sirolimus and 3-aminobenzamide

Poly(ADP-ribosyl)ation (PARylation) acts as a modulator of selective autophagic degradation of ubiquitinated aggregates for cellular quality control, functioning in pro-survival role. It was reported previously that the inhibition of PARylation resulted in autophagy defects leading accumulation of ubiquitinated aggregates SQSTM1/p62 and apoptosis in porcine blastocysts. Thus, this study aims to investigate the mechanism between PARylation and autophagy in porcine blastocysts. In vitro produced (IVP) embryos were treated with 3-aminobenzamide (3ABA, poly (ADP-ribose) polymerase inhibitor) and/or rapamycin (RAPA, an mTORC1 inhibitor) during blastocyst formation. Then, these treated blastocysts were analyzed by real-time PCR, immunocytochemistry and TUNEL Assay. We found that the 3ABA treatment increased mTORC1 downstream target, phosphorylation of thr389 p70S6K (p-p70S6K-thr389), suggesting an increase in mTORC1 activity. Co-treatment with rapamycin (RAPA), mTORC1 inhibitor, restored the 3ABA-induced autophagy defects to those of the controls by normalizing mTORC1 activity. Moreover, autophagy induction, with only RAPA treatment, increased the rate of blastocyst development (70.05 ± 0.93 vs. 50.61 ± 3.49%), total cell number (58.48 ± 2.94 vs. 49.58 ± 2.43) and blastomere survival, but decreased the accumulation of SQSTM1/p62 aggregates. In summary, mTORC1 signaling is a key mechanism of PARylation-autophagy and its inhibition improved developmental ability and embryo quality by promoting selective autophagic degradation of ubiquitinated aggregates in porcine blastocysts. Therefore, these findings have significant implications for understanding the importance of autophagy regulation for successful in vitro production of porcine embryos.

Topics: Animals; Autophagy; Benzamides; Blastocyst; Cells, Cultured; Gene Expression Regulation, Developmental; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Poly(ADP-ribose) Polymerases; Sirolimus; Swine; TOR Serine-Threonine Kinases

The aim of the present study was to investigate the role of autophagy in response to ionizing radiation (IR) in CNE-2 human nasopharyngeal carcinoma cells and to demonstrate the function of poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of IR-induced autophagy. Microtubule-associated protein 1 light chain 3 (LC3) and poly(ADP-ribose) (PAR) were assessed using western blotting. Ultrastructural analysis was performed using transmission electron microscopy (TEM). The percentage of apoptotic cells was assessed by flow cytometry. The MTT method was used to detect cell viability of CNE-2 cells at different time points after IR. Clonogenic survival assays were used to evaluate the radiosensitivity of nasopharyngeal carcinoma cells treated with IR and IR combined with autophagy inhibitor (chloroquine phosphate), with autophagy inducer (rapamycin) or with PARP-1 inhibitor 3-amino benzamide (3AB). IR induced a massive accumulation of autophagosomes detected by TEM and intensified the conversion of cytosolic LC3-I to LC3-II. PARP-1 activation was accompanied by strong upregulation of PAR and LC3-II expression in CNE-2 cells. Compared with radiation alone, chloroquine phosphate (CDP) or 3AB combined with IR significantly decreased cell viability, as well as the autophagic ratio and LC3-II protein levels. Inhibition of autophagy increased radiation-induced apoptosis; rapamycin (RAPA) significantly decreased cell viability as well, but RAPA increased the autophagic ratio and LC3-II protein levels; induction of autophagy increased radiation-induced apoptosis. To conclude, PARP-1 regulates IR-induced autophagy, and PARP-1 inhibitor contributes to the radiation sensitization of CNE-2 cells. Blockade of autophagy with CDP enhanced the cytotoxicity of radiotherapy in CNE-2 cells. This suggests that inhibition of autophagy or PARP-1 may be used as an adjuvant therapy to treat nasopharyngeal carcinoma.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Benzamides; Carcinoma; Cell Line, Tumor; Cell Survival; Chloroquine; Humans; Microtubule-Associated Proteins; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Phagosomes; Poly (ADP-Ribose) Polymerase-1; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Radiation Tolerance; Radiation-Sensitizing Agents; Sirolimus