Regulation of autophagy and apoptosis by Dp44mT-mediated activation of AMPK in pancreatic cancer cells

Journal Pre-proof Regulation of autophagy and apoptosis by Dp44mT-mediated activation of AMPK in pancreatic Cancer cells

S. Krishan, S. Sahni, L.Y.W. Leck, P.J. Jansson, D.R. Richardson PII:

S0925-4439(19)30388-6

DOI:

https://doi.org/10.1016/j.bbadis.2019.165657

Reference:

BBADIS 165657

To appear in:

BBA - Molecular Basis of Disease

Received date:

18 October 2019

Revised date:

16 December 2019

Accepted date:

20 December 2019

Please cite this article as: S. Krishan, S. Sahni, L.Y.W. Leck, et al., Regulation of autophagy and apoptosis by Dp44mT-mediated activation of AMPK in pancreatic Cancer cells, BBA - Molecular Basis of Disease(2019), https://doi.org/10.1016/ j.bbadis.2019.165657

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© 2019 Published by Elsevier.

Journal Pre-proof Regulation of Autophagy and Apoptosis by Dp44mT-Mediated Activation of AMPK in Pancreatic Cancer Cells

Krishan, S.,*1 Sahni, S.,*1 Leck, L.Y.W.,1 Jansson, P.J.,1 and Richardson, D.R.1,2 *Equal first authors 1

Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute,

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University of Sydney, Sydney, New South Wales, 2006, Australia; 2Department of Pathology and

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Biological Responses, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

To whom correspondence should be addressed: Dr. Des R. Richardson, Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales, 2006 Australia. Ph: +61-2-9036-3026; Email: [email protected].

Journal Pre-proof Abstract Upon activation, the 5ʹ-adenosine monophosphate-activated protein kinase (AMPK) increases catabolism, while inhibiting anabolism. The anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3thiosemicarbazone (Dp44mT), activates AMPK in multiple tumor cell-types (Biochim. Biophys Acta 2016;1863:2916-2933). This acts as an initial cell “rescue response” after iron-depletion mediated by Dp44mT. Considering Dp44mT-mediated AMPK activation, the role of AMPK on Dp44mT

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cytotoxicity was examined. Dp44mT increased the p-AMPK/AMPK ratio in multiple tumor cell-types over short (24 h) and longer (72 h) incubations. Notably, Dp44mT was more effective in inhibiting

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tumor cell proliferation after AMPK silencing, potentially due to the loss of AMPK-mediated metabolic

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plasticity that protects cells against Dp44mT cytotoxicity. The silencing of AMPK-increased cellular

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cholesterol and stabilized lysosomes against Dp44mT-mediated lysosomal membrane permeabilization.

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This was substantiated by studies demonstrating that the cholesterol-depleting agent, methyl-cyclodextrin (MCD), restores Dp44mT-mediated lysosomal membrane permeabilization in AMPK

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silenced cells. The increased levels of cholesterol after AMPK silencing were independent of the ability

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of AMPK to inhibit the rate-limiting step of cholesterol synthesis via the inactivating phosphorylation

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of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) at Ser872. In fact, Dp44mT did not increase phosphorylation of HMGCR at (Ser872), but decreased total HMGCR expression similarly in both the presence or absence of AMPK silencing. Dp44mT was demonstrated to increase autophagic initiation after AMPK silencing via an AMPK- and Beclin-1-independent mechanism. Further, there was increased cleaved caspase 3 and cleaved PARP after incubation of AMPK silenced cells with Dp44mT. Overall, AMPK silencing promotes Dp44mT anti-proliferative activity, suggesting a role for AMPK in rescuing its cytotoxicity by inhibiting autophagy and also apoptosis.

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Journal Pre-proof 1. Introduction The enzyme, 5ʹ-adenosine monophosphate-activated protein kinase (AMPK), forms part of a crucial cellular energy-sensing pathway, which is up-regulated under energy-deplete conditions [1, 2]. This upregulation results in the activation of catabolic pathways, such as autophagy, and inhibition of anabolic pathways, such as fatty acid synthesis [1]. It is also known that AMPK can play a role in pathways

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involving apoptosis [3].

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Our laboratories developed an innovative class of di-2-pyridylketone thiosemicarbazones (DpTs) that demonstrate marked anti-tumor activity in vitro and in vivo leading to autophagy and also the induction

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of apoptosis [4-8]. In our previous studies, it was observed that the potent DpT analogue, di-2-

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pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Fig. 1A), activates the AMPK-dependent

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stress pathway in a variety of tumor cell-types [9]. The mechanism of this activation occurs through the disruption of ATP-generation pathways, namely mitochondrial respiration and glycolysis. This effect

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was mediated by metal chelation, as well as the redox properties of the thiosemicarbazones [9].

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Activation of AMPK by Dp44mT led to an increase in the activity of regulators of downstream

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catabolic pathways, such as autophagy. This activation includes the up-regulation of the important autophagy regulator, Unc-like kinase 1 (ULK1), by Dp44mT in an AMPK-dependent manner. Furthermore, the Dp44mT-mediated up-regulation of the AMPK pathway was also shown to inhibit protein biosynthesis (i.e., via Raptor) and fatty acid synthesis/metabolism (i.e., via acetyl CoA carboxylase 1) [9]. These responses were implicated as part of an initial rescue response by the cell caused by metal-ion depletion mediated by the thiosemicarbazones.

Key to the anti-tumor activity of this class of di-2-pyridylketone thiosemicarbazones is their ability to induce lysosomal membrane permeabilization (LMP) resulting in dysfunctional autophagy, which 3

Journal Pre-proof prevents the lysosome fusing with the autophagosome to generate an autolysosome that completes the autophagic process [8, 10, 11]. This is particularly potent for the Dp44mT-copper (Cu) complex (Fig. 1B), which forms after chelation of intracellular Cu and is more effective than Dp44mT at inducing LMP, probably due to its marked redox activity [10, 12]. In fact, Dp44mT is well known to avidly bind Cu(II) [13] with a formation constant (log β2*) of 12.49 at pH 7.4 [14]. Understanding the molecular mechanism of how this class of di-2-pyridylketone thiosemicarbazones initiates LMP is important for

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designing more effective therapeutic strategies, as this class of clinically-trialed agents [15] demonstrates: (1) potent anti-tumor activity in a broad spectrum of cancers [5, 11, 16, 17]; (2)

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overcomes P-glycoprotein-mediated resistance [5, 11, 18]; and (3) inhibits metastasis [19, 20].

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Considering the molecular mechanism of how Dp44mT induces lysosomal permeability, it is notable

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that AMPK could play an important role in lysosomal membrane stability, due to its known ability to regulate endogenous cholesterol synthesis [21]. Activation of AMPK leads to the inhibition of the key

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rate-limiting enzyme in the cholesterol biosynthesis pathway, namely 3-hydroxy-3-methylglutaryl CoA

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reductase (HMGCR), through phosphorylation at Ser872 [21]. One of the roles of cholesterol is to stabilize the lysosomal membrane [22] and decreased levels of lysosomal membrane cholesterol have

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been shown to result in decreased lysosomal membrane stability [23, 24].

