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

Biochimica et Biophysica Acta 1843 (2014) 976–984

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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamcr

Bovine seminal ribonuclease triggers Beclin1-mediated autophagic cell death in pancreatic cancer cells Claudia Fiorini a, Giovanni Gotte a,⁎, Federica Donnarumma b, Delia Picone b, Massimo Donadelli a,⁎⁎ a b

Department of Life and Reproduction Sciences, Biochemistry Section, University of Verona, Verona, Italy Department of Chemical Sciences, University of Naples “Federico II”, Naples, Italy

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Article history: Received 6 December 2013 Received in revised form 21 January 2014 Accepted 23 January 2014 Available online 31 January 2014 Keywords: BS-RNase RNase A Autophagy Pancreatic cancer RNase oligomers Beclin1

a b s t r a c t 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. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Pancreatic adenocarcinoma is one of the most aggressive and devastating human malignancies and the fourth leading cause of worldwide cancer-related deaths. Standard treatments for advanced disease include monotherapy with gemcitabine (2′,2′-difluoro-2′-deoxycytidine), which has, however, a response rate of less than 20% [1]. Therefore, during the last few years, the identification of valuable targets [2,4] and novel efficient therapeutic strategies against pancreatic adenocarcinoma has been extensively investigated [5,7]. Among the biological events associated with the response of cancer cells to therapeutics [8], autophagy is acquiring a growing interest. It is a highly conserved cellular process in

Abbreviations: RNase, ribonuclease; BS-RNase, bovine seminal ribonuclease; WT, wild-type; CQ, chloroquine diphosphate; 3MA, 3-methyladenine; FBS, fetal bovine serum; SDS, sodium dodecyl sulphate; MDC, monodansylcadaverine; siRNA, small interfering RNA; D, dimers; T, trimers; TT, tetramers; LO, larger oligomers; RI, RNase inhibitor ⁎ Correspondence to: G. Gotte, Department of Life and Reproduction Sciences, Biochemistry Section, University of Verona, Strada Le Grazie 8, I-37134, Verona, Italy. Tel.: +39 045 8027694; fax: +39 045 8027170. ⁎⁎ Correspondence to: M. Donadelli, Department of Life and Reproduction Sciences, Biochemistry Section, University of Verona, Strada Le Grazie 8, I-37134, Verona, Italy. Tel.: +39 045 8027281; fax: +39 045 8027170. E-mail addresses: [email protected] (G. Gotte), [email protected] (M. Donadelli). 0167-4889/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamcr.2014.01.025

which cytoplasmic materials, including organelles, are sequestered into double-membrane vesicles called autophagosomes and delivered to lysosomes for degradation or recycling. Besides its cytoprotective role in cellular homeostasis, autophagy can be a form of programmed cell death, designated as “autophagic cell death” [9]. Physiologically, autophagy has the role to preserve the balance between organelle biogenesis, protein synthesis and their clearance [10]. More recently, autophagy has been shown to be an important mediator of pathological responses and to be engaged in the cross-talk with reactive oxygen species involved in cell signaling and protein damage [11]. Interest in the role of autophagy in cancer research starts from the discovery that BECN1 (the gene encoding the pro-autophagic protein Beclin1, also known as ATG6) is also a tumor suppressor gene [12], revealing that autophagy is under the control of a large panel of oncogenes and products of tumor suppressor genes [13]. The link between autophagy and antitumor activity prompted us to investigate the possible role of ribonucleases in this process. It is well known, in fact, that many ribonucleases display, besides their enzymatic activity, other remarkable biological activities [14,15], among which emerges a relevant cytotoxic effect against tumor cells in vitro and in vivo [16,19]. These findings aroused new interest in the fields of potential anticancer therapy related to this class of enzymes. In this work, we focused our attention in particular on two variants belonging to the secretory pancreatic-type RNase super-family: the proto-type bovine pancreatic ribonuclease (RNase A), which is monomeric in its native state, and the

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bovine seminal RNase (BS-RNase), a natively dimeric protein constituted by two identical subunits covalently bound through two antiparallel disulfide bridges. Each BS-RNase subunit shares 82% sequence identity and very close 3D structural and dynamical features in solution with RNase A [20]. In solution, BS-RNase is an equilibrium mixture containing 70% of N-terminal-swapped [21] and 30% of unswapped isoforms, respectively called MxM and M=M [22]. Notably, swapped MxM BS-RNase evades RI because of its dimericity, which persists also under the cytosol reducing environment [24], and can consequently bear a remarkable antitumor action [25], while RNase A, like most monomeric RNases, is strongly bound and inactivated in mammalian cells by the RNase Inhibitor (RI) [23]. Anyway, native RNase A can be induced [26] to form various N- or C-terminal domain-swapped dimers [27,29], trimers [30], and larger

