Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.806654
Title: The role of ATP6V1E1 in the control of apoptosis, survival and motility of breast cancer cells
Author: Mohammed, Saja Nadhem
Awarding Body: Keele University
Current Institution: Keele University
Date of Award: 2020
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Abstract:
Background and aims: Aggressiveness and invasiveness of breast cancer are influenced by multiple cellular and molecular factors, some of which are yet to be elucidated. In normal cells, vacuolar H (+) -ATPase (V ATPases), which pump protons across the plasma membrane function to maintain electrochemical gradient and acidity in intracellular compartments. Thus, alteration of normal V-ATPase activity could be involved in the modification of cancerous and tumorigenic processes in breast cancer. The present study investigated the role of V-ATPase subunit E1 (ATP6V1E1) — the major isoform (77%) of the five human ATPases (subunit B, C, D, E, and G) — in the apoptosis, survival, and motility of breast cancer cells. Materials and methods: The alteration of the expression of ATP6V1E1 in MCF7 and MDA-MB-231 cancer cells was achieved using recombinant plasmid DNA carrying fulllength ATP6V1E1 (pcDNATM 3.1-ATP6V1E1; for upregulation) and RNA interference (RNAi) technology (siRNA1-4; for downregulation). The cell lines with altered ATP6V1E1 expression were studied for cellular ATP6V1E1 protein localization using immunofluorescence confocal microscopy. They were also examined for altered apoptosis rate using cellular apoptosis assays including Annexin V, Multi-Caspase and Acridine orange. Cell viability was probed using fluorescence-activated cell sorting (FACS) analysis. The cells were investigated for intracellular and extracellular acidity (pHi, and pHe) using pH-sensitive LysoSensorTM Green DND-189 dye (Ambion® Life Technologies Ltd Paisley, UK) and pH changes of growth media, respectively. Lastly, the effect of altered ATP6V1E1 expression on actin-microtubule cytoskeleton by immunofluorescence and electron microscopy and the subsequent influence on cell motility was investigated. The motility (migration, and invasion) and colony-forming abilities of the cancer cells were investigated using a wound scratch closure assay with the aid of the ImageJ software. Statistical analysis was performed using GraphPad InStat software. Results: Immuno-labelling of the target ATP6V1E1 protein using the anti-ATP6V1E1 monoclonal antibody was comparable between two dilutions tested (1:100 and 1:250). A lower antibody concentration (1:250) was deemed cost-effective. MCF7 and MDA-MB- 231 cells exhibited comparable ATP6V1E1 immunolabelling. Cytoplasmic microtubules were visualized in both cells, while actin filaments were clearly visualized in only MCF7 cells. ATP6V1E1 overexpression significantly decreased cell survival and increased apoptosis rates in both cell lines, compared to controls (P<0.0001). By contrast, ATP6V1E1 down-expression significantly increased cell survival, especially cells in S phase and G2/M phase 48 post-plating. ATP6V1E1 overexpression appeared to have increased actin content in MCF7 cells more than in MDA MB-231 cells, which instead revealed higher microtubule labeling than the former. ATP6V1E1 downregulation resulted in faster wound closure rate in both cell lines. The intensity of LysoSensorTM-labelling (pHi) cells was significantly higher in ATP6V1E1-overexpressing cells compared to controls (p<0.01). Cells were grown in the acidic pH and alkaline pH exhibited an increased and decreased time-dependent survival from 2 h to 24 h post plating compared to controls (p<0.001), respectively. ATP6V1E1 upregulation reduced the actinmicrotubule cytoskeleton. Conclusions: Overexpression of ATP6V1E1 appears to repress breast cancer cell activities by promoting apoptosis, reducing actin-microtubule cytoskeleton (which decreases cancer cell motility) and decreasing pHi. Recombinant ATP6V1E1 gene products could be useful for breast cancer therapy warranting further studies.
Supervisor: Furness, D. N. ; Hoole, D. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.806654  DOI: Not available
Keywords: QH301 Biology
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