Hence, in the present investigation, it was hypothesized that AMPK plays a role in lysosomal membrane stabilization. As such, this study examined the role of AMPK in Dp44mT-mediated LMP in tumor cells. This is important, as lysosomes play an essential role in a variety of cell survival processes e.g., the autophagic pathway [20], development and programmed cell death [25]. Moreover, agents of the thiosemicarbazone class, such as Dp44mT, induce their anti-proliferative activity, at least in part, through LMP [10, 11]. Autophagy is a catabolic process that involves the degradation of cytoplasmic 4

Journal Pre-proof contents, including proteins and organelles via the lysosome, that can be initiated for example by micro-environmental stresses, such as nutrient deprivation and oxidative stress [26-28]. This process is controlled by autophagy-related genes (ATGs) [29-33] that encode proteins responsible for the formation of the double membrane autophagosome, to which damaged proteins and organelles are sequestered [32, 33]. The autophagosome loaded with cargo, fuses with the lysosome, resulting in the formation of an autolysosome [34]. The autolysosome contains hydrolases, which degrade the contents

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and release the nutrients back into the cytoplasm for recycling [34]. Autophagy can act as a survival mechanism during cellular stress and can lead to resistance to anti-cancer agents [35]. As

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chemotherapeutic agents generally mediate their activity by increasing intracellular damage due to

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intracellular stress, up-regulation of the autophagic pathway helps cancer cells to sustain and repair

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themselves under stressful conditions. This occurs via the recycling of constituents such as damaged

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proteins and organelles, leading to the development of chemotherapeutic resistance. Studies have shown that Dp44mT can overcome the pro-survival response of autophagy [8]. This latter effect is

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requires intact lysosomes [8].

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attributed to the ability of Dp44mT to induce LMP, thus resulting in dysfunctional autophagy, which

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One of the well-characterized targets of AMPK is ULK1, an important initiator of autophagy [36]. When activated, AMPK is able to phosphorylate ULK1 leading to autophagic initiation [36]. Previously, our studies demonstrated that ULK1 activation and expression was increased by Dp44mT, indicating this agent stimulated autophagic initiation [8, 9, 37]. The current investigation examined the effects of Dp44mT on cellular mechanisms involved in the rescue response mediated via its ability to induce AMPK activation. To examine this, AMPK was silenced and the effects of Dp44mT on cellular proliferation, LMP, cholesterol synthesis, autophagy and apoptosis, were then examined. Herein, it is demonstrated that AMPK silencing increases Dp44mT anti-proliferative activity, potentially through 5

Journal Pre-proof the loss of metabolic plasticity mediated by AMPK. Furthermore, AMPK silencing increased the expression of autophagic and apoptotic markers, suggesting that AMPK suppresses autophagy and apoptosis probably through its ability to aid metabolic plasticity, that inhibits the cytotoxicity of

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Dp44mT.

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Materials and Methods

2.1 Cell Culture and Treatments The human pancreatic cancer cell-types, PANC-1, AsPC-1, and MIAPaCa-2 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were used within 2 months of

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purchase after resuscitation of frozen aliquots. Cell lines were authenticated by the provider and this

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was based on viability, recovery, growth, morphology, cytogenetic analysis, antigen expression, DNA profile and iso-enzymology. Cells were routinely examined for mycoplasma contamination using

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standard methods [38].

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PANC-1, and MIAPaCa-2 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Melbourne, Victoria, Australia), while AsPC-1 cells were grown in RPMI-1640 medium (Gibco). All

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media was supplemented with 10% fetal bovine serum (FBS), 1% (v/v) non-essential amino acids, 1%

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sodium pyruvate, and 1% penicillin/streptomycin/glutamine. All supplements were obtained from Life

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Technologies (Carlsbad, CA).

The thiosemicarbazone, Dp44mT (Fig. 1A), and its copper(II) complex ([Cu(Dp44mT)Cl] from hereon referred to as Cu-Dp44mT; Fig. 1B) were synthesized and characterized by standard methods [12, 39]. Cells were seeded into 35 mm tissue culture plates and incubated until approximately 80% confluent. These cells were then treated with Dp44mT at the indicated concentrations for 24-, 48- or 72-h/37°C. For studies examining acridine orange staining to study LMP, cells were treated with Cu-Dp44mT (2.5 M) for 2.5 h/37°C, unless otherwise indicated.

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2.2 siRNA Transfection Cells were seeded on to 6- or 96-well plates for 24 h/37°C, under the conditions described in Section 2.1. The cells were then incubated with either 150 pmoles of negative control siRNA (Cat.# AM4637: ThermoFisher Scientific) or AMPKα1 siRNA (hereon referred to as AMPK siRNA; Cat.# AM16704

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(143192), ThermoFisher Scientific) and Lipofectamine 2000 in Opti-MEM for 6 h/37 °C. The media

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was then replaced and the cells incubated for 48 h/37°C, prior to treatment.

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2.3 MTT Proliferation Assay

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Cellular proliferation was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

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bromide (MTT) assay via standard protocols in our laboratory, where cellular proliferation was demonstrated to be directly proportional to viable cell counts as demonstrated via Trypan blue staining

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[40]. The cells (5 × 103/well) were seeded in 96-well plates and then transfected with either negative

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control siRNA or AMPK siRNA. This medium was subsequently replaced, and the cells incubated with

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Dp44mT for 24 h/37°C or 72 h/37°C and processed.

2.4 Protein Extraction and Western Analysis Cells were harvested and suspended in lysis buffer (150 mM NaCl, 10 mM Tris-HCl [pH 7.4], 0.5% (w/v) sodium dodecyl sulphate (SDS), 1 mM EDTA, 40 μM NaF, 1% (v/v) Triton X-100). The lysate was then sonicated, centrifuged (16,000 x g/40 min/4°C), and the supernatant collected for further analysis. The lysates were electrophoresed on an SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was fixed by soaking it in 100% methanol for 30 s at room temperature. 8

Journal Pre-proof The primary antibodies used included those against: AMPKα1 (Cat #2793; Cell Signaling Technology, Danvers, MA), p-AMPKα1 (Cat #2535, Cell Signaling Technology), HMGCR (Cat #ab174830, Abcam), p-HMGCR (Cat #ab215437, Abcam), LC3 (Cat #PM036, MBL International, Woburn, MA), Beclin-1 (Cat #3495, Cell Signaling Technology), caspase 3 (Cat #9665, Cell Signaling Technology), cleaved caspase 3 (Cat #9664, Cell Signaling Technology), PARP (Cat #9542, Cell Signaling Technology) and cleaved PARP (Cat #5625, Cell Signaling Technology). All primary antibodies were

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diluted and used at 1:1,000 in 5% (w/v) bovine serum albumin (BSA) in Tris-buffered saline (TBS; Sigma-Aldrich, St. Louis, MO) containing 0.1% Tween-20 (TBST). As an assessment of equal protein-

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loading, membranes were probed for β-actin (Cat #A1978; Sigma-Aldrich). The antibody against β-

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actin was diluted to 1:10,000 in 5% (w/v) non-fat milk in TBST. None of the agents tested had any

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effect on β-actin expression, demonstrating its utility as an appropriate loading control.

Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG from Sigma-Aldrich

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were used as secondary antibodies. These were diluted to 1:10,000 in 5% non-fat milk in TBST. The

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membranes were incubated with Luminata Crescendo Western HRP substrate (Cat. WBLUR0100; Millipore, Billerica, MA), or Luminata Forte Western HRP substrate (Cat. WBLUF0100). The

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chemiluminescence signals were captured using a ChemiDoc MP Imaging System (BioRad; Hercules, CA). Densitometric analysis was performed using ChemiDoc Image Lab Software (BioRad). Data were normalized to the corresponding β-actin standards.

2.5 Assessment of Lysosomal Membrane Permeabilization (LMP) Cells were seeded and transfected with siRNA, as described in Section 2.2. Cells were incubated with Cu-Dp44mT (2.5 μM) for 2.5 h/37°C unless otherwise specified. Acridine orange (Sigma-Aldrich) was used to determine LMP using standard procedures [41]. Briefly, cells were incubated with acridine 9

Journal Pre-proof orange (20 nM) for 15 min/37°C following incubation with the chelators. Stained samples were examined with a Zeiss Axio Observer.Z1 microscope equipped with an AxioCam camera (Zeiss, Oberkochen, Germany) and Zeiss Axiovision co-localization software (Zeiss).

1.6 Measurement of Cellular Cholesterol Cells were harvested after centrifugation (1000 rpm/5 min/20°C) and resuspended in lysis buffer (150

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mM NaCl, 10 mM Tris-HCl [pH 7.4], 0.5% (w/v) sodium dodecyl sulphate (SDS), 1 mM EDTA, 40

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μM NaF, 1% (v/v) Triton X-100). The lysate was then sonicated on ice, centrifuged (16,000 x g/40

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min/4°C), and the supernatant collected for further analysis. Intracellular cholesterol was measured using the Amplex® Red cholesterol assay kit (Invitrogen) using the manufacturer’s instructions with

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some modifications. Briefly, washed cell pellets were resuspended in 500 µL of hexane/isopropyl

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alcohol (3:2, v/v), nitrogen flushed, vortexed at 3,000 rpm/1 h and centrifuged at 14,000 rpm/5

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min/4°C. The clear lipid extract was dried under nitrogen and assayed based on the manufacturer's

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2.7 Statistics

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instructions. The above protocol was based on that described by [42].

Experimental data were compared using the Student’s paired t-test. Results were considered statistically significant when p < 0.05. Results are presented as the mean ± standard error of the mean (SEM).

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Journal Pre-proof 2.

Results

3.1 The Anti-Cancer Agent, Dp44mT, Increases AMPK Activation Over a Prolonged Incubation Period. Our previous investigation demonstrated that the thiosemicarbazone, Dp44mT, and other iron chelators, potently up-regulated the AMPK-dependent energy homeostasis pathway [9]. This latter study was limited to assessing short-term incubations (up to 24 h) of these agents on this pathway. The

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current research initially examined the effects of Dp44mT on AMPK phosphorylation over a considerably longer incubation periods (24-72 h), which are more appropriate to clinically relevant,

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pharmacological treatment.

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First, PANC-1 cells were incubated with Dp44mT (1.25- and 2.5-μM) for 24-, 48- and 72-h/37℃, with

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the levels of phosphorylated and total AMPK being examined at each time point using western blotting (Fig. 1C, D). Of note, phosphorylation of AMPK at T172 is well known to activate the protein [1, 2].

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There was a significant (p < 0.05) increase in p-AMPK (T172) levels at both concentrations of

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Dp44mT after a 24 h incubation relative to the control (i.e., 0 μM Dp44mT), which generally became more pronounced and significant (p < 0.001-0.05) after 48 h and particularly 72 h (Fig. 1C, Di). In

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contrast, both concentrations of Dp44mT significantly (p < 0.01-0.05) decreased total AMPK levels relative to the control after 24 h and 48 h, while only the higher concentration of Dp44mT (2.5 μM) significantly (p < 0.05) decreased total AMPK levels after 72 h (Fig. 1C, Dii). Furthermore, there was also a significant (p < 0.001-0.05) increase in the p-AMPK/AMPK ratio in PANC-1 cells after all incubation times with Dp44mT (1.25- and 2.5-µM; 24-72 h) compared to the corresponding controls at each time point (Fig. 1C, Diii).

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Journal Pre-proof Of note, at the higher Dp44mT concentration of 2.5 µM after 48- and 72-h, there was decrease in pAMPK levels relative to that observed at Dp44mT at 1.25 µM (Fig. 1C, Di). Similarly, at a Dp44mT concentration of 2.5 µM, a slight decrease in total AMPK was observed after 48 h, with a more pronounced decrease being demonstrated after 72-h relative to that demonstrated using Dp44mT at 1.25 µM (Fig. 1C, Dii). Nonetheless, the p-AMPK/AMPK ratio at a Dp44mT concentration of 2.5 µM after 48- and 72-h, still remained significantly (p < 0.05-0.01) increased at 2.5 µM relative to the

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control and not significantly different versus Dp44mT at 1.25 µM (Fig. 1C, Diii). It could be speculated that this decrease in pAMPK and total AMPK observed at the highest Dp44mT

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concentration may be due to a cellular feedback mechanism to “fine tune” the robust AMPK stress

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response to Dp44mT.

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In conclusion, incubation of PANC-1 cells with Dp44mT induces marked and sustained activation of

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AMPK over incubation periods of 24-, 48- and 72-h.

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cancer cell-types

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3.2 Dp44mT generally increases p-AMPK and decreases total AMPK levels in multiple pancreatic

The observed effects of Dp44mT on AMPK and its phosphorylation were also examined in two additional pancreatic cancer cell-types, namely AsPC-1 and MIAPaCa-2 cells (Fig. 2). The AsPC-1 cells were incubated with Dp44mT (2.5 μM), for 24-, 48- and 72-h/37℃, and the levels of phosphorylated and total AMPK were then examined at each time point (Fig. 2A, Bi, ii). Similar to the observations in PANC-1 cells (Fig. 1C, D), there was a significant (p < 0.01) increase in p-AMPK levels in AsPC-1 cells after 24 h relative to the control. Unlike PANC-1 cells (Fig. 1C, Di), there was no significant change in p-AMPK levels in AsPC-1 cells after a 48- or 72-h incubation with Dp44mT compared to the respective controls at each time point (Fig. 2A, Bi). 12

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However, as observed for PANC-1 cells (Fig. 1C, Dii), there was a significant (p < 0.01-0.05) decrease in total AMPK levels in AsPC-1 cells after incubation with Dp44mT at all timepoints (Fig. 2A, Bii). In contrast to PANC-1 (Fig. 1C, Dii), there was a pronounced and significant (p < 0.001) decrease in total AMPK levels in control AsPC-1 cells after 48 h compared to 24 h (Fig. 2A, Bii). The effect of Dp44mT after 48 h on decreasing total AMPK was significantly (p < 0.05) enhanced relative to that

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observed with this agent after 24 h. After a 72 h incubation, Dp44mT again markedly and significantly (p < 0.01) decreased total AMPK relative to the respective control at this timepoint (Fig. 2A, Bii).

