Bovine seminal ribonuclease triggers Beclin1-mediated autophagic cell death in pancreatic cancer cells

Abstract

Among the large number of variants belonging to the pancreatic-type secretory ribonuclease (RNase) superfamily, bovine pancreatic ribonuclease (RNase A) is the proto-type and bovine seminal RNase (BS-RNase) represents the unique natively dimeric member. In the present manuscript, we evaluate the anti-tumoral property of these RNases in pancreatic adenocarcinoma cell lines and in nontumorigenic cells as normal control. We demonstrate that BS-RNase stimulates a strong anti-proliferative and pro-apoptotic effect in cancer cells, while RNase A is largely ineffective. Notably, we reveal for the first time that BS-RNase triggers Beclin1-mediated autophagic cancer cell death, providing evidences that high proliferation rate of cancer cells may render them more susceptible to autophagy by BS-RNase treatment. Notably, to improve the autophagic response of cancer cells to BS-RNase we used two different strategies: the more basic (as compared to WT enzyme) G38K mutant of BS-RNase, known to interact more strongly than wt with the acidic membrane of cancer cells, or BS-RNase oligomerization (tetramerization or formation of larger oligomers). Both mutant BS-RNase and BS-RNase oligomers potentiated autophagic cell death as compared to WT native dimer of BS-RNase, while the various RNase A oligomers remained completely ineffective. Altogether, our results shed more light on the mechanisms lying at the basis of BS-RNase antiproliferative effect in cancer cells, and support its potential use to develop new anti-cancer strategies.

Onconase induces autophagy sensitizing pancreatic cancer cells to gemcitabine and activates Akt/mTOR pathway in a ROS-dependent manner

“…In the present manuscript, we demonstrate that ONC strongly affects the proliferation of pancreatic adenocarcinoma cells, while it is significantly less active in normal and non-tumoral cells, as we previously observed for bovine seminal RNase (BS-RNase) [26,27]. This result can be explained with the higher capability of these RNases to interact with the very acidic membrane lipids of cancer cells than with those of normal cells [28][29][30].…”

“…This result can be explained with the higher capability of these RNases to interact with the very acidic membrane lipids of cancer cells than with those of normal cells [28][29][30]. Furthermore, interaction of RNases with cell membrane is augmented upon oligomerization increasing their basic charge density [31][32][33] or when some key residues are mutated to more basic ones, such as for G38K-BS-RNase [26,28].…”

“…Our results, obtained with knockdown assay, reveal that Beclin1 (also called Atg6) is functionally involved in the formation of autophagosomes induced by ONC. According to these data, we previously demonstrated that BS-RNase triggers Beclin1-dependent autophagic cell death in cancer cells providing evidence that high proliferation rates of cancer cells may render them more susceptible to autophagy by BS-RNase treatment [26]. Moreover, autophagy variations in live cells can be monitored by using a GFP-fused LC3 protein (GFP-LC3) analyzing the presence of GFP-LC3 puncta which rapidly increase when autophagy is activated.…”

Fungal proteinaceous compounds with multiple biological activities

“…Fungal ribosome-inactivating proteins and their plant counterparts (Stirpe andBattelli, 2006, Stirpe 2013) have dissimilar N-terminal sequences but have biological activities in common like antimicrobial, anticancer and antiviral activities (Akkouh et al 2015). Fungal ribonucleases display anticancer and HIV-1 reverse transcriptase activities like ribonucleases of other origins (Fiorini et al, 2014(Fiorini et al, , 2015. Ribosome-inactivating proteins and laccases have not been reported from mammals.…”

Approaches to Endow Ribonucleases with Antitumor Activity: Lessons Learned from the Native Cytotoxic Ribonucleases

“…Like for ONC, rRNA is a target of BS-RNase and its cleavage induces apoptosis [64], but the enzyme has also been found in the nucleolus of cancer cells [65], and although it is not known how it reaches the cell nucleus, a correlation between cytotoxicity and a decrease of telomerase activity and its associated RNA has been found [70]. Recently, it has been described that BS-RNase triggers Beclin1-mediated autophagy in treated cancer cells being ineffective in normal cells, suggesting that autophagy more than apoptosis can be the mechanism of cancer cell death induced by BS-RNase [71]. Comparable to ONC, this selective autophagy for cancer cells seems to be related to the basic charge distribution in the surface of these RNases [36,71].…”