Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumour which secretes ACTH and other related peptides. Contrary to normal production by the pituitary, ACTH production is not inhibited by glucocorticoids (Gcs) in SCLC. This insensitivity to Gc action can be attributed to impaired Gc receptor (GR) expression in these cells. Over-expression of the GR induces apoptosis both in vitro and in vivo. Evasion of GR signalling thus confers a significant survival advantage to SCLC cells. Re-expression of endogenous GR in SCLC cells may provoke the same effect. Many tumours silence the expression of tumour suppresser genes by epigenetic mechanisms. Recent evidence suggests that the GR in SCLC cells is epigenetically silenced by hypermethylation of its promoter.
The overall aim of this study was to determine whether endogenous GR re-expression induces apoptosis of SCLC cells. The DMS 79 SCLC cell line, and the control HEK and non-SCLC A549 cell lines were treated with the DNA methyltransferase inhibitor (DNMTi), 5-aza-2′-deoxycytidine (5-aza), to determine whether treatment with 5-aza results in re-expression of endogenous GR. Conflicting results were thought to result from the use of possibly degraded 5-aza. However, a quantitative real-time PCR analysis using newly purchased, freshly prepared 5-aza indicated that 5-aza treatment up-regulated GR mRNA expression in the DMS 79 cells (p<0.0005). No significant changes in GR expression were seen in the HEK and/or A549 cells, suggesting that the GR in these cell lines is not methylated. Contrary to expectations and possibly due to the use of degraded stock, Western blot analysis revealed that 5-aza had no effect on GR protein expression in DMS 79 cells, yet affected GR protein expression in HEK and A549 cells (p=0.003 and p=0.042, respectively).
Cell viability assays indicated that treatment with varying concentrations of 5-aza had no effect on the viability of DMS 79 and A549 cells, but had a minimal effect on HEK cell (p<0.0005) viability. These data reinforce the hypothesis that stock 5-aza had degraded as 5-aza is known to exert cytotoxic effects at higher concentrations.
Using newly purchased, freshly prepared 5-aza, flow cytometry and/or microscopy were performed to establish whether endogenous GR re-expression was sufficient to kill the SCLC cells by apoptosis. FITC Annexin V staining and nuclear morphology showed that significant
proportions of the 1 μM (p=0.010 and p=0.027) and 5 μM (p=0.002 and p=0.018) 5-aza treated DMS 79 cells were apoptosing, with little apoptosis seen in HEK cells. 5-Aza induced negligible HEK cell death, as determined by microscopic analyses.
The effect of dexamethasone (Dex; a synthetic Gc) on HEK and DMS 79 cells was examined to determine whether Gc treatment could enhance apoptosis. Treatment with Dex alone, and in combination with 5-aza, resulted in significant HEK cell death (p=0.046 and p=0.005 respectively), but not apoptosis. This was unexpected as HEK cells express very little unmethylated GR, and may be due to excessive drug exposure or combined drug toxicity. The same effect was observed with DMS 79 cells (p=0.003 and p<0.0005 respectively), with 5-aza appearing to enhance cell death induced by Dex. No effects on apoptosis were seen confirming earlier reports that GR-mediated apoptosis is ligand-independent.
As 5-aza does not selectively demethylate the GR, cells were exposed to the GR antagonist, RU486, to establish whether apoptosis associated with 5-aza treatment is specifically due to demethylation and subsequent expression of the GR. Treatment with RU486 in conjunction with 5-aza induced cell death (p=0.014), but not apoptosis, of HEK cells. Again, this may have been due to excessive drug exposure or combined drug toxicity. Flow cytometric data showed that DMS 79 cell death was induced by both RU486 (p=0.004), and RU486 in combination with 5-aza (p=0.003). Furthermore, although not significant, RU486 treatment appeared to inhibit apoptosis induced by 5-aza in the DMS 79 cells. The data suggest that re-expression of the GR may be responsible for apoptotic induction. Our findings, although not significant, hint that endogenous re-expression of the GR leads to apoptosis.
Unlike mutations, epigenetic marks are reversible and clinical trials with DNMTis have shown promising results. The identification of a novel endogenous mechanism that specifically induces apoptosis of SCLC cells offers great promise for the development of targeted therapeutics for the treatment of this deadly disease. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/9855 |
| Date | 01 November 2013 |
| Creators | Houston, Kerryn. |
| Contributors | Sommer, Paula. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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