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Although an increase in p-AMPK levels was only observed after a 24 h incubation of AsPC-1 cells

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with Dp44mT (Fig. 2A, 2Bi), there was a significant (p < 0.01-0.05) increase in the ratio of

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phosphorylated AMPK to total AMPK at all timepoints after Dp44mT versus the respective controls

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(Fig. 2A, Biii). This was mediated by the decrease in total AMPK expression upon Dp44mT treatment. Due to the pronounced decrease in total AMPK in the control at 48 h relative to 24 h (Fig. 2A, Bii),

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24 h (Fig. 2Biii).

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there was a significant (p < 0.05) increase in the p-AMPK/AMPK ratio of the control at 48 h relative to

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As for PANC-1 and AsPC-1 cells, the MIAPaCa-2 cell-type was also incubated with Dp44mT (2.5 μM), for 24-, 48- and 72-h/37℃, and the levels of phosphorylated and total AMPK were again examined at each time point (Fig. 2C, Di-iii). There was a significant (p < 0.05) increase in p-AMPK levels after incubation with Dp44mT for 24 h compared to the respective control (Fig. 2C, Di). The increase in p-AMPK became more pronounced and was significantly increased (p < 0.01-0.05) after both 48- and 72-h compared to the respective controls at each time point (Fig. 2C, Di). Similarly to PANC-1 and AsPC-1 cells, the levels of total AMPK were significantly (p < 0.01) reduced after incubation of MIAPaCa-2 cells with Dp44mT for 24 h and there was also a pronounced and significant 13

Journal Pre-proof (p < 0.001) decrease in total AMPK after 48- and 72-h versus the respective controls (Fig. 2C, Dii). Due to these changes in p-AMPK and total AMPK, there was a significant increase (p < 0.001-0.01) in the p-AMPK/AMPK ratio after incubation of MIAPaCa-2 cells with Dp44mT, which increased as a function of incubation time (Fig. 2C, Diii).

Overall, these results demonstrate that Dp44mT generally increases p-AMPK and decreases total

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AMPK levels in multiple pancreatic cancer cell-types, with this effect being prominent after a

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prolonged incubation.

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3.3 AMPK silencing increases the anti-proliferative activity of Dp44mT in pancreatic cancer cells.

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In order to initially determine the effect of Dp44mT-mediated activation of the AMPK pathway in

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cancer cells, studies examined the effects of AMPK silencing on the anti-proliferative activity of increasing concentrations of Dp44mT against the PANC-1, AsPC-1 and MIAPaCa-2 pancreatic cancer

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cell-types (Fig. 3A-C). PANC-1 cells were first pre-incubated with either negative control (NC) siRNA

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or AMPK siRNA for 48 h/37°C. These siRNA-transfected cells were then treated with increasing concentrations of Dp44mT (2.5-20 µM) for 24- or 72-h/37°C. Cellular proliferation was then

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measured, as described in the Materials and Methods section.

Silencing of AMPK in PANC-1 cells had no significant (p > 0.05) effect on proliferation under control conditions (i.e., 0 µM Dp44mT) after a 24 h incubation (Fig. 3Ai). Incubation of either NC siRNA or AMPK siRNA-treated cells with Dp44mT at all concentrations resulted in a significant (p <0.001-0.05) dose-dependent decrease in cellular proliferation over 24 h relative to the 0 µM Dp44mT controls (Fig. 3Ai). Furthermore, at all Dp44mT concentrations, this agent was significantly (p < 0.05) more effective at inhibiting cellular proliferation in the presence of AMPK siRNA relative to NC siRNA (Fig. 3Ai). 14

Journal Pre-proof The extent of this inhibition of proliferation of PANC-1 cells by Dp44mT was markedly enhanced after a 72 h incubation relative to the 24 h incubation (cf. Figs. 3Ai to 3Aii). After a 72 h incubation, there was a pronounced and significant (p < 0.001-0.01) decrease in proliferation at all Dp44mT concentrations in AMPK- or NC-siRNA treated cells versus the 0 µM Dp44mT controls (Fig. 3Aii). However, after a 72 h incubation, significantly (p < 0.05) increased susceptibility of AMPK silenced cells towards Dp44mT relative to the NC siRNA was only observed at the highest Dp44mT

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concentration of 20 µM (Fig. 3Aii).

Examining AsPC-1 cells, there was a significant (p < 0.001-0.05) decrease of proliferation in the NC

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and AMPK silenced cells after incubation with all Dp44mT concentrations for 24 h relative to the

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respective control (i.e., Dp44mT; 0 µM; Fig. 3Bi). However, while the proliferation of AMPK siRNA-

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treated cells was lower at all Dp44mT concentrations than their NC siRNA treated counterparts, the difference was only significant (p < 0.05) at Dp44mT concentrations of 2.5- and 20-µM (Fig. 3Bi). The

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AsPC-1 cells treated with either NC siRNA or AMPK siRNA were more susceptible to Dp44mT after

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72 h, with a marked and significant (p < 0.001-0.01) decrease in proliferation being evident at all Dp44mT concentrations relative to the 0 µM Dp44mT control (Fig. 3Bi). However, there was no

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significant (p > 0.05) difference between NC siRNA and AMPK siRNA-treated cells at almost all Dp44mT concentrations, except for the significant (p < 0.05) difference observed at a Dp44mT concentration of 20 µM (Fig. 3Bii).

Similarly to PANC-1 and AsPC-1 cells, assessing MIAPaCa-2 cells, there was a significant (p < 0.0010.05) decrease in cellular proliferation in the NC and AMPK silenced cells after incubation with Dp44mT after a 24- or 72-h incubation at all Dp44mT concentrations compared to the respective control (i.e., Dp44mT at 0 µM; Fig. 3Ci,ii). Moreover, AMPK silencing led to significantly (p < 0.0115

Journal Pre-proof 0.05) greater inhibition of proliferation by Dp44mT at all concentrations relative to the NC siRNA after an incubation of 24 h. In contrast, no significant (p > 0.05) difference in proliferation was observed between AMPK siRNA-treated cells and the NC siRNA after 72 h (Fig. 3Cii).

Generally, these results indicate that AMPK expression acts to induce a rescue response against Dp44mT-mediated cellular stress especially after a 24 h incubation, and thus, acts antagonistically

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against its anti-cancer activity. In contrast, after a 72 h incubation with Dp44mT, this rescue effect of

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AMPK is lost, probably due to the pronounced cytotoxicity induced by this compound.

