Pharmacological studies of voltage-gated sodium channel expression in human breast cancer cells: Control of metastatic cell behaviours

Abstract

The overall aim of this PhD was to improve our understanding, including the clinical potential, of neonatal Nav1.5 (nNav1.5) expression in human metastatic breast cancer. Mainly, the strongly metastatic MDA-MB-231 cells were used throughout the studies. The specific aims were threefold, as follows: 1) To test the effects of several types of voltage-gated sodium channel (VGSC) blocker on nNav1.5 mRNA and protein expression and metastatic cell behaviours (MCBs); (2) to determine the effects of hypoxia on the drug treatments and MCBs; and (3) to elucidate a possible association of carbonic anhydrase-9 (CA9) and nNav1.5 expression/activity. There are three main Results chapters. Results-1 demonstrates the effects of the drugs on MCBs of MDA-MB-231 cells under normal oxygen level (normoxia). Two classes of blocker were used: a) Local anaesthetics (lidocaine and procaine) and (b) blockers of persistent current (INaP) (ranolazine and riluzole). In addition, a specific VGSC blocker, tetrodotoxin (TTX), was incorporated as a control. At concentrations not affecting the cells‘ viability or proliferative activity, 24 h treatment with all the drugs tested significantly reduced MCBs (lateral motility, transverse migration and Matrigel invasion). Concurrently, the treatments reduced nNav1.5 mRNA and protein levels. TTX had a similar effect. Overall, this chapter showed (i) that MCBs in MDA-MB-231 cells were enhanced by VGSC activity and (ii) that INaP played a significant role in the enhancement. Results-2 shows that viability and proliferative activity of MDA-MB-231 cells were not affected by hypoxia (mostly 2 % oxygen applied for 24 h). However, hypoxia increased the cells‘ invasiveness and this was accompanied by upregulation of HIF-1α (protein), nNav1.5 (mRNA) and CA9 (both mRNA and protein). Treatment for 24 h with INaP blockers; ranolazine and riluzole under hypoxia reduced lateral motility, transverse migration and Matrigel invasion. At concentrations not affecting cell viability and proliferation, the effects of ranolazine and riluzole in suppressing MCBs were generally greater under hypoxia compared to normoxia. It was concluded (i) that hypoxia enhanced VGSC-mediated MCBs and (ii) that the enhancements were likely to be increase in INaP amplitude induced by hypoxia. Results-3 examined a possible functional link between hypoxia (HIF-1α and CA9) and VGSCs. Acetazolamide was used as a general inhibitor of CAs and siRNA was used to silence specifically CA9. Under hypoxia, treatment with acetazolamide for 24 h had no effect on invasion, and treatment with TTX was without effect on CA9 expression. In contrast, silencing CA9 using siRNA (siCA9) reduced CA9 and nNav1.5 expression and Matrigel invasion was also significantly inhibited. It was concluded (i) that CA9 played a role in cellular invasion and (ii) that nNav1.5 was down-stream to CA9 in the control of MCBs. The Thesis is concluded with a General Discussion and Conclusion chapter integrating the findings and highlighting their clinical potential.