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oligomers [31,32], all reconstituting the active site [26,28] and augmenting the enzymatic activity against dsRNA with respect to the native monomer [33]. Despite depending on the tumor type tested [34,36], RNase A oligomers can also acquire cytotoxic activity both in vitro and in vivo [37]. Also BS-RNase is known to oligomerize through the 3D domain swapping of both N- and C-termini, and its multimers show to be enzymatically and biologically more active than the native dimer(s) [38]. Furthermore, BS-RNase activity can be increased also by mutations which augment its basicity and capability to interact with cell membranes, as it occurs for the G38K mutant [39] that has been tested in this work. Thus, in the present manuscript we investigated whether the two mentioned secretory RNases (i.e., RNase A and BS-RNase), and their respective oligomeric aggregates, could display an efficient anti-proliferative activity against pancreatic adenocarcinoma cells, as compared to nontumorigenic cell lines. Furthermore, considering its relevant role in the cell lifebalance, we explored if autophagy could have a role in the RNasemediated cancer cell death.

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Fig. 1. Effect of RNase A and BS-RNase on growth inhibition and apoptosis in Panc1, fibroblasts, and HaCaT cells. (A and B) Cells were seeded in 96-well plates, incubated overnight, and treated with increasing concentrations of RNase A or BS-RNase for 72 h. Cell proliferation was determined using the Crystal Violet colorimetric assay. Values are the means of three independent experiments, each performed in triplicate. Statistical analysis: (#) p b 0.05 Panc1 vs fibroblasts or HaCaT. (C) Cells were seeded in 96-well plates, incubated overnight, and treated with increasing concentrations of RNase A or BS-RNase for 72 h. Apoptosis was analyzed using the annexinV/FITC binding assay. Values are the means (±SD) of three independent experiments, each performed in triplicate. Statistical analysis: (*) p b 0.05.

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Fig. 2. Effect of RNase A and BS-RNase on autophagy in Panc1, fibroblasts, and HaCaT cells. (A and B) Cells were seeded in 96-well plates, incubated overnight, and treated with increasing concentrations of RNase A or BS-RNase for 72 h. Autophagosome formation assay was analyzed using the incorporation of MDC probe. Values are the means of three independent experiments, each performed in triplicate. Statistical analysis: (#) p b 0.05 Panc1 vs fibroblasts or HaCaT. (C) Cells were seeded in 60-mm diameter culture dishes, incubated overnight, and treated with 100 μg/ml RNase A or BS-RNase for 32 h. Whole-cell extracts were used for Western blot analysis of the autophagy-related proteins Beclin1 and LC3. Ponceau S staining was used as control loading. (D) Quantitative analyses of LC3-II/LC3-I ratio. The bands were scanned as digital peaks and the areas of the peaks were calculated in arbitrary units, as described in the “Materials and methods” section. The value of Ponceau S dye was used as normalizing factor. Values are the means of three independent experiments (±SD). Statistical analysis: (*) p b 0.0.

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2. Materials and methods 2.1. Chemicals Chloroquine diphosphate [CQ; N4-(7-chloro-4-quinolinyl)-N1,N1dimethyl-1,4-pentanediamine], and 3-methyladenine (3MA) were obtained from Sigma (Milan, Italy). Recombinant RNase A and BS-RNase were produced and purified from Escherichia coli as unfolded monomers, extracted from inclusion bodies and then refolded as reported in [40]. In the case of BS-RNase, and of its G38K mutant [39], Cys-31 and 32 were linked to two glutathione molecules [40]. After selectively reducing the mixed disulfides, the protein was dialyzed against 0.1 M Tris/acetate, pH 8.4, followed by SEC onto a Sephadex G-75 column to obtain dimers [41]. The protein solution was incubated at 37 °C for at least 72 h to reach the equilibrium between the two MxM and M=M isoforms [41]. RNase A and BS-RNase were treated with Aeromonas proteolytica aminopeptidase (Sigma) to remove Met-1 before cytotoxicity assays. 2.2. RNase A or BS-RNase oligomeric products RNase A and BS-RNase oligomers were prepared by lyophilizing 5 to 50 mg/ml protein samples dissolved in 40% acetic acid [26], and then re-dissolving them in 0.2 M NaPi, pH 6.75 [26,33]. Purification of the two mentioned RNases, or separation of their oligomers, were performed, respectively, through SEC using a Sephadex G-75 column (70 × 1.5 cm), in 0.1 M Tris/acetate, pH 8.4, flow rate 0.4–0.5 ml/min, or with a Superdex 75 HR 10/300 column, attached to an ÄKTA FPLC system (GE-Healthcare), and equilibrated with NaPi 0.20 M [33] or 0.40