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3.4 AMPK Silencing Ablates the Ability of Dp44mT to Cause Lysosomal Membrane

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Permeabilization (LMP).

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Lysosomal membrane stability plays a major role in the anti-tumor activity of Dp44mT and its redox active analogues [8, 10, 11]. In fact, Dp44mT forms a redox-active Cu complex in lysosomes that

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generates reactive oxygen species (ROS), resulting in LMP and tumor cell death [10, 43, 44]. The

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current investigation examined the role of AMPK expression in LMP. Considering that the induction of

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the AMPK pathway is well known to inhibit the activity of the anabolic, cholesterol synthesis enzyme, HMGCR [22], silencing AMPK may increase HMGCR activity. This could result in cholesterol accumulation in cells, which is known to increase lysosomal membrane stability and prevent LMP by Dp44mT [43].

Of note, Dp44mT rapidly permeabilizes tumor cells and chelates intracellular Cu forming a redoxactive complex, which then induces LMP [10, 43]. Considering this, the Cu complex of Dp44mT, i.e., Cu-Dp44mT (2.5 µM), was utilized to examine the role of the AMPK pathway in Dp44mT-mediated LMP. PANC-1 cells were transfected with either NC siRNA or AMPK siRNA, followed by incubation 16

Journal Pre-proof with Cu-Dp44mT for 1-2.5 h (Fig. 4). The lysosomes were visualized using the well characterized acridine orange assay described in the Materials and Methods [43, 45-48]. Using this assay, the acidic lysosomal vesicles fluoresce red, while the cytosol fluoresces green, with a decrease in the red/green ratio of fluorescence indicating LMP [43, 45-48]. We have previously performed extensive studies using Lysotracker, Pepstatin A-BODIPY FL conjugate and acridine orange as lysosomal markers for examining Dp44mT-mediated lysosomal membrane permeabilization [10, 11]. Notably, acridine

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orange demonstrated exactly analogous results to both Lysotracker and Pepstatin A BODIPY FL

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conjugate, justifying the use of acridine orange in this investigation.

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In these studies, lysosomes were visualized under control conditions (i.e., at 0 time) by red acridine

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orange staining, using cells transfected with NC siRNA or AMPK siRNA (Fig. 4A). There was a

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significant (p < 0.05) decrease in red lysosomal staining indicating induction of LMP, after incubation of NC siRNA-treated cells with Cu-Dp44mT (2.5 µM) for 2.5 h/37°C (Fig. 4B). Furthermore, under

na

this latter condition, there was a substantial decrease in cell size, indicating cellular damage by the

ur

compound, which could be consistent with apoptosis [49] that is induced by Dp44mT [50]. Similar

laboratories [10].

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results with Cu-Dp44mT examining LMP using AO staining have been previously reported by our

In contrast, after AMPK silencing, Cu-Dp44mT was unable to cause LMP in AMPK silenced cells even after 2.5 h incubation and cell size remained constant relative to the 0 h control (Fig. 4A, B). These results demonstrate that AMPK silencing resulted in resistance to Dp44mT-mediated LMP, potentially due to inhibition of cholesterol synthesis via activated AMPK [21, 51, 52]. In fact, since AMPK inhibits HMGCR activity [21], it can be hypothesized that silencing AMPK may result in increased

17

Journal Pre-proof cholesterol levels, which is known to maintain lysosomal membrane stability [22], particularly against the activity of Cu-Dp44mT [43].

To test this latter hypothesis, PANC-1 cells were incubated with either NC siRNA or AMPK siRNA, and then treated in the presence or absence of the cholesterol-depleting agent, methyl--cyclodextrin (MCD;10 mM; [24, 43]), for 1 h/37°C, followed by incubation with Cu-Dp44mT (2.5 µM) for 2.5

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h/37°C in the presence or absence of MCD. Of note, MCD has been demonstrated to extract

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cholesterol from biological membranes [24] and sensitize cells to the ability of the Cu-Dp44mT complex to induce LMP [43]. There was a significant (p < 0.05) decrease in the red/green fluorescence

-p

in the NC siRNA-treated cells after incubation with Cu-Dp44mT (2.5 µM) compared to the control

re

(Fig. 5A). However, in contrast, this latter effect with Cu-Dp44mT was not observed with AMPK

na

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silenced cells, which were resistant to this agent (Fig. 5A).

When AMPK silenced cells were pre-incubated with MCD (10 mM/1 h/37°C) prior to incubation with

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Cu-Dp44mT, there was a significant (p < 0.05) decrease in the red/green fluorescence compared to Cu-

Jo

Dp44mT alone (Fig. 5A, B). These results suggest that MCD had removed cellular cholesterol and sensitized these cells to LMP induced by Cu-Dp44mT. This was consistent with the hypothesis that the resistance to Cu-Dp44mT-mediated LMP caused by AMPK silencing was due to AMPK’s known catabolic role in decreasing cholesterol levels [21]. Examining either NC siRNA or AMPK siRNA silenced cells and preincubating them with MCD (10 mM) alone for 1 h/37°C also decreased the red/green fluorescence ratio relative to the respective controls, although this was not significant.

Overall, the studies in Figure 5 demonstrate that AMPK siRNA enhances lysosomal stability to CuDp44mT, while the cholesterol-depleting agent, MCD, increases lysosomal sensitivity to this agent. 18

Journal Pre-proof 3.5 Incubation of Cells with High Glucose Levels Ablates Dp44mT-Mediated LMP As AMPK silencing decreased the ability of Cu-Dp44mT to induce LMP (Figs. 4, 5), potentially via inhibition of cholesterol synthesis, studies then examined other protocols that increase cellular cholesterol levels. As such, it is well known that prolonged incubation of cells in culture with high concentrations of glucose increases cholesterol synthesis [53-55]. In these studies, PANC-1 cells were cultured in media containing standard (25 mM) or high (50 mM) glucose concentrations for 2

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weeks/37°C.

Initial studies were performed to examine if PANC-1 cells cultured with high glucose medium resulted

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in increased cholesterol levels relative to standard control medium. There was a significant (p < 0.05)

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increase in cholesterol levels in cells incubated under high glucose conditions compared to cells

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incubated under standard control glucose levels (Fig. 6A). Next, to assess the effect of elevated cholesterol levels on Dp44mT-mediated LMP, both standard control and high glucose cultured cells

na

were then incubated with Cu-Dp44mT (2.5 µM) for 2.5 h/37°C and LMP examined using acridine

ur

orange staining (Fig. 6B, C). Incubating cells cultured under standard control glucose conditions (i.e., 25 mM glucose) with Cu-Dp44mT significantly (p < 0.01) decreased acridine orange lysosomal

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staining relative to the control (i.e., 0 µM Cu-Dp44mT; Fig. 6B, C). In contrast, in cells cultured under high glucose condition, there was no apparent Cu-Dp44mT-induced LMP, and in fact, there was a significantly (p < 0.001) increased acridine orange staining after incubation with Cu-Dp44mT compared to control conditions. Thus, pre-incubation of cells with high glucose (50 mM) inhibited CuDp44mT-mediated LMP, suggesting higher cholesterol levels (Fig. 6), which resulted in resistance to Cu-Dp44mT-mediated LMP.

