Electrochemotherapy with Mitomycin C Potentiates Apoptosis Death by Inhibiting Autophagy in Squamous Carcinoma Cells

SIMPLE SUMMARY: Internationally, electrochemotherapy is considered an innovative therapeutic strategy capable of increasing the efficacy of lyophobic drugs in many solid tumors. Our study aimed to verify the potentiating effect of electroporation (EP), in combination with the drug mitomycin C (MMC), in oral and laryngeal cancer cells that exhibited intrinsic drug resistance. Our study showed that EP increased MMC cytotoxicity on both lines compared to single treatment; the mechanism of action was due to EP inhibition of the autophagic process induced by MMC, thus favoring apoptosis. EP plus MMC treatment was also performed on two veterinary patients demonstrating its efficacy, tolerability, and clinical feasibility. ABSTRACT: We investigated the chemosensitizing effect of electroporation (EP), which, using electrical pulses, permeabilizes cancer cells to drugs. The study involved two human hypopharyngeal and tongue carcinoma cell lines. The surface and intracytoplasmic expression of P-gp were evaluated by flow cytometry, demonstrating that both lines were intrinsically resistant. After establishing the optimal dose of mitomycin C (MMC) to be used, in combination with EP, we showed, by both MTT assay and optical and electron scanning microscopy, the potentiating cytotoxic effect of EP with MMC compared to single treatments. Flow cytometry showed that the cytotoxicity of EP + MMC was due to the induction of apoptosis. In addition to verifying the release of cytochrome C in EP + MMC samples, we performed an expression analysis of caspase-3, caspase-9, Akt, pAkt, HMGB1, LC3I, LC3II, p62, Beclin1, and associated proteins with both apoptotic and autophagic phenomena. Our results were confirmed by two veterinary patients in whom the EP + MMC combination was used to control margins after the resection of corneal squamous carcinoma. In conclusion, we affirmed that the effect for which EP enhances MMC treatment is due to the inhibition of the autophagic process induced by the drug in favor of apoptosis.