M [38], pH 6.75, flow rate 0.08–0.10 ml/min, at room temperature. Besides native BS-RNase dimer (D), its domain-swapped oligomers were purified as N-swapped-only or NCN-swapped tetramers (TTN and TTC, respectively) [38,42], and larger oligomers (LO) [38]. Concerning RNase A, beyond native monomer (M), also N- or C-swapped dimers (DN, DC), trimers (N + C-swapped TNC; C-swapped-only, TC), tetramers (NCN-tetramer, TTN, and CNC-tetramer, TTC) or a mixture of larger oligomers (LO) [32,43] were chromatographed through a Source 15S 16/10 cationexchange column with a 0.09–0.20 M NaPi gradient, pH 6.75 [33]. The purified oligomers of both RNases were kept at 4 °C until use, or concentrated against NaPi 0.010 M, pH 6.75, in Centricon Ultra-filters (C.O. 10 kDa, Millipore), just before use. The concentration of BS-RNase and RNase A species was spectrophotometrically measured at 278 nm with a ε1%278 of 4.65, and at 280 nm, ε1%280 of 7.3, respectively [38]. 2.3. Cell culture Panc1 and PaCa44 human pancreatic adenocarcinoma cell lines were grown in RPMI 1640 supplemented with 2 mM glutamine (Life Technologies, Milan, Italy), 10% FBS, and 50 μg/ml gentamicin sulfate (BioWhittaker, Lonza, Bergamo, Italy). Normal primary fibroblasts (PromoCell, PBI, Milan, Italy) and HaCaT nontumorigenic keratinocytes were grown in DMEM supplemented with 2 mM glutamine (Life Technologies, Milan, Italy), 10% FBS, and 50 μg/ml gentamicin sulfate. All cell types were incubated at 37 °C with 5% CO2. In serum starvation experiments, cells were plated in culture medium containing 10% FBS. The day after the medium was replaced with one containing a reduced percentage of FBS (1%, 0.1% or 0%) in the absence or presence of the various compounds.

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Fig. 3. Effect of autophagy inhibition on the cytotoxic and apoptotic outcomes of RNase A and BS-RNase in Panc1 cells. (A and C) Cells were seeded in 96-well plates, incubated overnight, and treated with increasing concentrations of RNase A or BS-RNase in the absence or presence of 5 μM CQ or 1 mM 3MA for 72 h. Cell proliferation was determined using the Crystal Violet colorimetric assay. Values are the means of three independent experiments each performed in triplicate. Statistical analysis: (#) p b 0.05 BS-RNase vs BS-RNase + CQ or BS-RNase + 3MA. (B and D) Cells were seeded in 96-well plates, incubated overnight, and treated with increasing concentrations of RNase A or BS-RNase in the absence or presence of 5 μM CQ or 1 mM 3MA for 72 h. Apoptosis was analyzed using the annexinV/FITC binding assay. Values are the means of three independent experiments each performed in triplicate. Statistical analysis: (#) p b 0.05 BS-RNase vs BS-RNase + CQ or BS-RNase + 3MA.

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Cells were seeded in 96-well plates (2.5 × 103 cells/well) and, the day after, at the times indicated in the figure legends, they were incubated with RNase A or BS-RNase in the absence or presence of autophagy inhibitors. The protein concentrations are expressed as μg/ml to better compare the effects of the two RNases, and of their oligomers, taking into account the different MWs of the native monomeric RNase A and dimeric BS-RNase. For example, 100 μg/ml corresponds to 7.3 μM for RNase A, while to 3.65 μM for BS-RNase. At the end of the treatments, cells were stained with Crystal Violet solution (Sigma, Milan, Italy). The dye was solubilized in PBS containing 1% SDS and photometrically measured (A595nm) to determine cell growth. 2.5. Apoptosis assay Cells were seeded in 96-well plates (2.5 × 103 cells/well) and, the day after, treated with the various RNase compounds at the indicated concentrations for 72 h. At the end of the treatment, cells were fixed with 2% paraformaldehyde in PBS at room temperature for 30 min, then washed twice with PBS and stained, for 10 min in the dark, at room temperature, with annexinV/FITC (Bender MedSystem, Milan, Italy) in binding buffer (10 mM HEPES/HCl pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). Finally, cells were washed with binding buffer solution and fluorescence was measured by using a multimode plate reader (λexc 485 nm and λem 535 nm) (GENios Pro, Tecan, Milan, Italy). The values were normalized on cell proliferation by Crystal Violet assay.