3.6 Dp44mT Inhibits Total HMGCR Expression and Decreases Cholesterol Levels 19

Journal Pre-proof Activated AMPK is known to suppress cholesterol synthesis through the phosphorylation of HMGCR at Ser872, which inhibits the activity of HMGCR, and thus, cholesterol biosynthesis [21]. To determine if HMGCR is regulated by Dp44mT and to determine if it is AMPK-dependent, PANC-1 cells were pre-incubated with either NC siRNA or AMPK siRNA for 48 h/37°C prior to a 24 h/37°C incubation in the presence or absence of Dp44mT (5 µM; Fig. 7A, B).

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Examining first the incubation of NC siRNA-treated PANC-1 cells with Dp44mT, there was a

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significant (p < 0.05) increase in p-AMPK relative to the respective control (Fig. 7A, Bi). In contrast, after AMPK siRNA treatment of cells, a significant (p < 0.001-0.01) decrease in p-AMPK levels were

-p

observed in cells treated with the control or Dp44mT relative to the NC siRNA-treated control (Fig.

re

7A, Bi). Assessing total AMPK levels in the NC siRNA-treated cells, Dp44mT resulted in a significant

lP

(p < 0.01) decrease in total AMPK relative to the respective control (Fig. 7A, Bii), as shown previously [9]. Silencing AMPK resulted in a significant (p < 0.001-0.01) decrease in total AMPK levels in the

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absence and particularly the presence of Dp44mT, relative to the NC siRNA control (Fig. 7A, Bii).

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As a result of the increase in p-AMPK and decrease in total AMPK after incubation with Dp44mT, the p-AMPK/AMPK ratio increased significantly (p < 0.05) relative to the control after NC siRNA (Fig. 7A, Biii). A more pronounced and significant (p < 0.05) increase in the p-AMPK/AMPK ratio was observed after incubation with Dp44mT in AMPK siRNA-treated cells versus the respective NC siRNA control (Fig. 7A, Biii). However, there was no significant difference in the Dp44mT-induced pAMPK/AMPK ratio between the NC- or AMPK-siRNA treated cells (Fig. 7A, Biii). In summary, AMPK silencing significantly decreased the Dp44mT-induced p-AMPK levels, probably due to the decrease in total AMPK.

20

Journal Pre-proof Examining p-HMGCR and HMGCR, 2 bands were observed at 80- and 97-kDa (Fig. 7A) [56], which are consistent with isoforms of the protein previously reported by others [57]. Of note, AMPK siRNA had no significant effect on total HMGCR under control conditions relative to the respective control incubated with NC siRNA. However, there was a significant (p < 0.01) decrease in the levels of total HMGCR after incubation of cells with Dp44mT in both NC siRNA and AMPK siRNA-treated cells (Fig. 7A, Biv). Incubation of PANC-1 cells with Dp44mT (5 µM) had little effect (p > 0.05) on p-

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HMGCR levels in both NC siRNA- and AMPK siRNA-treated cells relative to their respective controls (Fig. 7A, Bv). This was especially notable considering the activation of AMPK after Dp44mT

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particularly in NC siRNA-treated cells, and the increase in the p-AMPK/AMPK ratio after incubation

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with Dp44mT. Hence, Dp44mT regulated HMGCR by decreasing its overall expression, but it did not

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alter HMGCR phosphorylation at Ser872.

Due to the decrease in total HMGCR expression after Dp44mT treatment, there was a significant (p <

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0.01-0.05) increase in the p-HMGCR/HMGCR ratio in cells incubated with NC siRNA or AMPK

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siRNA and treated with Dp44mT compared to the respective NC siRNA control (Fig. 7A, Bvi). After Dp44mT treatment, there was a slight, but significant (p < 0.05) decrease in the p-HMGCR/HMGCR

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ratio in AMPK-siRNA treated cells, relative to the corresponding cells treated with Dp44mT and incubated with NC siRNA (Fig. 7A, Bvi). However, examining the fold increase in the pHMGCR/HMGCR ratio after Dp44mT versus the control, there was no significant difference observed (i.e., 2.20 ± 0.79 (3) and 2.93 ± 1.21 (3)-fold) under either NC siRNA or AMPK siRNA-treated conditions, respectively (Fig. 7A, Bvi). These studies indicate that despite the increase in p-AMPK and the p-AMPK/AMPK ratio after Dp44mT treatment, there was little effect on the p-HMGCR/HMGCR ratio under NC siRNA or AMPK siRNA-treated conditions.

21

Journal Pre-proof Considering the above results, studies were then conducted to examine cholesterol levels under the incubation conditions described above in Figure 7A, B. There was a slight, but significant (p < 0.01) suppression in cholesterol levels in NC siRNA cells treated with Dp44mT compared to control cells (Fig. 7C). This observation may be explained by the marked decrease in total HMGCR expression after Dp44mT that resulted in an increase in the p-HMGCR/HMGCR ratio (Fig. 7Bvi), which is inhibitory in terms of cholesterol synthesis [21]. Silencing AMPK resulted in a slight, but significant (p < 0.05)

of

increase in cholesterol levels in the control relative to corresponding control cells incubated with NC siRNA (Fig. 7C). In AMPK silenced cells, treatment with Dp44mT led to a slight, but not significant (p

-p

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> 0.05) decrease in cholesterol levels compared to corresponding control cells (Fig. 7C).

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In conclusion, Dp44mT decreased total HMGCR expression without affecting HMGCR

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phosphorylation, leading to a similar increase in the p-HMGCR/HMGCR ratio in both NC siRNA- and AMPK siRNA-treated cells. The decrease in total HMGCR expression after Dp44mT treatment could

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have decreased the cholesterol levels in NC siRNA-treated cells, while in AMPK silenced cells, it was

ur

unable to significantly decrease cholesterol levels (Fig. 7C). Considering these observations, AMPK silencing slightly increased cholesterol levels (Fig. 7C) by a mechanism that appears independent of

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the effect of AMPK on HMGCR. The increased cholesterol levels in PANC-1 cells after AMPK silencing may explain their increased resistance to Dp44mT-induced LMP (Figs. 4, 5).

3.7 Dp44mT Activates Autophagic Initiation in AMPK-Silenced PANC-1 Cancer Cells The studies above demonstrated that under control conditions, Dp44mT treatment induced: (1) activation of the AMPK pathway; (2) down-regulation of total HMGCR expression; and (3) a decrease in total cellular cholesterol, which renders lysosomes susceptible to LMP by this agent [43]. Lysosomes are critically involved in autophagy [58], which is an important cell survival pathway [35]. Moreover, 22

Journal Pre-proof AMPK is also a direct activator of autophagic initiation via its ability to phosphorylate ULK1 [36]. We have previously demonstrated that Dp44mT can initiate the autophagic pathway in PANC-1 cancer cells [59], but it can also lead to LMP which could inhibit autophagy [8]. Hence, studies were initiated to examine the role of AMPK in Dp44mT-mediated autophagic initiation (Fig. 8A).

