survivin expression. however, it has not been ruled out yet that there may be other possible epigenetic mechanisms that may be responsible for a high level of survivin expression observed in cancer cells and not in normal cells. for example, the acetylation profile of the survivin promoter region can also be looked at. different cancer and tissue types may have slight or significant differences in the way survivin expression is regulated in the cell, and, thus, the methylation status or genetic differences in the survivin promoter may be observed to be different in different tissues. thus, further experiments assessing the epigenetic and genetic profile of different tumour types must be investigated. as a drug target expression in cancer as a tool for cancer-directed therapy survivin is known to be highly expressed in most tumour cell types and absent in normal cells, making it a good target for cancer therapy. the exploitation of survivin's over-active promoter in most cancer cell types allows for the delivery of therapeutics only in cancer cells and removed from normal cells. small interfering rna (sirna) are synthetic antisense oligonucleotides to the mrna of the gene of interest that works to silence the expression of a particular gene by its complementary binding. sirnas, such as ly2181308, bound to the respective mrna results in disruption of translation of that particular gene and thus the absence of that protein in the cell. thus, the use of sirnas has great potential to be a human therapeutic, as it can target and silence the expression of potentially any protein you want. a problem arises when sirna expression in a cell cannot be controlled, allowing its constitutive expression to cause toxic side-effects. with regard to practical treatment of cancer, it is required to either deliver the sirnas specifically into cancer cells or control the sirna expression. previous methods of sirna therapy employ the use of sirna sequences cloned into vectors under the control of constitutively active promoters. this causes a problem, as this model is non-specific to cancer cells and damages normal cells too. knowing that survivin is over-expressed specifically in cancer cells and absent in normal cells, one can imply that the survivin promoter is active only in cancer cells. thus, the exploitation of this difference between cancer cells and normal cells will allow appropriate therapy directed only at the cells in a patient that are harmful. in an experiment to demonstrate this idea, trang et al. have created a cancer-specific vector expressing sirna for green fluorescent protein (gfp) under the human survivin promoter. mcf7 breast cancer cells were cotransfected with this vector and a gfp-expressing vector as well. their major finding was that mcf7 cells transfected with the sirna vector for gfp under the survivin promoter had a significant reduction in gfp expression then the cells transfected with the sirna vector under a cancer non-specific promoter. moreover, normal non-cancerous cells transfected in the same way mentioned above showed no significant reduction in gfp expression. this is implying that, in normal cells, survivin promoter is not active, and, thus, the sirna will not be expressed under an inactive survivin promoter. antisense oligonucleotides targeting survivin mrna as it is known that survivin is over-expressed in most cancers, which may be contributing to the cancer cells' resistance to apoptotic stimuli from the environment. the use of antisense survivin therapy hopes to render cancer cells susceptible to apoptosis by eliminating survivin expression in the cancer cells. olie et al. developed different 20-mer phosphorothioate antisense oligonucleotides that target different regions in the mrna of the survivin gene. the antisense function of the oligonucleotides allows binding to surviving mrna and, depending on the region on which it binds, might inhibit surviving mrna from being translated into a functional protein. real-time pcr was used to assess the levels of mrna present in a lung adenocarcinoma cell line a549 that overexpresses survivin. the best antisense oligonucleotide was identified that effectively down-regulated survivin mrna levels and resulted in apoptosis of the cells. survivin's role in cancer development in the context of a signaling pathway is its ability to inhibit activation of downstream caspase-3 and -7 from apoptosis inducing stimuli. the overexpression of survivin in tumors may serve to increase the tumors resistance to apoptosis and, thus, contribute to cell immortality even in the presence of death stimuli. in this experiment, the oligonucleotide 4003 that targets nucleotides 232-251 of survivin mrna was found to be the most effective at down-regulating the levels of survivin mrna in the a549 tumour line. the 4003 oligonucleotides were introduced into the tumour cells by transfection. further experiments were then conducted on 4003. one of the additional experiments involved determining the dose-dependent effect of 4003 on the down-regulation of survivin mrna levels. it was found that a concentration of 400 nm resulted in a maximum down-regulation of 70% of the initial survivin mrna present. another experiment on 4003 involved assessing any biological or cytotoxic effect 4003 down-regulation of survivin mrna has on a549 cells using the mtt assay. the numbers of a549 cells transfected with 4003 significantly decreased with increasing concentration of 4003 compared to cells transfected either with a mismatch form of the 4003 or lipofectin control. many physical observations that confirmed the induction of apoptosis by 4003 were made. for example, lysates of the 4003-treated cells showed increased levels of caspase-3-like protease activity; nuclei were observed to be condensed and chromatin was fragmented. cancer immunotherapy survivin has been a target of attention in recent years for cancer immunotherapy, as it is an antigen that is expressed mostly in cancer cells and absent in normal cells. this is because survivin is deemed to be a crucial player in tumour survival. there has been much evidence accumulated over the years that shows survivin as a strong t-cell-activating antigen, and clinical trials have already been initiated to prove its usefulness in the clinic. activation of the adaptive immune system a. cellular t cell response the first evidence of survivin-specific ctl recognition and killing was shown in an assay wherein cytotoxic t cells (ctls) induced lysis of b cells transfected to present survivin peptides on its surface. the