centrifuged at 14,000 ×g for 10 min at 4 °C and the supernatant used for Western blot. Protein concentration was measured with the Bradford protein assay reagent (Pierce, Milan, Italy) using bovine serum albumin as a standard. Protein extracts (50 μg/lane) were electrophoresed through a 12% SDS-polyacrylamide gel and electroblotted onto PVDF membranes (Millipore, Milan, Italy). Membranes were incubated in blocking solution [5% BSA in TBST (50 mM Tris pH 7.5, 0.9% NaCl, 0.1% Tween 20)] for 1 h at room temperature and probed overnight at 4 °C with a rabbit monoclonal anti-LC3 (1:1000) (Cell Signaling) or a rabbit polyclonal anti-Beclin1 (1:1000) (GeneTex, cat. n° GTX113039). Horseradish peroxidase conjugated anti-rabbit IgGs (1:8000 in blocking solution) (Upstate Biotechnology, Milan, Italy) were used to detect specific proteins. Immunodetection was carried out using chemiluminescent substrates (Amersham Pharmacia Biotech, Milan, Italy) and recorded using a HyperfilmECL (Amersham Pharmacia

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2.8. Immunoblot analysis Cells were harvested, washed in phosphate-buffered saline, and resuspended in RIPA Buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% Igepal CA-630, 0.5% Na-Doc, 0.1% SDS, 1 mM Na3VO4, 1 mM NaF, 2.5 mM EDTA, 1 mM PMSF, and 1 × protease inhibitor cocktail). After three freeze/thaw cycles and incubation on ice for 30 min, lysate was

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Exponentially growing cells were seeded at a density of 2.5 × 103 cells/well in 96-well plates for proliferation assays and at 2.5 × 105 cells/plate in 60 mm cell culture plates for protein extraction. Twentyfour hours later, transfections were carried out with a specific small interfering (si) RNA duplex targeting Beclin1 mRNA (5′-ACAGUGAA UUUAAACGACAGCAGCU-3′ and 5′-AGCUGCUGUCGUUUAAAUUCAC UGU-3′) and a non-targeting (NT) siRNA (5′-CAGUCGCGUUUGCGAC UGG-3′) purchased by Life Technologies (Monza MB, Italy). Cells were transfected with siRNAs at a final concentration of 50 nM using Lipofectamine 2000 (Life Technologies) for 24 h. At the end of the transfection time, cell medium was changed and cells were treated with BS-RNase under the conditions indicated in Fig. 4.

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To quantify the induction of autophagy, cells were incubated with the fluorescent probe monodansylcadaverine (MDC; Sigma, Milan, Italy). MDC is a selective marker for acidic vesicular organelles (AVOs), such as autophagic vacuoles, and especially autolysosomes. Briefly, cells were seeded in 96-well plates (2.5 × 103 cells/well) and treated with the various compounds as indicated in figure legends. At the end of the treatments, cells were incubated in culture medium with 50 μM MDC at 37 °C for 15 min. After incubation, cells were washed with Hanks buffer (20 mM Hepes pH 7.2, 10 mM glucose, 118 mM NaCl, 4.6 mM KCl, and 1 mM CaCl2) and fluorescence was measured by using a multimode plate reader (λexc 340 nm and λem 535 nm) (GENios Pro, Tecan, Milan, Italy). The values were normalized on cell proliferation by Crystal Violet assay.

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Fig. 4. Effect of Beclin1 knock-down on autophagy and growth inhibition by BS-RNase in Panc1 cells. (A and B) Cells were seeded in 96-well plates, incubated overnight, transfected with Beclin1 siRNA or non-targeting (NT) siRNA, and/or treated with 200 μg/ml BS-RNase for 72 h. Autophagosome formation assay was analyzed using the incorporation of MDC probe, while cell proliferation was determined using the Crystal Violet colorimetric assay. Values are the means (±SD) of three independent experiments each performed in triplicate. Statistical analysis: (*) p b 0.05. (C) Cells were seeded in 60-mm diameter culture dishes, incubated overnight, transfected with Beclin1 siRNA or non-targeting (NT) siRNA, and treated with 100 μg/ml BS-RNase for 32 h. Beclin1 expression was analyzed by western blot using whole-cell extracts. Ponceau S staining was used as control loading.

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Biotech). The bands were scanned as digital peaks, and the areas of the peaks were calculated in arbitrary units using the public domain NIH Image software (http://rsb.info.nih.gov/nih-image/). The value of Ponceau S dye was used as a normalizing factor.

2.9. Statistical analysis ANOVA analysis was performed by GraphPad Prism 5 software. p value b0.05 was indicated as being statistically significant.

