzymatic properties and anti-cataract effects. We finally have succeeded in optimizing one peroxidase mimic, DhHP-6 (Dh-AlaHisThrValGluLys). DhHP-6 exhibited high peroxidase activity (93% of MP-11). The optimal pH and temperature of its catalytic activity were close to those of in vivo condition. In addition, DhHP-6 was cell-permeable and very potent at reducing intracellular H2O2. At concentrations of higher than 7.5μM, DhHP-6 totally blocked caspase activation and apoptotic body formation in both H9c2 and HeLa cells. Importantly, DhHP-6 didn't affect cell viability even at 20 fold higher than its working concentration. Taken together, this small artificial microperoxidase may be beneficial in the treatment of aging and diseases associated with oxidative damage such as heart failure, Parkinson's disease, cancers. |
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