The contribution of sodium to the bioelectric behaivour of ovarian cancer

McBride, Mark (2021) The contribution of sodium to the bioelectric behaivour of ovarian cancer. PhD thesis, University of Nottingham.

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Ovarian cancer (OvCa) is the most lethal gynaecological malignancy worldwide and while less prevalent than other malignancies among women, 5-year survival is low and has remained relatively unchanged over the past 40 years. In recent decades the im-portance of bioelectric properties, in particular the plasma membrane potential (Vm),have become increasingly recognised as playing central roles in the regulation of nu-merous biological processes and disease states, including cancer. Recent studies have demonstrated alterations of Vmimpact critical properties of cancer hallmarks, namely sustained proliferation and metastasis. Na+channels play critical roles in regulating Vmand recent evidence has accrued implicating voltage gated Na+channels in metastatic progression of numerous cancers. Limited information however exists for the role of the epithelial Na+channel (ENaC) a constitutively open channel ubiquitously expressed across numerous cell types.

The aim of this study was to investigate the involvement of Na+ and ENaC inmodulating the proliferative and migratory potential in OvCa. The contribution of Na+to Vmin SKOV-3 and primary cells derived from the ascites of patients with FIGO3−4cancer OvCa (OvCaasc) cells were assessed by patch clamp studies. Vm values in both cell types were markedly depolarised in physiological solutions (≈-5 to -10 mV) and hyperpolarised markedly by≈20 to 25 mV (p<0.05) upon removal of extracellular Na. The contribution of ENaC to Vmwas assessed using the blocker amiloride. Wash-in induced a mild hyperpolarisation of≈5 mV in SKOV-3 cells.

The contribution of ENaC to the proliferative capacity of SKOV-3 and OVCAR-3 cell lines was assessed using the resazurin reduction assay. Amiloride resulted in a marked reduction in cell proliferation in SKOV-3 and a mild reduction in OVCAR-3 cell lines (IC50≈60 and M respectively at 72 hours post-treatment). The faster growing SKOV-3 cell line exhibited a dose dependent increase in apoptosis when treated with amiloride whereas OVCAR-3 cells demonstrated a cytostatic response. Mild increases in [Na+]eup to 30 mM resulted in a significant reduction in the rate of proliferation of SKOV-3 cells. Conversely, elevated [Na+]e resulted in a mild increase (≈20%, p<0.05) detectable at 72 hours. Since cell volume (Vcell) changes are required in proliferating cells, Vcell of amiloride treated SKOV-3 populations were assessed using imaging flow cytometry. Cells incubated in lower doses of amiloride (10 & 50 µM) yielded no significant change in volume relative to control conditions. Cells cultured in the presence of 100 µM amiloride resulted in a significant increase in cell volume. This however only equates to an≈1% change relative to control and significance is likely attributable to high sensitivity of non-parametric rank tests.

Gene expression studies of ENaC- α and delta were conducted between normal ovarian (NOv), FIGO3-4 primary tumours (FIGO3−4T) & OvCaasc tissue. ENaC-α demonstrated an ≈60-fold up-regulation in FIGO3-4T & OvCaasc groups relative to NOv(p<0.05). Conversely ENaC- δ demonstrated a significant down-regulation between NOv and OvCaasc tissue. Functional expression was confirmed by western blot (WB)and immunofluorescence. Differential expression of ENaC- α was assessed in distinct OvCaasc sub-populations using flow cytometry. Approximately 80% of CD117+/CD44+were positive for ENaC- α, whereas only≈50% of EpCam+cells exhibited ENaC- α +staining.

The role of ENaC in cell migration in SKOV-3, OVCAR-8 & A2780 cells was next assessed using scratch assays. Amiloride reduced wound closure in all three cell lines, with the maximal effects observed in SKOV-3 cells (3-fold reduction in wound closure relative to control). In a wound-stimulated environment, SKOV-3 cells exhibited afunctional up-regulation of ENaC- α (≈1.5-fold increase) as assessed by WB. Since cell deformability is important in cancer migration, the role of ENaC in modulating cell stiffness was assessed using atomic force microscopic (AFM). Amiloride resulted in an≈4-fold increase in cell stiffness in A2780 cells.

Finally, since long-distance endogenous electric fields (EF) have been implicated in directing metastatic dissemination of cancer, it was assessed whether OvCa cells exhibited galvanotaxis. OvCaasc cells were strongly galvanotactic, exhibiting highly directional migration towards the anode. Amiloride and phenamil (a derivative of amiloride) resulted in a mild reduction in anodal migration, however, cells still exhibited the ability to ‘sense’ an external EF. Depletion of [Na+]e with impermeant replacements abolished the anodal migration in an external EF.

In conclusion this study demonstrated that ENaC may have an important role in contributing to the depolarised phenotype and modulating critical hallmarks in OvCa.ENaC contributes to sustained proliferation, potentially by persistent facilitative de-polarisation of Vm. ENaC- α is up-regulated in OvCa progression and potentially con-tributes to sustained Na+influx in OvCa, and stemness. ENaC facilitates OvCa migration and modulates critical cell properties essential for migrating in heterogeneous environments. ENaC and Na-mediated depolarisation seem to be central to controlling the galvanotactic response in OvCa dissemination. The contribution of the galvano-tactic cue in OvCa metastasis still needs to identified. Altered Na+homeostasis and subsequent modulation of Vm is thus a promising area for further research in OvCa

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Khan, Raheela
Bali, Anish
Pavlovskaya, Galina
Rauch, Cyril
Keywords: sodium, Na, membrane potential, ENaC, ovarian cancer, galvanotaxis
Subjects: W Medicine and related subjects (NLM Classification) > WP Gynecology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Graduate Entry Medicine and Health
Item ID: 65491
Depositing User: McBride, Mark
Date Deposited: 04 Aug 2021 04:42
Last Modified: 04 Aug 2021 04:42

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