REPETITIVE MEMBRANE POTENTIAL OSCILLATIONS ENHANCE METFORMIN¿S ANTIPROLIFERATIVE EFFECT ON GLIOBLASTOMA CANCER STEM CELLS

Glioblastoma (GB) is the most common brain tumor with an extremely poor prognosis. Although the current standard of care is a combination of surgery, chemo, and radiotherapy, their effectiveness remains extremely poor in terms of patients’ survival. Recent publications highlight the presence of a functional subset of cells that may be responsible for tumor recurrence and resistance to conventional therapies. This subset is known as glioblastoma stem cells (GSCs). New strategies selectively targeting GSCs and/or their microenvironmental niche should be designed. The chloride intracellular channel 1 (CLIC1) represents a fruitful research topic to achieve this purpose since its functional activity was reported to be related to glioblastoma aggressiveness. CLIC1 is a peculiar protein that coexist in two isoforms. In normal condition it’s mostly cytoplasmic, while in response to persistent stress translocates to the plasma membrane (tmCLIC1) determining a chloride conductance. tmCLIC1 was found to be chronically expressed in GSCs sustaining their abnormal in vitro proliferation rate. Given this, tmCLIC1 could be considered a promising pharmacological target to counteract glioblastoma progression. Our laboratory has recently found tmCLIC1 protein as an extracellular target of the antidiabetic drug metformin. Despite it is well known the antineoplastic effect of metformin, the mechanism of action remains unclear. It was proposed that the binding between metformin and tmCLIC1 occurs at the level of Arg29 only when the channel is in the open state. However, metformin impairs GSCs proliferation at a millimolar range, a concentration unattainable in the brain upon metformin oral administration. The purpose is to decrease metformin’s working concentration, enhancing its action on tmCLIC1. tmCLIC1 is a voltage dependent channel and its open probability increases under depolarization. We propose to enhance CLIC1-metformin interaction using repetitive membrane potential oscillations provided by field potential, optogenetics and electromagnetic field stimulations. In this work, we confirm that metformin inhibits CLIC1 channel by binding to the R29 amino acid, which is localized inside the pore region. We show that by applying stimulation to GSCs the operative metformin concentration is reduced up to 10-fold. In addition, we demonstrate that the phenomenon is specifically confined to metformin. The combination of metformin treatment with repetitive membrane depolarizations produce an average 30% decrease of GB progression compared to metformin itself in vitro as well as in vivo. Taken together, we provide insights of a new possible therapeutic approach to face glioblastoma progression by specifically targeting GSCs.