The Maintenance of Homeostasis in a Constantly Regenerating Population of Cells - Lab Report Example

?Lab report An important characteristic of most cancers is a defect in apoptosis signaling. The gradual transformation of colorectal epithelium to carcinomas is associated with a series of accumulated genetic alterations which give rise to unregulated epithelial cell proliferation as a result of a progressive inhibition of apoptosis (Bedi et al., 1995). Apoptosis is a morphologically distinct form of cell death which is under genetic control. It is also well recognized that apoptosis and the genes that control it have a strong effect on the malignant phenotype. The genes found mutated most frequently in colon cancer are adenomatous polyposis coli (APC), deleted in colon cancer (DCC), K-Ras, and p53 (Bhatnagar et al., 2009). Apart from playing an important role in the maintenance of homeostasis in a constantly regenerating population of cells, such as the colonic epithelium, apoptosis also functions to destroy cells with DNA damage and prevent neoplasia. Inhibitor of apoptosis or IAP proteins are a class of anti-apoptotic regulator proteins which are characterized by the presence of baculoviral IAP repeat (BIR) domains. IAPs inhibit the activation caspases which are the key effector proteases of apoptosis, by directly binding to them through the BIR domains. Extensive evidence exists to show the involvement of the IAP family of proteins in oncogenesis, via their efficient suppression of apoptosis (Hunter et al., 2007). Survivin is a novel member of the IAP family of proteins with a potential dual role in apoptosis inhibition and regulation of mitosis. Survivin inhibits apoptosis by interfering with the function of caspase-3, caspase-7 and caspase-9 (Shin et al., 2001; Dohi et al., 2004). In addition to apoptosis inhibition, survivin is implicated in the regulation of the mitotic spindle checkpoint and the promotion of angiogenesis, and chemoresistance (Altieri, 2003; Mita et al., 2008). Several mitotic kinases, including the three Aurora kinases, Aurora-A, -B and –C kinases regulate the progression of the cell through mitosis. Lens et al. (2006) have provided evidence to show that survivin acts as a mitotic regulator. It functions as a subunit of the chromosomal passenger complex, which is essential for proper chromosome segregation and cytokinesis. In this complex, Aurora B acts as the enzymatic core, while survivin dictates chromosomal passenger complex localization. Survivin is uniquely placed at the border of both the cell-death machinery and mechanisms of cell cycle progression and microtubule stability (Dan et al., 2004). Survivin is a highly conserved 16.5 kDa protein with 142 amino acids. X-ray crystallography has revealed the protein to be an unusual bow tie-shaped dimer with two ?-helical extensions (Chantalat et al., 2000). It interacts with the microtubules through the ?-helical extension at the carboxyl terminal. Expressed in the G2/M phase (Li et al., 1998), survivin is up-regulated in almost all cancers, including colon cancer, but has low or no expression in most normal, differentiated adult tissues (Duffy et al., 2007). Expression of survivin in cancer cells has been shown to promote tumorigenesis (Li, 2005), cancer progression, poor prognosis, shortened patient survival and resistance to chemo- and radiation therapies (Li and Ling, 2006). A number of signaling molecules, transcription factors and other ligands modulate survivin expression and/or function in cancer cells through transcriptional and/or posttranscriptional mechanisms (Zhang et al., 2006). Of these, regulation of its gene transcription is an important mechanism for the modulation of survivin expression. Sp1, a transcription factor is reported to be involved in the transcriptional activation of survivin (Li and Altieri, 1999). According to Kim et al. (2003), an aberrant TCF/? catenin might cause the stimulation of survivin expression leading to enhanced cell proliferation and resistance to apoptosis, thereby promoting the molecular pathogenesis of colorectal cancer. Zhang et al. (2000) demonstrated that APC mutation causes constitutive expression of survivin. At the protein level, phosphorylation of survivin is important for its biological activity and function (O’Connor et al., 2000). The expression of survivin during mitosis occurs in a cell cycle dependent manner and is localized to different components of the mitotic apparatus (Carrasco et al., 2011). The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays an antiapoptotic role while the p53 pathway plays a proapoptotic role in cell death. p53 has been shown to suppress survivin expression by directly interacting with the survivin promoter (Raj et al., 2008). Thus, cancer cell growth