- Vivi Padova
- Il BO
This project continues previous AIRC-funded research to seek the validation of mitochondrial Kv channels as oncological targets.
In our cell models, upon induction of apoptosis, Kv1.3 located in the Inner Mitochondrial Membrane (IMM) is blocked by outer membrane-inserted Bax acting like a K+ channel-blocking toxin. This results in a transient hyperpolarization, production of ROS, cytochrome c release and apoptosis (Szabò et al, PNAS, 2008). A point mutation of Bax eliminating its ability to block Kv1.3, turns the protein non-apoptotic in a cellular context, i.e. when expressed in Bax/Bak-less embryonic fibroblasts (MEF) (Szabò et al, CDD, 2011). Kv1.3 is present in several tissues, but normally it is expressed at high levels mainly in proliferating lymphocytes and in the brain. Its expression has been shown to be upregulated in various cancer cell lines and tissues. A mitochondrial population was discovered by us in lymphocytes (Szabò et al, JBC, 2005), and we have now detected it also in various cancerous cell lines (Gulbins et al, BBA, 2010).
Our data indicate that death can be induced by treating CTLL-2 lymphocytes (normally Kvless) transfected to express Kv1.3 with membrane-permeable Kv1.3 inhibitors (PAP-1, Psora-4 and clofazimine) applied together with MDR efflux pump inhibitors (Leanza et al, submitted). Other mtKv1.3-expressing cells behave similarly while cells not expressing mtKv1.3 do not respond. The effect is not produced by impermeant inhibitors of Kv1.3. We have developed also other cellular models, which can form solid tumours, stably transfecting GL261 murine glioma cells to express Kv1.3 either in both the PM and the IMM, or only in the IMM. These cells will be used in in vitro assays to confirm the involvement of mtKv1.3 in death induced by various pharmacological agents, including psoralen derivatives, and to characterize the process as apoptotic.
To make sure of the oncological relevance of our findings we shall rely on murine cancer models, implanting GL261 glioma cells as well as mtKv1.3 expressing B16F10 melanoma cells in mice. We shall use also Eu-myc mice. The growth of the tumours will be monitored as a function of treatment with chemotherapeutic drugs already in use and with irradiation, and upon administration to the animals of Psora-4, clofazimine, PAP-1 and of the derivatives we plan to develop. In preliminary experiments, clofazimine significantly reduced lung metastases of engrafted B16F10 melanoma (61 ±4 metastases in non-treated mice (n=6) versus 9 ±13 in clofazimine-treated mice (n=6)).
The effect of the above compounds will be investigated also in specific human cancer cells of lymphoid origin, known to express high levels of Kv1.3. Expression will be confirmed by patch clamp.
Mitochondria; Apoptosis; Small molecule inhibitors; Animal models; Chemistry