Autophagy as a common cell death mechanism during the pharmacological treatment of Echinococcus granulosus larval stage.

LOOS JULIA A; CUMINO ANDREA C

Guayaquil

Congreso; XXI Congreso Latinoamericano de Parasitología; 2013

Federación Latinoamericana de Parasitología

Autophagy as a common cell death mechanism during the pharmacological treatment of Echinococcus granulosus larval stage Julia Alexandra Loos, Pedro Caparros, María Celeste Nicolao, Andrea Carina Cumino CONICET - Laboratorio de Zoonosis Parasitarias, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3350 Nivel Cero, CP7600, Mar del Plata, Argentina. Introduction. Autophagy is a conserved lysosomal pathway in which cells degrade organelles to generate energy. A key regulator of autophagy is TORC1 and its pharmacological inhibition, through rapamycin or metformin, induces this catabolic process. Previously we verified that rapalogs and metformin increased the accumulation of cytoplasmic acidic organelles, suggesting autophagy induction as cell-death mechanism. Additionally, the detection of autophagosomes is a common denominator in antiechinococcal drug screening reports. Here, we evidenced the molecular components of the autophagy machinery in basal conditions and demonstrated its overexpression under pharmacological treatment in Echinococcus granulosus. Materials and methods. We tested As2O3, potential autophagic inductor, as a new compound against the larval stages. Using tBLASTn we have identified orthologs to all the proteins involved in the autophagosome formation. Expression of different autophagy-related genes (atg1 to atg18), Tor and raptor were analyzed by RT-PCR and qPCR from drug-treated protoscoleces and metacestodes. SEM and TEM were used to investigate ultrastructural damage and autophagosome progress. Also, we performed in toto immunofluorescence and western blot assays in protoscoleces to determine the expression and localization of Eg-Atg8/LC3. Finally, we analyzed by MEB-EDX the composition, structure and Eg-Atg8/LC3 expression in protoscolex calcareous corpuscles. Data were compared using the Student´s t-test. Results. In this work treatment with As2O3 inhibited viability in a dose-dependent manner in protoscoleces and metacestodes. Cyst ultrastructural damages appeared earlier with detachment of the germinal layers and autophagic structures were evidenced. Rapamicyn and As2O3 upregulated the LC3II expression, Eg-atg6-8-12-16 and Eg-atg18 in both larval stages. Eg-Atg8/LC3 protein was immunodetected in the tegument, the excretory system and the calcareous corpuscles of control and treated protoscoleces. Colocalization of arsenic with the principal ions found in calcareous corpuscles was detected, and different degrees of development of the corpuscles were identified. Conclusion. In Echinococcus either the induction or the inhibition of autophagy can provide therapeutic benefits to hydatidosis treatment.