3. Results 3.1. BS-RNase, but not RNase A, inhibits pancreatic adenocarcinoma cell growth and stimulates apoptosis To analyze the effect of the pancreatic-type secretory ribonucleases RNase A and BS-RNase on pancreatic adenocarcinoma cell growth, we used Panc1 cell line which we previously demonstrated to be highly resistant to chemotherapy as a cellular model [44]. Panc1 cancer cells were resistant to RNase A treatment (Fig. 1A), while they were sensitive to BS-RNase, with a ~ 60% cell growth inhibition after treatment with 200 μg/ml of enzyme (Fig. 1B). Similar data have been observed also with another pancreatic adenocarcinoma cell line, PaCa44, establishing that these events were not restricted to the specific cancer cell line chosen (Supplemental Fig. 1A). Intriguingly, normal primary fibroblasts and HaCaT nontumorigenic keratinocytes were significantly less affected by BS-RNase than Panc1 cancer cells (Fig. 1B). Accordingly with cell proliferation results, BS-RNase, but not RNase A, strongly induced apoptosis in Panc1 cancer cells, while non-tumoral cells were significantly less affected (Fig. 1C).

To further characterize the biological events regulated by the two mentioned RNases in pancreatic adenocarcinoma cells, we investigated whether autophagy could be a mechanism involved in cell proliferation inhibition. Fig. 2A clearly shows that RNase A did not significantly affect autophagosome formation, while in cancer cells this event was strongly stimulated by BS-RNase in a concentration-dependent manner (Fig. 2B). Similar results have been obtained in pancreatic adenocarcinoma PaCa44 cell line (Supplemental Fig. 1B). Conversely, BS-RNase was able to induce autophagy only slightly either in normal fibroblasts or in HaCaT keratinocytes. To confirm and further characterize the autophagy induced by BS-RNase in cancer cells, we performed Western blot analyses of the autophagy-related proteins Beclin1 and LC3. Accordingly with previous data, RNase A did not affect expression of both Beclin1 and LC3 isoforms (Fig. 2C). Conversely, BS-RNase strongly induced the expression of both Beclin1 and LC3-II, the phosphoethanolaminated and functionally active form of the autophagosome protein LC3-I. Since the conversion of LC3-I in LC3-II is required for autophagosome formation, we analyzed the LC3-II/LC3-I ratio after treatment with ribonucleases, revealing that BS-RNase strongly increased this ratio (~ 4-fold) in cancer cells (Fig. 2D). This result reveals the formation of the intracellular autophagosomes, which, after fusion with lysosomes, constitute acidic vesicular organelles (AVOs) detectable by monodansylcadaverine staining (see Fig. 2B). Since autophagy has been described as a double-edged sword event, i.e., a protective mechanism of cells in response to stress stimuli or, conversely, a mortal mechanism, named autophagic cell death [9,10], we analyzed the impact of autophagy stimulation on Panc1 cell growth inhibition and apoptosis induced by ribonucleases. To do so, we used the autophagy inhibitors chloroquine (CQ) or 3methyladenine (3MA). No alteration on cell growth and apoptosis was

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Fig. 5. Effect of autophagy inhibition on the cytotoxic and apoptotic outcomes of RNase A and BS-RNase in fibroblasts and HaCaT cells. (A and B) Fibroblasts and HaCaT cells, respectively, were seeded in 96-well plates, incubated overnight, and treated with increasing concentrations of RNase A in the absence or presence of 5 μM CQ or 1 mM 3MA for 72 h. (C and D) Fibroblasts and HaCaT cells, respectively, were seeded in 96-well plates, incubated overnight, and treated with increasing concentrations of BS-RNase in the absence or presence of 5 μM CQ or 1 mM 3MA for 72 h. Cell proliferation was determined using the Crystal Violet colorimetric assay. Values are the means of three independent experiments each performed in triplicate.

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3.3. G38K BS-RNase mutant or BS-RNase oligomerization represent efficient strategies to enhance autophagic cell death in cancer cells To analyze different strategies addressed to enhance the response of cancer cells to BS-RNase, we first compared the cytotoxic and autophagic outcome induced by WT or G38K BS-RNases in Panc1 cancer cells and normal fibroblasts. Although the G38K mutant determined a significant increase of the cytotoxic effect in both Panc1 cells and fibroblasts as compared to WT BS-RNase, the amount of the antiproliferative potentiation in Panc1 cancer cells was higher than that obtained in normal cells (Fig. 7A and B), further amplifying the differential response between cancer and normal cells to BS-RNase treatment. Intriguingly, G38K mutant very strongly enhanced also the autophagic response of cancer cells as compared to that obtained with WT BS-RNase, without improving the autophagic outcome in normal fibroblasts (Fig. 7C). In addition, since it is well known that oligomerization of ribonucleases enhances their enzymatic activity [33,38,46] we investigated whether both RNase A or BS-RNase domain-swapped oligomers could further stimulate autophagic cell death in Panc1 cancer cells. Fig. 8A and B show that, besides native RNase A monomer, its dimers (DN and DC), trimers (TNC, TC), tetramers (TTN and TTC) [33] or larger oligomers (LO) [31] were not able to significantly induce autophagy and cell growth