As autophagy is a dynamic process that involves both formation and degradation of autophagosomes

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[26-28, 32-34], studies were performed in the presence or absence of the late-stage autophagic

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inhibitor, Bafilomycin A1 (Baf A1) [60]. The level of autophagosomes is an indicator of autophagic initiation, and this was assessed by examining the expression of the classical marker, microtubule-

-p

associated protein 1 light chain 3 (LC3-II), which is present on the autophagosomal membrane

re

throughout its life [61]. Initially, PANC-1 cells were incubated with either NC siRNA or AMPK siRNA

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for 48 h/37°C, followed by incubation with Dp44mT (2.5 µM), Baf A1 (100 nM), or both, for 24 h/37°C (Fig. 8A, Bi-iii). These conditions with Baf A1 have been demonstrated by our laboratory to

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1 cells [59].

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inhibit lysosome-mediated autophagosome degradation in the same cell-type used here, namely PANC-

First, assessment of AMPK levels in NC siRNA-treated cells in the absence of Baf A1 demonstrated that Dp44mT (p < 0.001) decreased total AMPK relative to the respective control (Fig. 8A, Bi). Incubation of cells with AMPK siRNA significantly (p < 0.001) decreased AMPK levels after incubation with either the control or Dp44mT relative to the NC siRNA treated control. Comparable results were demonstrated in the presence of Baf A1 under all conditions (Fig. 8A, Bi).

Next, examining LC3-I levels in the absence of Baf A1 in cells incubated with NC siRNA or AMPK siRNA, there was no significant change in LC3-I levels comparing the control or Dp44mT-treated 23

Journal Pre-proof groups (Fig. 8A, Bii). In contrast, examining LC3-II levels in cells without Baf A1 and incubated with NC siRNA or AMPK siRNA, there was a significant (p < 0.001-0.01) increase in LC3-II levels after Dp44mT-treatment relative to the respective control (Fig. 8A, Biii). Of note, there was a decreased effect of Dp44mT on up-regulating LC3-II after AMPK silencing relative to that observed after NC siRNA treatment (Fig. 8A, Biii). This observation is in good agreement with the known ability to

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AMPK to induce autophagy [36].

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Assessing LC3-I levels in presence of Baf A1, after NC siRNA treatment, its levels remained low and comparable to those observed in the absence of Baf A1 either with or without Dp44mT (Fig. 8A, Bii).

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In contrast, after AMPK silencing, there was a significant (p < 0.01-0.05) increase in LC3-I in the

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presence of Baf A1 with or without Dp44mT, relative to the respective NC siRNA treatments with Baf

lP

A1 (Fig. 8A, Bii). This latter finding indicates that after AMPK silencing, there is inhibition in the

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conversion of LC3-I to LC3-II, leading to the increased LC3-I levels.

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In the presence of Baf A1, LC3-II levels in NC siRNA-treated control cells were increased significantly

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(p < 0.001) relative to the respective control cells incubated with NC siRNA without Baf A1 (Fig. 8A, Biii). Upon incubation with Dp44mT and Baf A1 in NC siRNA-treated cells, there was a significant (p < 0.05) increase in LC3-II levels relative to the respective control (Fig. 8A, Biii). Furthermore, examining the effect of Dp44mT in the presence of Baf A1 after AMPK silencing, again, there was a significant (p < 0.05) increase in LC3-II levels in cells relative to the corresponding control (Fig. 8A, Biii). These latter results demonstrate that Dp44mT can still activate autophagic initiation in the absence of the important autophagic regulator, AMPK.

24

Journal Pre-proof Overall, Figure 8A, B demonstrates Dp44mT can activate autophagic initiation in the absence of AMPK. However, the rate of autophagic initiation is slightly retarded in the case of AMPK silenced cells, as indicated by the presence of higher levels of LC3-II precursor, LC3-I, in the presence of Baf A1.

3.8 Dp44mT Can Still Increase Autophagic Initiation after AMPK Silencing via an AMPK and

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Beclin-1 Independent Mechanism

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The results above in Figure 8A, B demonstrated that Dp44mT could still induce autophagic initiation after silencing of the autophagy regulatory gene, AMPK. Considering this, it was hypothesized that up-

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regulation of another important autophagic regulator, namely Beclin-1 [62], could be responsible for

re

the autophagic initiation observed. To examine this, cells were again treated with NC siRNA or AMPK

lP

siRNA and incubated in the absence or presence of Dp44mT (2.5 µM) for 24 h/37°C and Beclin-1

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expression assessed by western blot analysis (Fig. 8C, Di,ii).

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In NC siRNA-treated cells, Dp44mT significantly (p < 0.001) decreased Beclin-1 relative to the control

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(Fig. 8C, Dii), as demonstrated by our laboratory previously in another cell-type [8]. On the other hand, there was a significant (p < 0.001) increase in Beclin-1 levels in control cells incubated with AMPK siRNA compared to control cells incubated with NC siRNA (Fig. 8C, Dii). Thus, these results indicate a compensatory increase in another autophagic regulator (i.e., Beclin-1) in cells upon AMPK silencing. Upon incubation of AMPK siRNA-treated cells with Dp44mT, there was a significant (p < 0.05) decrease in Beclin-1 levels relative to the respective control (Fig. 8C, Dii). However, the level of Beclin-1 after incubation of AMPK siRNA-treated cells with Dp44mT was not significantly (p > 0.05) greater than NC siRNA control cells. As such, although Beclin-1 levels were increased after AMPK

25

Journal Pre-proof silencing under control conditions, the Dp44mT-mediated increase in autophagic initiation after silencing AMPK was not due to Beclin-1 up-regulation (Fig. 8C, Di,ii).

Taken together, Dp44mT was able to initiate autophagy in cancer cells, independent of AMPK and Beclin-1 regulation. However, as Dp44mT results in LMP [43], this will probably result in

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dysfunctional autophagy.

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3.9 AMPK Silencing Increases Susceptibility of PANC-1 Cells to Dp44mT-Mediated Apoptosis. Studies above demonstrated that incubation of cells with Dp44mT leads to the activation of AMPK

-p

(Figs. 1, 2) and a decrease in the total levels HMGCR (Fig. 7A,B) that could lead to the decreased

re

cholesterol levels (Fig. 7C), which facilitates LMP by Dp44mT [43]. Silencing of AMPK does not

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affect the ability of Dp44mT to initiate autophagy, as demonstrated by increased levels of LC3-II in the presence of Dp44mT and the autophagic inhibitor, Baf A1 (Fig. 8A, Biii). However, AMPK silencing

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also increased the susceptibility of cells towards Dp44mT anti-proliferative activity (Fig. 3). There is

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well-established cross-talk between autophagy and the apoptotic pathway, and Beclin-1 is known to

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play a pivotal role in this process [37, 63]. As Beclin-1 is increased under AMPK silenced conditions, it could lead to an autophagy-apoptosis switch under these conditions. Collectively, these results and previous literature indicate that Dp44mT could potentially increases apoptosis after AMPK silencing. Hence, studies were performed to examine the effect of AMPK on apoptotic cell death pathways.

