Browsing by Author "Maumela, Lutendo"
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- ItemInvestigating the effect of simulated Ischemia on phosphatases : comparing a cardiomyocytes (H9c2) cell line with a breast cancer (MDA-MB 231) cell line(Stellenbosch : Stellenbosch University, 2020-03) Maumela, Lutendo; Van Vuuren, Derick; Lopes, John; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.Background: Reversible phosphorylation is responsible for an estimated 30% of protein regulation. Protein phosphorylation is catalysed by protein kinases and dephosphorylation by protein phosphatases. Ischemia, which is characterised by reduced blood flow to tissue and hypoxia has been implicated in causing pathology in the regulation of proteins involved in signalling both in the heart and in cancer cells. Methods: 10000 cells/100µl/well of H9c2 and 25000 cells/100µl/well of MDA-MB 231 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10 % Fetal Bovine Serum (FBS) and 1 % Penicilin Streptomycin (PenStrep). Incubation was done in a sterile environment in 95 % atmospheric air in 5 % CO2 at 37 ºC in a humidified atmosphere. At 70 % - 80 % confluence, ischemia was simulated by Modified Esumi Buffer in conjugation with exposure to a hypoxic gas mixture [0% O2/5 % CO2/95 % N2 (H9c2) or 0.5 % O2/5 % CO2/balance N2 (MDA-MB 231)]. Incubation for simulated ischemia (SI) was done for 2 hours and acidic reperfusion for 30 minutes. Phosphatase activity was measured using both a p-nitrophenol phosphate (pNPP), as well as a 6,8-difluro-4methylumbelliferyl phosphate (DiFMUP) assay. Phosphatase expression was measured by Western blotting. Cell viability was measured using an ATP assay, as well as both propidium iodide (PI) and JC-1 staining. Cells were pharmacologically manipulated by administering cantharidin (2 µM and 5 µM), an inhibitor of both PP2A and PP1 and FTY 720 (1 µM and 5 µM), an activator of PP2A. All experiments were repeated three or four times on three or four different days. All data was analysed using Graphpad Prism version 5. All the data was expressed as mean ± standard error of the mean (SEM). For comparison between two groups, the student T-test was used. For multiple comparisons, one-way ANOVA with Bonferroni post hoc test was used. Differences were considered statistically significant at p < 0.05. Results: Energy status was measured as ATP levels revealing that SI reduced ATP levels in both H9c2 cells (control: 1.000 ± 0.000 Arbitrary Unit (AU) vs SI: 0.597 ± 0.042 AU; n = 3; p < 0.001) and MDA-MB 231 cells (control: 1.000 ± 0.000 AU vs SI: 0.458 ± 0.025 AU; n = 4; p < 0.01). JC-1 stain showed no mitochondrial impairment and PI staining detected no significant cell membrane breakage in both H9c2 and MDA-MB 231 cell lines treated with SI. pNPP and DiFMUP assays showed no noticeable change in the activity of protein phosphatases in both the MDA-MB 231 and H9c2 under SI conditions. The total expression of Akt/PKB in MDA-MB 231 cell line showed a statistically significant reduction following 2 hours SI (control: 1.000 ± 0.000 AU vs SI: 0.556 ± 0.027 AU; n = 3; p < 0.01). In addition, the expression of PP2Ac was also inclined in MDA-MB 231 cell line due to SI (control: 1.482 x 107 ± 8.715 x 105 AU vs SI: 1.964 x 107 ± 1.406 x 106 AU; n = 3; p < 0.05). Protein phosphatase 2A was elected for pharmacological manipulation in both H9c2 and MDA-MB 231 cell lines under SI conditions. Normoxic MDA-MB 231 cell line was treated with 1 µM FTY 720 to show an increase in PI fluorescence (normoxia: 199.5 ± 8.381 relative fluorescence units (RFU) vs normoxia + 1 µM FTY 720: 228.1 ± 2.902 RFU; n= 4; p < 0.05). Data assessed by JC-1 staining showed that 5 µM cantharidin treatment under SI conditions reduced the mitochondrial function and integrity of the MDA-MB 231 cell line (SI: 1.000 ± 0.000 RFU vs SI + 5 µM cantharidin: 0.625 ± 0.112 RFU; n= 4; p < 0.01). JC-1 image analysis showed that SI + 5 µM cantharidin also reduced mitochondrial function and integrity in H9c2 cells (SI: 1.000 ± 0.000 RFU vs SI + 5 µM cantharidin: 0.285 ± 0.059 RFU; n= 3; p < 0.01). Discussion and conclusion: Two hours SI did not significantly influence phosphatase activity in both H9c2 and MDA-MB 231 cell lines. In MDA-MB 231 cell line SI was associated with an increase in the expression of PP2Ac. SI caused a significant reduction in ATP levels in both H9c2 and MDAMB 231 cell lines as expected. However, 2 hours SI did not induce cell death as measured by PI and JC-1 staining. Pharmacological inhibition of PP2A with 5 µM cantharidin in combination with SI protected the cells by reducing the consumption of ATP in H9c2 cells. Other studies showed that the inhibition of PP2A indeed protects heart cells from ischemia whereas, studies done in cancer report that the inhibition of PP2A induces cell death. Surprisingly, even though the inhibition of PP2A by 5 µM cantharidin under SI conditions reduced the consumption of ATP in H9c2 cells, JC-1 image analysis reported that it also reduced mitochondrial function and integrity. SI + 5 µM cantharidin also reduced mitochondrial function and integrity in MDA-MB 231 cell line. As stated, in the heart the inhibition of PP2A has been shown by other researchers to be protective. The activation of PP2A by 1 µM FTY 720 in MDA-MB 231 cell line under normoxic conditions induced cell death as measured by PI. Other studies have showed that it is indeed the activation of PP2A that induces cell death in cancer cells, making it a tumour suppressor. Interestingly, an increasing body of evidence shows that the inhibition of PP2A can also lead to cell death, making it a tumour promotor in other types of cancer. It is therefore concluded that PP2A may play a dual role in cell death depending on the targeted holoenzyme and maybe also cell type. More studies must be done to investigate the role of PP2A in cell death in different cell types, as well as to identify which holoenzymes should be targeted for a desired outcome. PP2A remains of great interest in the field of research for cancer therapy as well as cardioprotection.