inhibition in Panc1 cells. Conversely, BS-RNase tetramers (TTN and TTC) [38] or larger oligomers (LO) [38] significantly potentiated both autophagy and inhibition of cancer cell growth with respect to the native dimer (D) [38]. Interestingly, this phenomenon is well correlated with the dimension of BS-RNase multimers (D b TTN ≈ TTC b LO), suggesting their potential use to enhance the autophagic response of cancer cells to BS-RNase treatment. 4. Discussion At its endogenous basal rate, autophagy exercises quality control of the cytoplasm of most cells by removing damaged organelles and protein aggregates [47,48]. Advances in understanding of the autophagic process paved the way for the discovery of the importance of autophagy in many physiological events, such as development, tissue homeostasis, metabolism, immune response and various diseases, including cancer [47,49]. However, the role of autophagy in tumors is complex and ranges from being a tumor suppressor role to exerting an action in cell

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observed after treatment with RNase A either in the absence or presence of the autophagy inhibitors CQ or 3MA (Fig. 3A and B). Conversely, Fig. 3C shows that BS-RNase-mediated cancer cell growth inhibition was strongly reduced in the presence of CQ or 3MA, which were instead ineffective when used alone (Supplemental Fig. 2A). Consistently, Fig. 3D reports that apoptosis induced by BS-RNase was significantly attenuated by autophagy inhibitors. The efficiency of CQ and 3MA in the autophagy inhibition event was checked by their relative inhibitory effect on the autophagosome formation stimulated by BS-RNase treatment (Supplemental Fig. 2B). Altogether, these data indicate a key role played by autophagy on the cytotoxic effect of BS-RNase in pancreatic cancer cells. To further characterize the mechanism of autophagy induction triggered by BS-RNase, we performed knock-down experiments of Beclin1 by siRNA transfection, showing that Beclin1 siRNA significantly rescued autophagy induced by BS-RNase alone or by BS-RNase in the presence of non-targeting siRNA as negative control (Fig. 4A). Accordingly with the cell death feature of autophagy shown in Fig. 3C and D, Beclin1 knock-down determined a significant reduction of the cytotoxic effect of BS-RNase (Fig. 4B). Western blot analysis was used to check Beclin1 silencing after siRNA transfection (Fig. 4C). Since BS-RNase was able to stimulate low levels of autophagosome formation in normal fibroblasts and HaCaT cells, we investigated whether this event could have a role on cell growth inhibition by BS-RNase in these non-tumoral cells. Concerning RNase A, which was not able to stimulate autophagy, the addition of the autophagy inhibitors CQ or 3MA to RNase A treatment did not determine any alteration on fibroblast and HaCaT cell growth (Fig. 5A and B). Interestingly, autophagy inhibitors failed to modify also BS-RNase-mediated growth inhibition in these normal cell types (Fig. 5C and D), suggesting that autophagy stimulated by BS-RNase may be considered a cancer-related cell death mechanism. To analyze the role of cancer cell proliferation rate in response to BS-RNase, we analyzed Panc1 cell growth and autophagy affected by BS-RNase in serum starvation, a well described condition favoring a quiescent state of the cells [45]. The decrease of serum percentage in culture medium strongly reduced the cytotoxic activity of BS-RNase in Panc1 cancer cells, as compared to their relative untreated cells (Fig. 6A). Furthermore, the rescue of cell growth inhibition by autophagy inhibitors CQ or 3MA progressively disappeared under serum starvation conditions and, accordingly, autophagy induced by BS-RNase treatment gradually decreased (Fig. 6B). Altogether, these data strongly suggest that the intrinsic higher proliferation rate of cancer cells with respect to normal cells may play a crucial role in their preferential vulnerability to BS-RNase treatment.