and progression are associated with high levels of PI3K/Akt activation and inactivation of p53. As opposed to cancer cells with mutant p53 that upregulates survivin expression, normal cells possess the wild-type p53 gene that downregulates survivin expression (Zhang et al., 2006). The increase in survivin expression has been found to be maintained throughout the different stages of colorectal cancer i,e., during the mucosa-adenoma-carcinoma sequence (Hernandez et al., 2011). Besides, the expression levels of survivin strongly correlate with the proliferative activity of the tumours indicating a possible role in cell cycle regulation and cancer progression (Martinez et al., 2004). The prognostic importance of measuring survivin expression at the level of protein as well as mRNA is evident from the study of Sarela et al. (2000), who showed that survivin mRNA was associated with decreased overall survival of patients with colon cancer after treatment. Oxaliplatin, a third-generation diaminocyclohexane (DACH)-platinum complex, is a chemotherapeutic drug which induces DNA double-strand breaks and apoptosis in colorectal cancer cells. The biological activity of oxaliplatin is mainly based on its ability to form lethal DNA lesions, including interstrand DNA crosslinks and DNA–protein crosslinks (Woynarowski et al., 2000). According to Chiu et al. (2008), oxaliplatin induces persistent gamma-H2AX production, leading to apoptosis in colorectal cancer cells. Oxaliplatin is a potent inhibitor of survivin. Recently, Liu et al. (2010) showed that survivin protein expression is significantly reduced in human colon cancer cells exposed to oxaliplatin whereas the survivin gene expression changes slightly. However, the primary mechanism through which cell death occurs with oxaliplatin depends on the cell type (Ngan et al., 2007). Vinflunine is a second generation Vinca alkaloid showing anti-neoplastic activity in a wide spectrum of solid tumours. It acts by binding to tubulin and causing the subsequent cellular arrest in mitosis (Yip et al., 2008). Vinflunine administration has been found to potentiate the anti-cancer activity of the platinum-based chemotherapeutic drug, cisplatin, in colon adenocarcinoma (Schnyder et al., 2003). The aim of this study was to investigate the effects of oxaliplatin and vinflunine treatment on survivin protein expression in lymphocytes from colon cancer patients. Results Survivin expression inhibition by oxaliplatin in lymphocytes from colon cancer patients Survivin protein expression in lymphocytes from colon cancer patients treated with 0.02?M, 0.2?M and 2?M oxaliplatin was evaluated by Western blot assay. Untreated cells were used as control to determine the cut-off value of survivin overexpression for comparison. Survivin expression in normal cells could not be detected. As depicted in Fig. 1, at all the concentrations of oxaliplatin tested, the expression of survivin was significantly reduced. The reduction was dose dependent. Survivin expression in 0.02?M oxaliplatin-treated lymphocytes was nearly 10% less than in the control cells, about 39% less in 0.2?M oxaliplatin-treated cells and 72% less in 2?M oxaliplatin-treated cells. Thus, oxaliplatin at 0.02?M, 0.2?M and 2?M concentration caused, respectively, 1.3–fold, 1.6-fold and 3.6-fold decrease in survivin expression. Survivin expression inhibition by vinflunine in lymphocytes from colon cancer patients The values of relative reduction in survivin expression in lymphocytes from colon cancer patients treated with 0.06?M, 0.6?M and 6?M vinflunine in comparison with the control (untreated) lymphocytes are plotted in Fig. 2. Vinflunine only at higher concentrations, that is, 0.6?M and 6?M, produced statistically significant reduction in survivin expression, of around 39% (i.e., ~1.6-fold) and 85% (i.e., ~6.7-fold), respectively. There was no statistical difference between the survivin expression levels of untreated and 0.06?M vinflunine-treated lymphocytes. Discussion Clinical studies have demonstrated the efficacy of oxaliplatin monotherapy in several cancers, including colon cancer (Cvitkovic and Bekradda, 1999). Some desirable factors that are specific to oxaliplatin are its high potency even in sensitive cell lines (Woynarowskiet al., 2000), and the absence of resistance due to mismatch repair deficiency (Sergent et al., 2002). Cyclin-dependent kinase inhibitor p21waf1/cip1 has been observed to play a crucial role in the oxaliplatin-mediated regulation of cell cycle progression in both p53-dependent and -independent pathways (Hata et al., 2005). For instance, in a p53-dependent pathway oxaliplatin causes induction of p21waf1/cip1 which leads to G1 arrest and, thereby, contributes to the inhibition of cell proliferation. On the other hand, in a