In this novel study, the effects of newly in silico designed bromodomain 4 and sirtuin 1 inhibitors on cellular growth and death was investigated in the neuroblastoma SH-SY5Y and acute myeloid leukemia U937 cell lines. The investigated bromodomain 4 compounds were found to be not as potent when compared to the sirtiun 1 inhibitor, W137. Therefore, the anti-proliferative effects and mechanistic action of W137 on the two human cancer cell lines were investigated.
The regulation of cancer associated gene expression may be possible in SIRT1-advantaged cancers such as in neuroblastoma and acute myeloid leukemia through the inhibition of SIRT1, which functions by deactivating or down-regulating anti-cancer proteins through deacetylation. Crystal violet DNA staining was applied to study the effect of W137 on cell numbers of SH-SY5Y cells and inhibition of cell proliferation was achieved in a concentration dependent manner. An IC50(SIRT1) value of 20 µM for the SH-SY5Y cells and 25 µM for U937 cells was determined after 48 h exposure. The IC50(SIRT1) values obtained for the compound as tested on the two cell lines were comparable to other well-known SIRT 1 and 2 inhibitors and low enough to merit further experimentation. Qualitative studies on cell morphology employing fluorescent microscopy and triple dye staining indicated a decrease in cell density and loss of cellular membrane integrity. A slight increase in propidium iodide staining of DNA after 48 h exposure to the W137 compound suggested either necrosis and/or late stages of apoptosis.
Employing flow cytometry and propidium iodide to study the progression of the cell cycle revealed increased cell numbers in the sub-G1 phase in SH-SY5Y and U937 cells, suggesting cell death is induced by W137 after 24 and 48 h respectively. No significant cell cycle block in either the G1 or G2/M phase was observed, suggesting that the compound does not work by modulating the cycle in SH-SY5Y cells. The efficacy of W137 does not vary significantly after 24 h exposure compared to 48 h exposure for the SH-SY5Y cell line, while an increase in compound efficacy seems to be observed after an extended exposure period (48 h) for the U937 cell line.
Flow cytometry employing propidium iodide in conjunction with Annexin V was used to analyse cell death induced via apoptosis and/or necrosis and 24 h as well as 48 h exposure to the W137 compound revealed an increase in early and late apoptosis with a slight increase in necrosis, in SH-SY5Y cells. Analysis of the U937 cell line indicated a slight population shift towards early apoptosis after 24 h exposure and a slight shift towards late apoptosis and necrosis after 48 h. These findings confirm that cell death occurs via apoptosis in the SH-SY5Y as well as in the U937 cell line when exposed to W137. SH-SY5Y (Control: 22.32% vs exposed: 26.53%) and U937 (Control: 24.52% vs exposed: 43.07%) cells exhibited increased mitochondrial membrane depolarization and therefore the intrinsic mitochondrial pathway is the likely means by which apoptosis is achieved. Therefore, apoptosis is the mechanism by which cell death is achieved in both cell lines, as confirmed by cell cycle studies, analyses of cell death and mitochondrial membrane permeability studies in both cell lines. Levels of hydrogen peroxide (H2O2) generated within W137-exposed cells after 48 h was measured using DH2CF-DA and flow cytometry. After 48 h exposure, a statistically significant decrease in DCF-fluorescence SH-SY5Y cells (Control: 40 vs exposed: 13.4) but not U937 cells (Control: 251.4 vs exposed: 259.8) was observed, suggesting that increased ROS levels was not induced in response to W137 exposure after 48 h.
To study the effect of exposure to the W137 compound on the expression of the p53 and C-MYC genes, both key factors in healthy and cancerous cells, the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) technique was employed. The SH-SY5Y cell line indicated a decrease in C-MYC levels upon Actinomycin D and W137 exposure after 24 h, while expression of the p53 gene was decreased after 24 h Actinomycin D exposure. Exposure to W137, however, increased p53 expression after 24 h as well as 48 h. p53 is crucial in programming cells for death and probably induces SH-SY5Y cells to enter apoptosis. Significant increase in C-MYC gene expression was observed in U937 cells after Actinomycin D exposure after 24 h and 48 h (p-value <0.05). Repression of SIRT1 therefore possibly resulted in the activation or increased expression of the pro-apoptotic gene p53, possibly enhancing cancer cell susceptibility to death and repair mechanisms. MYC oncoproteins, which are commonly up-regulated in human cancers of different organ origins, exert oncogenic effects by modulating gene and protein expression.
ELISA (Enzyme-linked Immunosorbent Assay) was employed to test for change in p53 protein K382 acetylation and activity following 48 h exposure to W137. The active, acetylated p53 protein is a crucial component in cell maintenance, damage detection and programmed cell death, and inhibition or down-regulation of p53 has been detected in many cancer types (1-3). Deacetylation of p53 by SIRT1 results in protein deactivation or down-regulation and therefore SIRT1 inhibition is expected to result in p53 being in the acetylated stage and active (4). Following 48 h exposure, analyses of the SH-SY5Y cells indicated a small but statistically significant increase in the expression of K382 acetylated p53 protein in Actinomycin D (1.17-fold) and W137 exposed cells (1.12-fold) (p-value <0.05).
In conclusion, the novel dual SIRT1 and 2 inhibitor W137 therefore inhibited cell proliferation in both the U937 and SH-SY5Y cell lines in a dose-dependent manner through the inhibition of cell growth and the induction of cell death in vitro. The two cell lines exhibited different reactions to the compound in some experiments but similar reactions in others, motivating the importance of further study into the cell line specificity and mechanistic variation of W137. The confirmation of these results by that obtained from the fluorescent microscopy study leads to the conclusion that apoptosis is the most likely cause of cell death for these cell lines. The current study contributes to the unravelling of the in vitro molecular mechanisms associated with and influenced by SIRT1 and SRT2 providing a basis for further research on this multi-functional cellular component and its diverse role in disease regulation. The W137 compound could potentially be an effective component in a drug designed to treat more than one type of cancer.
Keywords: Anti-cancer, acute myeloid leukemia (AML), neuroblastoma, sirtuin 1 (SIRT1), U937, SH-SY5Y, flow cytometry, apoptosis, cell cycle, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), gene expression, qRT-PCR, protein acetylation, ELISA, p53, C-MYC.