| Popis: |
Metastasis is the major problem in clinical management of cancer, including prostate cancer (PCa). Biochemical conditions in tumour microenvironment (especially O2 and Ca2+ levels) are important for modulating metastatic cell behaviours (MCBs). Ionic mechanisms, in particular voltage-gated sodium channel (VGSC) and intracellular Ca2+ activities, were shown previously to be involved in MCBs in PCa. This PhD tested the hypotheses (1) that MCBs would be sensitive to hypoxia and be inhibited by VGSC blockers including those selective for the persistent current and (2) that VGSC activity would control intracellular Ca2+ signalling. Experiments were carried on a variety of model cell lines in a comparative approach: strongly metastatic Mat-LyLu and weakly metastatic AT-2 rat PCa cells, and analogous human PCa cell lines (PC-3M and LnCaP, respectively). Experiments on Mat-LyLu cells showed that hypoxia (2 % O2, 24 h) increased mRNA expression of Nav1.7, the predominant VGSC α-subunit expressed in PCa; however, both plasma membrane and intracellular VGSC protein levels were reduced. There was no change in cellular proliferation, Matrigel invasion or lateral motility, whilst transverse migration increased significantly. Treatment (24 h) with the VGSC blockers ranolazine, riluzole, lidocaine and procaine (micromolar concentrations) decreased Nav1.7 mRNA and total VGSC protein levels, and suppressed Matrigel invasion in both normoxia (~21 % O2) and hypoxia. PC-3M (but not LNCaP or Mat-LyLu) cells showed spontaneous, transient elevations of intracellular Ca2+ (“Ca2+ oscillations”). Ionic substitution and pharmacological experiments suggested that Ca2+ influx and release from intracellular stores both contributed to the oscillations. Importantly, acute treatment with tetrodotoxin and ranolazine decreased the oscillation amplitude and frequency. Thus, both hypotheses were confirmed, i.e. (1) that hypoxia increases PCa (Mat-LyLu) cell migration and VGSC expression and (2) that intracellular Ca2+ oscillations are downstream to VGSC activity. Overall, the thesis concludes that VGSC blockers could serve clinically as anti-metastatic agents. |
