Inhibition and Apoptotic Responses of Human Colorectal Carcinoma Cells by Anti-Survivin Small Interfering RNA
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Title: Inhibition and Apoptotic Responses of Human Colorectal Carcinoma Cells by Anti-Survivin Small Interfering RNA
Series Title: Journal of Undergraduate Research
Physical Description: Serial
Language: English
Creator: Lin, Philip W.
Liu, Chen ( Mentor )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: Fall 2009
Abstract: As evidenced by the 940,000 cases and 655,000 deaths worldwide per year, colon cancer is one of the most prominent diseases in the world. Within the United States alone, colon cancer has been linked to over 56,000 deaths and is considered the second leading cause of cancer death. Colon cancer, or colorectal cancer, develops from the growth of adenomatous polyps in the colon due to mutations in the DNA of the gastrointestinal epithelial cells. The advancement of the formation of polyps in the colon results in cancer that has the ability to metastasize to adjacent organs, such as the liver. Currently 3 different forms of treatment are used in combination. Surgical removal of the cancerous growth is the primary treatment for colon cancer. Through the colectomy procedure, the cancerous mass is removed and the colon resected, restoring the original purpose of the colon. Colostomy surgery also excises the cancerous growth, but the colon is instead attached to the anterior abdominal wall due to the unrestricted and increased manifestation of the cancerous cells. Despite the advancements in surgery, approximately 40-50% of patients will relapse and require additional therapies and treatments (2). Subsequent applications of chemotherapy or radiation are used to treat metastasized cancer and prevent such tumor recurrence after surgery. These treatments stimulate cancer cells to enter apoptotic pathways, causing cell death in these targeted cells. There have been well-documented cases of resistance to apoptotic stimuli. This resistance to chemotherapy and radiation could result from the over-expression of anti-apoptotic factors. These factors, such as survivin, are predominately expressed in cells of common human cancers. Cell division is coupled with checkpoints along the different stages of cell life. These checkpoints allow the prevention of the developments of abnormalities such as mutations, by inducing the abnormal cell to enter the apoptotic pathway. By this definition, apoptosis is a state of programmed cell death due to DNA damage or cellular aging. The balance between cell growth and checkpoints is an essential feature in preventing the development of any form of cancer. Failure of the induction of apoptosis in cells that contain abnormalities in the mitotic spindle, DNA structure, and mutations in oncogenes could result in increased resistance to chemotherapy and radiation. There are two major apoptotic pathways. The extrinsic pathway involves extracellular inducers such as toxins, hormones and growth factors that induce cellular signals for apoptosis. The intrinsic pathway results in the mitochondrial release of cytochrome c and a caspase cascade that mediates cellular apoptosis. Survivin is a protein belonging to the inhibitor of apoptosis protein (IAP) family and is an important regulator of the intrinsic apoptotic pathway. The survivin gene is located on chromosome 17, which encodes a 16.5 kD protein containing 142 amino acids and a Baculovirus IAP Repeat (BIR). The survivin protein regulates the intrinsic apoptotic pathway by interfering with caspase-3, caspase-7, and caspase-9 activity. Also, the association of survivin with the cell cycle and mitotic spindle is consistent with the 40-fold increase in expression levels during the G2/M phase in HeLa cells. Its expression is upregulated in most human cancer cells, but undetectable or found at very low levels in normal adult tissues. The increased activity of survivin in cancer cells in contrast to normal somatic cells provides an ideal target for cancer treatment. Current research provides the technology to synthetically produce a 21-23 RNA nucleotide molecule that would be able to effectively inhibit specific gene expression by RNA interference. This small interfering RNA (siRNA) disrupts the expression of a gene by down-regulating the desired target protein. The siRNA is designed by selecting target sequences from the RNA of a gene of interest. The sense and anti-sense templates are placed on both sides of a loop sequence in a plasmid DNA. The transcription of siRNA uses an RNA Polymerase III promoter (U6 or H1), which allows the transcription of short hairpin RNA. The single strand complementarily binds to the target mRNA and consequently down-regulates targeted gene expression through the RNA-induced silencing complex (RISC). The transfection of siRNA through plasmids is limited to in vitro use. In vivo delivery of siRNA may be optimized through the use of a viral vector such as an adeno-associated virus (AAV). The adenovirus (AV) and the adeno-associated virus (AAV) have been used successfully for delivery in gene therapy. Examples of this include the successful delivery of a survivin mutant to breast cancer cells by using adenoviruses, resulting in apoptosis in cancerous cells and inhibited growth of tumors (14). We hypothesize that by introducing siRNA that specifically inhibits survivin expression, cancer cells will be more susceptible to apoptotic stimuli and ultimately result in apoptosis. A vector will be constructed with four targets of the survivin gene. These 4 targets will be analyzed for their efficacy in in-vitro down-regulation of survivin protein in cancerous cell lines. The optimal siRNA target will then be cloned into an adeno-associated virus (AAV) for further in vivo studies. The inhibition of colorectal carcinoma cells by siRNA survivin could prove to be a potential therapy for colon cancer.
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Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UF00091523:00006

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