p53-independent pathway oxaliplatin degrades the p21waf1/cip1 protein and thereby causes the abrupt progression of the cells from G0-G1 phase to S phase, finally resulting in G2-M arrest (Hata et al., 2005). The primary components of cell cycle progression are cyclin-cyclin dependent kinases which undergo activation in an ordered manner to initiate DNA replication, nuclear envelope breakdown, spindle formation, and chromosome segregation (Maity et al., 1994). Timely activation of the cyclin-dependent protein kinase, Cdc2, a heterodimeric protein complex consisting of Cdc2 and cyclin B1 controls the transition from the G2 phase of the cell cycle into mitosis (Yu et al., 1998). Translocation of cyclin B1 to the nucleus is essential for the G2/M transition (Badie et al. (2000) and suppression of Cdc2 activity for DNA damage-induced G2 arrest. Oxaliplatin has been observed by Hata et al. (2005) to down-regulate Cdc2 protein and decrease nuclear translocation of cyclin B1. The anti-apoptotic protein survivin is a potential tumor marker which can aid the early diagnosis of bladder cancer, determine prognosis in multiple cancer types and predict response to a variety of anti-cancer therapies (Duffy et al., 2007). A possible mechanism that explains the efficacy of oxaliplatin against colorectal cancer is its inhibition of expression of survivin (Liu et al., 2010). Sohn et al. (2010) have reported that oxaliplatin exposure causes changes in the intracellular localization of survivin in cancer cells as well. Consistent with the findings of Liu et al. (2010), the present study also revealed significant reduction in the expression of survivin protein ranging from 10% to 72% in lymphocytes from colon cancer patients (Fig.1). Overexpression of survivin protein is a hallmark of most cancers which also generally denotes poor prognosis and chemo-resistance in some types of cancer (Schultz et al., 2006). On the other hand, it has also been reported that a nuclear localization of survivin could potentially slow down tumor progression (Okada et al., 2001). Since survivin is reported to inhibit apoptosis by binding to caspases, downregulation of survivin by oxaliplatin would help re-establish the apoptosis process. Oxaliplatin-aided phosphorylation of p38 mitogen-activated protein (MAP) kinase which down-regulates survivin is another mechanism that has been described to explain the cytotoxic effects of oxaliplatin (Liu et al., 2010). In addition to considerable survivin downregulation, oxaliplatin has been found to cause drastic mitotic catastrophe in adenocarcinoma and gastric cancer (Gu et al., 2006). Mitotic catastrophe is a defined process of cell-death that occurs in addition to or as an alternative to apoptosis (Okada and Mak, 2004). In colon cancer cells, oxaliplatin was found to decrease survivin expression as well as the mitosis-commencing protein cdc2 and anti-apoptotic proteins, phospho-Bcl(2) and Bcl-xl (Fujie et al., 2005). In combination with an apoptosis-inducing chemoreagent (paclitaxel), oxaliplatin caused marked growth inhibition of the colon cancer cells by enhancing both apoptosis and mitotic catastrophe. Studies show that oxaliplatin can induce different modes of cell death. For instance, following cell-cycle arrest at G(2) phase after oxaliplatin treatment of two esophageal cancer cell lines, the squamous cell carcinoma cells died via caspase- dependent apoptosis, and adenocarcinoma cells died via mitotic catastrophe (Ngan et al., 2008). Interestingly, a stronger inhibition of survivin expression occurred in the adenocarcinoma cells while survivin promoter activity was inhibited by oxaliplatin in both cell lines. Survivin downregulation in squamous cell carcinoma was in part due to the proteasome-mediated protein degradation pathway and also due to the downregulation of Sp1 transcription factor. The action of the spindle checkpoint is crucial during mitosis to ensure proper segregation of chromosomes. Chromosomal passenger proteins Aurora B and survivin affect the spindle checkpoint. In simplistic terms, the function of the survivin/Aurora B complex is mainly to facilitate tension and attachment coordination of the cell (Lens and Medema, 2003). Survivin is important for Aurora B localization as well as for its kinase activity (Bolton et al., 2002). Anomalous expression of Aurora B and survivin results in chromosomal instability, aneuploidy and malignant transformation (Fu et al., 2007). Aurora kinases make a potential target for anticancer therapy, and several Aurora-A and Aurora-B kinase inhibitors are being synthesized (Kitzen et al., 2010). Discrimination of neoplastic cells from normal ones is critical for immunological control of malignancy (Troeger, 2007). The overexpression of survivin in cancer cells as compared to