naive cd8+ t cells were primed with dendritic cells and could therefore recognize the specific peptides of survivin presented on the surface major histocompatibility complex i (mhc i) molecules of the b cells. b. humoral antibody response taking blood samples from cancer patients, scientists have found antibodies that are specific for survivin. these antibodies were absent in the blood samples of healthy normal patients. therefore, this shows that survivin is able to elicit a full humoral immune response. this may prove useful, as one could measure the level of survivin-specific antibodies in the patient's blood as a monitor of tumour progression. in acquiring the humoral response to tumour antigens such as survivin, cd4+ t cells are activated to induce b cells to produce antibodies directed against the particular antigens. the isolation of the antibodies specific for survivin peptides is useful, as one can look at the structure and sequence of the epitope binding groove of the antibody and, therefore, deduce possible epitopes that may fit in that particular antibody groove. therefore, one can determine the particular peptide portion of the survivin protein that is bound most efficiently and most commonly by humoral antibodies generated against survivin. this will lead to the production of more specific survivin vaccines that contain a specific portion of the survivin protein that is known to elicit a good immune response, generate immune memory, and allow for protection from tumour development. over-expression in tumours and metastatic tissues xiang et al. found a new approach in inhibiting tumour growth and metastasis by simultaneously attacking both the tumour and its vasculature by a cytotoxic t cell (ctl) response against the survivin protein, which will later result in the activation of apoptosis in tumour cells. the idea and general principle behind his technique is described below. mice were immunized with the oral vaccination and then subjected to tumour challenges by injecting them in the chest with a certain number of tumour cells and a matrigel pre-formed extracellular matrix to hold the tumour cells together. the mice were sacrificed and the endothelium tissue was stained with a fluorescent dye that would aid in the quantification of tumour neovascularisation using a matrigel assay. there was found to be a significant difference between the control and test groups, whereby mice given the vaccine had less angiogenesis from the tumour challenge than the control mice that were not given any of the vaccine prior to tumour challenge. in vitro assays and other tests were also performed to validate the idea of the occurrence of an actual immune response to support what they observed in the mice. for example, the spleen on the challenged mice were isolated and measured for the presence of any cytokines, and specifically activated immune cell groups that would indicative that a specific immune response did occur upon vaccination. the isolated ctls specific for the survivin protein after vaccination of the mice were used in cytoxicity assays where mice tumour cells expressing survivin were shown to be killed upon incubation with the specific ctls. by using an oral dna vaccine carried in an attenuated non-virulent form of salmonella typhimurium, which co-encoded secretory chemokine ccl21 and survivin protein in c57bl/6j mice, xiang et al. have been able to elicit an immune response carried out by dendritic cells (dcs) and ctls to eliminate and suppress the pulmonary metastases of non-small cell lung carcinoma. the activation of the immune response is most likely taking place in the secondary lymphoid organ called the peyer's patch in the small intestine where dcs take up the survivin protein by phagocytosis and present them on their surface receptors to naive cd8+ t cells (uninactivated ctl) to achieve a specific immune response targeting survivin exclusively. activated ctls specific for a particular antigen kill their target cells by first recognizing parts of the survivin protein expressed on mhc i (immunohistocompatability) proteins presented on the surface of tumour cells and vasculature and then releasing granules that induce the tumour cells to undergo apoptosis. the dna vaccine contained the ccl21 secretory chemokine as a way to enhance the likelihood of eliciting the immune response by better mediating the physical interaction of the antigen-presenting dcs and the naive cd8+ t cells, resulting in a greater likelihood of immune activation. resveratrol-mediated sensitization it has been shown by fulda et al. that the naturally occurring compound resveratrol (a polyphenol found in grapes and red wine) can be used as a sensitizer for anticancer drug-induced apoptosis by the action of causing cell cycle arrest. this cell cycle arrest causes a dramatic decline in survivin levels in the cells, as it is known from the literature that survivin expression is highly linked with the cell cycle phase state. thus, the decrease in survivin, which is a contributing factor to chemotherapy resistance and apoptosis induction therapies, would render the cancer cells more prone to such cancer treatments. fulda et al. have demonstrated the benefits of resveratrol through a series of experiments. first, the authors of the paper tested the intrinsic cytotoxic effects of resveratrol. they found that it induced moderate apoptosis levels only in shep neuroblastoma cells. after, they tested resveratrol in combination with several different known anticancer agents. they found a consistent increase in the level of apoptosis induced by the drugs when resveratrol was also present. moreover, they varied the order with which either the drugs or resveratrol was introduced to the cancer cells to determine whether the sequence of treatment had any important effect. it was found that the highest levels of apoptosis induction were observed when resveratrol was added prior to anticancer drug treatment. next, the authors tested for any differential sensitivity to apoptosis linked to the phase of the cell cycle the cells were in. analysis by flow cytometry revealed an accumulation of cells in s phase upon treatment with resveratrol. the cells were also halted in different phases of the cell cycle using special compounds and then treated with the anticancer drugs. they found that cells halted in s phase were significantly more sensitive to the cytotoxic effects of the drugs. to determine the involvement of survivin in resveratrol-mediated sensitization, the authors decided to test whether downregulation of