Interazioni on target ed off target di farmaci e citossici con le subunità TRPV1, ABCC8, ABCC9, KCNJ8, KCNJ11, KCNMA1, KCNMB1-4: implicazioni terapeutiche e reazioni avverse.

The genes TRPV1, ABCC8, ABCC9, KCNJ8, KCNJ11, KCNMA1, KCNMB1-4 encode for macromolecular complexes that perform various functions in cells as subunits of ion channels and they are well known drug targets in pathological conditions. These genes owe their pathophysiological role to the function of membrane ion channels such as TRPV1, ATP-dependent potassium channels (KATP) and activated calcium potassium channels (BK). They also owe their pathophysiological role to the function of soluble aberrant cytosolic proteins that interfere with intracellular processes such as proliferation and inflammatory processes. For this reason, these channels and their genes have recently emerged as potential drug targets in channelopathies and in the treatment of various proliferative diseases including neoplastic pathologies. At the same time, they are the target of off-target interaction for many molecules already used, requiring pharmacovigilance investigations. These macromolecular complexes are increasingly considered in the biology of tumors, and in pediatric brain tumors, where they are up regulated in 33% of cases and downregulated in 48% of cases. No data is currently available on the effects of ion channels in intrinsic diffuse pontine glioma (DIPG) cells. Diffuse intrinsic pontine glioma (DIPG) is a pediatric neoplasm that affects a region of the brainstem, the pons, in which reside regulatory centers of many vital functions. Actually, the only available treatment is radiotherapy. So, samples of neoplasms from kidney sections of male rats, treated locally with high doses of a hydroalcoholic solution of minoxidil, and samples of mammary neoplasms from canine biopsies were analyzed by immunohistochemistry. These experiments are followed by clinical and pharmacovigilance studies to obtain correlations between over-expression or down-regulation of previous genes and the progression of tumor pathologies or adverse cancerous reactions induced using potassium-dependent ATP channel modulators. In the DIPG study, the effects of TRPV1, BK, and KATP channel modulators, including staurosporine (STS) and midostaurine, were evaluated in vitro. The macroscopic current was characterized by using potassium channel modulators in patch clamp technique, while gene expression was analyzed to confirm the presence of target subunits. Protein expressions of H3K27ac, mTOR, and Caspase3 were also investigated through RT-PCR and western blot, using beta-actin as a control. Immunohistochemistry experiments revealed, for the first time, an increased reactivity of the SUR2A subunit in tumor samples from both minoxidil-induced renal cancer in male rats and spontaneous canine tumors. Omic data also showed upregulation of the ABCC9, KCNJ11, and KCNJ8 genes in renal carcinoma, linked to reduced survival probability in lung squamous cell carcinoma in men. In the DIPG study, patch clamp experiments showed that diazoxide did not activate K-ATP currents further, while glibenclamide reduced them by 30-40%, suggesting channel overactivity. Capsaicin did not enhance outward currents, but capsazepine reduced them, indicating TRPV1 channel activity. This suggests that these channels could be potential targets for DIPG therapy. Treatment with 2.14 μM STS inhibited K-ATP and TRPV1 currents in SU-DIPG-36 cells, reducing cell survival by 51%, 69%, and 100% after 6, 48, and 72 hours, respectively. RT-PCR revealed high expression of ABCC8 and ABCC9 (sulfonylurea receptors) in both cell lines, with ABCC8 higher in DIPG 50 and ABCC9 in DIPG 36. KCNJ11 and KCNJ8 were equally expressed, and TRPV1 expression was higher in DIPG 50. Western blotting showed that repaglinide increased H3K27ac expression, while mTOR and phospho-mTOR levels decreased in DIPG 36 but increased in DIPG 50. Cleaved Caspase-3 expression increased in both cell lines.