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serum starvation Fig. 6. Effect of serum starvation on growth inhibition and autophagy by BS-RNase in Panc1 cells. (A) Cells were seeded in 96-well plates, incubated overnight, and treated with 200 μg/ml BS-RNase in culture media containing percentages of FBS ranging from 10% to 0%, in the absence or presence of 5 μM CQ or 1 mM 3MA for 72 h. Cell proliferation was determined using the Crystal Violet colorimetric assay. Values are the means (±SD) of three independent experiments each performed in triplicate. Statistical analysis: (*) p b 0.05. (B) Cells were seeded in 96-well plates, incubated overnight, and treated with 200 μg/ml BS-RNase in cell culture media containing percentages of FBS ranging from 10% to 0% for 72 h. Autophagosome formation assay was analyzed using the incorporation of MDC probe. Values are the means (±SD) of three independent experiments each performed in triplicate. Statistical analysis: (*) p b 0.05.

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survival and environment adaptation [50,51]. Accordingly with this dual-role of autophagy, our research group recently demonstrated that pancreatic adenocarcinoma cell lines can react to various stimuli inducing a cyto-protective [52] or a cell death-related autophagic response [11,53,54]. Nonetheless, deeper efforts are needed to identify the mechanisms lying at the basis of this differential response of cancers cells. The present work represents the first strong evidence of a key role for autophagy played by the biological activity mediated by the secretory protein BS-RNase. Accordingly with our data, two very recent studies revealed that RNase L, a nearly ubiquitous enzyme present in mammalian cells that lies dormant until viral infections occur, triggers autophagy in response to the infections and affects the viral pathogenesis outcomes [55,56]. Here, we demonstrate for the first time that autophagy is induced in cancer cells by BS-RNase, but not by RNase A, and that this finding encloses a cancer cell death feature. Indeed, the addition of the autophagy inhibitors, CQ or 3MA, strongly reduced both pancreatic adenocarcinoma cytotoxicity and apoptotic cell death, suggesting that autophagy could represent a preparatory mechanism of apoptosis induced by BS-RNase treatment. Autophagy stimulation by BS-RNase is further recognized by the conversion of the autophagosome protein LC3-I in its phosphoethanolaminated form LC3-II, which is involved in the elongation of the autophagosomal membrane. LC3-II is often used as a marker of autophagy and accompanied by the induction of Beclin1, one of the main regulators of autophagosome formation. Beclin1 is a pro-autophagic protein that has been extensively studied as a tumorsuppressor factor and as an interactor of the anti-apoptotic Bcl-2 family members (Bcl-2/Bcl-XL) [57]. However, many studies have uncovered a number of Beclin1-dependent and -independent autophagy pathways, indicating that autophagy is a very complex process [58]. Our results of RNA silencing clearly demonstrate that Beclin1 plays a role in the autophagy induced by BS-RNase. Nonetheless, since Beclin1 silencing failed to completely abolish autophagy by BS-RNase, other Beclin1independent pathways could exist, such as MAPK (ERK, JNK, or p38)

A

induction or stimulation of different autophagy-related proteins such as, for example, ATG5, ATG7, and hVps34 [58]. Comparing the effect of the two pancreatic-type RNases tested (RNase A and BS-RNase) on pancreatic adenocarcinoma cells, we clearly revealed that, despite both enzymes displaying an appropriate enzymatic activity [33,59], cancer cells were unequivocally resistant to native RNase A, while they were highly sensitive to BS-RNase natural dimer. These data can be mainly explained by the inhibitory effect of the RNase inhibitor (RI), described to strongly bind most monomeric pancreatic-like RNases, including RNase A, in mammalian cells [23]. Conversely, BS-RNase, although sharing about 82% of sequence identity with RNase A and a closely conserved 3D structure of its subunit(s) [20], has the appropriate structural determinants which allow the protein to keep a dimeric structure able to evade RI interaction even under the reducing cytosolic environment [60]. This unique feature, together with its pI (10.3), higher than the one of RNase A (9.3), allows BS-RNase to bear a strong antitumor action [25,29,39]. Remarkably, we show that BS-RNase induced a quite low and ineffective level of autophagy stimulation in normal fibroblasts and nontumorigenic HaCaT cells, as compared to cancer cells. Indeed, autophagy inhibitors did not significantly modify cell growth of fibroblasts and HaCaT cells, suggesting autophagy to be a cancer-related mechanism of cell death induced by BS-RNase. To unravel this finding we investigated whether the high proliferation rate, a typical feature of cancer cells, may play a critical role in the stronger response to BS-RNase treatment of cancer than normal cells. Since serum starvation is a classic condition determining cell cycle arrest and quiescence [45], we analyzed autophagic cell death in Panc1 cells under gradual serum starvation demonstrating that this condition is able to completely abolish autophagy stimulation by BS-RNase strongly reducing its cytotoxic effect. In addition, we tested different approaches to enhance autophagic cell death in cancer cells by BS-RNase-related treatments. We reveal here that the G38K mutant BS-RNase, with an enhanced positive charge as compared with WT, determined a definitely higher autophagic cell