its near absence in normal tissue confers the required differentiation. Casati et al. (2003) have demonstrated a potential role for survivin as a tumor associated antigen (TAA) for colon carcinoma. Their study has confirmed that CRC patients can develop a survivin-specific immunity involving both a CD8+ and CD4+ T cell–mediated response which would be invaluable for the development of immune-based clinical approach to fight CRC. Earlier, TAAs such as mucins, carcinoembryogenic antigen and epithelial cell adhesion molecule have been tried for immunotherapy against CRC, but the trials were mostly inadequately associated with objective clinical responses (Casati et al., 2003). Numerous therapeutic techniques, including chemotherapeutic drugs, immunotechnology, antisense, dominant negative survivin mutant, RNA interference and peptide-based methods are being studied (Kanwar et al., 2010). To address the issue of selectively targeting survivin, nanotechnology-based platforms are being developed. Vinflunine is a novel microtubule inhibitor produced by semisynthesis using superacidic chemistry to selectively modify the catharanthine moiety of the Vinca alkaloid molecule (Bennouna et al., 2008). The optimal structural modification involves the introduction of two fluorine atoms which impart antitumor activity to the compound. Vinflunine exerts marked cytotoxicity against several human solid tumor cell lines of different origins including colon cancer (Kruczynski and Hill, 2001). The mitotic spindle which plays a key role in chromosome segregation during cell division is an important target for anticancer therapy. Specifically, microtubules present in the mitotic spindle are the focus of a number of microtubule inhibitors that have been developed as successful anticancer drugs. The vinca alkaloids including vinflunine are a class of microtubule-interfering agents (Bennouna et al., 2008). At high concentrations vinflunine causes extensive depolymerization of microtubules and formation of tubulin paracrystals. Used at moderately high concentrations, it induces depolymerization of the microtubule network. At the lowest effective concentrations, vinflunine suppresses microtubule dynamics (Pourroy et al., 2004). Vinflunine has been found to be moderately active with a manageable and noncumulative toxicity profile in urothelial carcinoma (Vaughn et al., 2009). The mechanism of vinflunine action consists of interaction with the Vinca alkaloid binding site on tubulin and causing suppression of the microtubule dynamics and microtubule treadmilling, causing G2/M phase arrest followed by accumulation of cells in mitosis, leading to apoptosis and cell death (Kruczynski et al., 1998). Apoptosis promoted by vinflunine likely follows a mitochondrial pathway (Pourroy et al., 2004). The present study revealed a strong inhibition of survivin protein expression by vinflunine (0.06?M, 0.6?M and 6?M) in lymphocytes from colon cancer patients (Fig. 2). Since survivin is a proven anti-apoptotic protein, the antitumor activity of vinflunine is probably also due to re-establishment of apoptosis as a consequence of inhibition of survivin expression by vinflunine. But literature is lacking on the aspect of vinflunine effect on survivin expression in cancer cells. In conclusion, significant reduction in survivin protein expression was observed in lymphocytes from colon cancer patients exposed to oxaliplatin or vinflunine. Treatment, especially with oxaliplatin and possibly with vinflunine, is likely to induce mitiotic catastrophe in the lymphocytes. References Altieri DC., 2003. Validating survivin as a cancer therapeutic target. Nat Rev Cancer, 3:46–54. Bedi A, Pasricha PJ, Akhtar AJ, et al. (1995) Inhibition of apoptosis during development of colorectal cancer. Cancer Res 55:1811–1816. Bennouna J, Delord J, Campone M, et al., 2008. Vinflunine: a new microtubule inhibitor agent. Clin Cancer Res., 14:1625. Bhatnagar N, Li X, Chen Y., et al., 2009. 3,3?-Diindolylmethane enhances the efficacy of butyrate in colon cancer prevention through down-regulation of surviving. Cancer Prev Res., 2:581. Bolton MA, Lan W, Powers SE, et al., 2002. 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Zhang M, Yang J. & Li F., 2006. Transcriptional and posttranscriptional controls of survivin in cancer cells: Essential interfaces for developing novel approaches for cancer treatment. J Exp Clin Cancer Res., 25(3): 391. Fig. 1. Survivin expression in lymphocytes from colon cancer patients following treatment with different concentrations of oxaliplatin. Control consisted of untreated lymphocytes. Fig. 2. Survivin expression in lymphocytes from colon cancer patients after treatment with different concentrations of vinflunine. Control consisted of untreated lymphocytes. Read More