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µg/ml Fig. 7. Effect of BS-RNase G38K mutant on growth inhibition and autophagy in Panc1 cells and fibroblasts. (A, B and C) Cells were seeded in 96-well plates, incubated overnight, and treated with increasing concentrations of BS-RNase WT (black circle) or BS-RNase G38K mutant (white square) for 72 h. Cell proliferation was determined using the Crystal Violet colorimetric assay, while autophagosome formation assay was analyzed using the incorporation of MDC probe. Values are the means of three independent experiments each performed in triplicate. Statistical analysis: (#) p b 0.05 BS-RNase WT vs BS-RNase G38K mutant.

C. Fiorini et al. / Biochimica et Biophysica Acta 1843 (2014) 976–984

A

oligomerization of the two RNases could be ascribable mainly to the following reasons: i) a lower basic net charge, or charge exposure, of RNase A with respect to BS-RNase [29,39]; ii) the relative higher stability in solution of domain-swapped BS-RNase oligomers with respect to RNase A ones. Anyway, a partial reconversion of RNase A or BS-RNase oligomers to their native species can lead to a monomer blocked by RI or to an active swapped dimer which can evade RI interaction, respectively.

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Our data indicate that BS-RNase, but not RNase A, strongly inhibits pancreatic adenocarcinoma cell proliferation triggering Beclin1mediated autophagic cell death. The potential use of the more active G38K mutant or BS-RNase oligomers may be considered as a novel promising therapeutic tool against pancreatic adenocarcinoma. This study provides the rational basis for future investigations which could be addressed to evaluate the anti-tumoral in vivo efficiency of nanovectors containing active RNases, as BS-RNase is. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbamcr.2014.01.025.

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Acknowledgements

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Fig. 8. Effect of 3D domain-swapped RNase A and BS-RNase oligomers on autophagy and growth inhibition in Panc1 cells. Oligomers are named as reported in the “Materials and methods” section. (A) Cells were seeded in 96-well plates, incubated overnight, and treated with 100 μg/ml (black histograms) or 200 μg/ml (white histograms) RNase A or BS-RNase for 72 h. Autophagosome formation assay was analyzed using the incorporation of MDC probe. Values are the means (±SD) of three independent experiments each performed in triplicate. Statistical analysis: (*) p b 0.05 D vs TTN, TTC, or LO; (#) p b 0.05 TTN vs D or LO; (§) p b 0.05 TTC vs D or LO; (†) p b 0.05 LO vs D, TTN, or TTC. (B) Cells were seeded in 96-well plates, incubated overnight, and treated with 100 μg/ml (black histograms) or 200 μg/ml (white histograms) RNase A or BS-RNase for 72 h. Cell proliferation was determined using the Crystal Violet colorimetric assay. Values are the means (±SD) of three independent experiments each performed in triplicate. Statistical analysis: (*) p b 0.05 D vs TTN, TTC, or LO; (#) p b 0.05 TTN vs D or LO; (§) p b 0.05 TTC vs D or LO; (†) p b 0.05 LO vs D, TTN, or TTC.

death level in cancer cells than WT BS-RNase. This result can be explained by the higher capability of G38K mutant to interact with the very acidic membrane lipids of cancer cells with respect to WT [39]. This effect is in line with the stronger interaction of G38K mutant with anionic phospholipid bilayers observed by SPR measurements and with its deeper insertion in liposomes that we previously reported [39]. In addition, we cannot exclude that the increased cluster of positive charges on protein surface can also enhance the capability of BS-RNase to evade RI binding, producing a 3–4 fold increase in autophagic cell death induced by G38K mutant. Furthermore, since we previously demonstrated that various artificial N- or C-terminal domain-swapped oligomers of both RNase A and BS-RNase displayed an increased enzymatic activity against dsRNA, as compared to the native enzymes [33,38], we investigated here whether this feature could determine a potentiated autophagic cell death in cancer cells. Intriguingly, we demonstrate that RNase A oligomers failed to stimulate autophagy and cell growth inhibition, while BS-RNase multimers strongly enhanced autophagic cell death in pancreatic cancer cells, with an activity increasing with the oligomers' size (D b TTN ≈ TTC b LO). Indeed, multimerization increases both catalytic and biological activities of RNases by enhancing substrate affinity and cell membrane interaction because of the augmented basic charge density of the oligomers [19,33,37,38]. Despite sensitivity of cells to RNase A oligomers may depend on many factors [19,34,35,61], the differential effect shown by the

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