PANC-1 cells were transfected with either NC siRNA or AMPK siRNA and pre-incubated for 48 h/37°C and then incubated in the presence and absence of Dp44mT (2.5 µM) for 24 h/37°C (Fig. 9A, Bi-iii). The samples were then assessed for two well-established apoptotic markers, i.e., cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) [64, 65], as well as their total levels. 26

Journal Pre-proof Incubation of NC siRNA-treated cells with Dp44mT resulted in a slight, but significant (p < 0.05) increase in cleaved caspase 3 relative to the respective control (Fig. 9A, Bii). This slight effect of Dp44mT was not surprising, especially considering the short period of incubation (24 h) and low concentration used (2.5 µM), which does not result in marked anti-proliferative activity (Fig. 3Ai). Notably, AMPK silencing in the presence or absence of Dp44mT led to a marked and significant (p < 0.001-0.05) increase in the levels of a single cleaved caspase 3 band at 17-kDa relative to the respective

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NC siRNA conditions (Fig. 9A, Bii). One major band of total caspase 3 at 17-kDa, and two minor bands at 21- and 32-kDa, were observed under all treatment conditions and were not significantly

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changed relative to the control (Fig. 9A, Biii).

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Examining cleaved PARP levels, again Dp44mT induced no effect in cells incubated with NC siRNA

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relative to the control (Fig. 9A, Biv). This observation is probably due to the short incubation and low concentration of Dp44mT used that results in a minimal cytotoxicity, as demonstrated previously [6,

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50, 59]. However, there was a marked and significant (p < 0.001-0.01) increase of cleaved PARP in

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cells incubated with AMPK siRNA after control and Dp44mT treatment (Fig. 9A, Biv). Furthermore, Dp44mT significantly (p < 0.05) enhanced cleaved PARP relative to the control after AMPK silencing

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(Fig. 9A, Biv). In contrast, total PARP levels remained relatively constant under all incubation conditions (Fig. 9A, Bv).

Overall, these results in Figure 9 indicated an increase in apoptotic marker expression in AMPK silenced cells, which was exacerbated by Dp44mT.

27

Journal Pre-proof 4. Discussion In this investigation, AMPK was demonstrated to play a protective role in the cellular response to the potent anti-tumor agent, Dp44mT [5, 20, 50], with AMPK silencing resulting in decreased proliferation and enhanced expression of markers of apoptosis and autophagy. Several studies have demonstrated that AMPK plays a role in tumor progression. For example, the AMPK agonist, A769662, was shown to promote tumor progression [66]. On the other hand, where AMPK agonists have been demonstrated

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to induce a tumor suppressive effect, this could be AMPK-independent, with AMPK activation occurring as an adaptive response to protect cells from the cytotoxicity of the agent [66-68]. The latter

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suggestion could equate to the effects observed herein for Dp44mT, which activated the AMPK

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pathway, while the anti-proliferative efficacy of Dp44mT was amplified upon AMPK silencing.

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Key to the anti-tumor activity of Dp44mT is its ability to alter autophagy and lysosomal metabolism [8, 59]. Dp44mT has a dual effect on autophagy, namely: (1) it increases the autophagic flux, resulting in

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enhanced generation of autophagosomes [8, 59]; and (2) Dp44mT upon entering the cell, binds copper

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to form the Dp44mT-Cu complex, which is markedly redox active, leading to permeabilization of the lysosomal membrane [8, 10, 11]. The latter effect prevents the fusion of the autophagosome and the

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lysosome and inhibits autophagic completion.

In the current studies, AMPK silencing resulted in decreased LMP after incubation of cells with Dp44mT, suggesting that the lysosomes were more stable under these conditions. It has been well characterized that LMP plays a significant role in the cytotoxic activity of Dp44mT [10, 43]. The decreased LMP observed after AMPK silencing was potentially through the increased cholesterol levels observed under these conditions (Fig. 7C). It is well known that cholesterol stabilizes lysosomal membranes [22] and treatment of cells with the cholesterol transport inhibitor, 3-β-[2-(diethyl28

Journal Pre-proof amino)ethoxy]androst-5-en-17-one (U18666A), has been demonstrated to increase cholesterol levels and prevent Dp44mT-mediated LMP [43]. The role of cholesterol in stabilizing lysosomes against the activity of Dp44mT after AMPK silencing was further confirmed by examining the effect of Dp44mTmediated LMP in cells incubated with high glucose, resulting in increased cholesterol levels and inhibition of LMP. This finding was further corroborated using the well-characterized, cholesterol-

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depleting agent, MCD [24], which restored the ability of Dp44mT to initiate LMP.

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Considering the mechanism of how AMPK silencing increased cholesterol levels, it is well known that activated AMPK suppresses cholesterol synthesis through an inhibitory phosphorylation of HMGCR at

-p

Ser872, which inhibits HMGCR that catalyzes the rate-limiting step of cholesterol synthesis [21]. Of

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note, Dp44mT had no significant effect on p-HMGCR levels in both NC siRNA- and AMPK siRNA-

lP

treated cells, even despite its ability to activate AMPK and significantly increase the p-AMPK/AMPK ratio. However, Dp44mT decreased overall protein expression of HMGCR, resulting in a similar

na

increase in the p-HMGCR/HMGCR ratio after treatment with either NC siRNA or AMPK siRNA. As

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such, Dp44mT could potentiate LMP via inhibition of cholesterol synthesis mediated by the decrease in

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HMCGR levels and increase in the p-HMGCR/HMGCR ratio, which is inhibitory in terms of cholesterol synthesis in cells treated with NC siRNA. However, another mechanism independent of the inhibitory effect of AMPK activation on HMGCR, was responsible for the increased cholesterol levels after AMPK silencing. Considering this, other AMPK-mediated mechanisms could be responsible for this effect. For instance, AMPK activation is known to stimulate cholesterol efflux via ABCG1 [69], and thus, silencing AMPK could possibly increase cellular cholesterol by this mechanism.

Silencing AMPK increased the anti-proliferative activity of Dp44mT, while also inhibiting LMP. These data suggest that Dp44mT possesses anti-tumor activity that occurs, at least in part, via an LMP29

Journal Pre-proof independent mechanism. This is consistent with the fact that Dp44mT possesses multiple mechanisms of inhibiting proliferation by effecting a variety of molecular pathways [15]. In fact, Dp44mT inhibits oncogenic signalling and the cell cycle and up-regulates metastasis and tumor suppressor proteins, like other metal ion chelators [15, 70-78]. AMPK is a catabolic enzyme that provides the cell with an alternate energy source under conditions of metabolic stress [2]. Therefore, silencing AMPK would result in cells losing this metabolic plasticity and becoming more susceptible to metabolic stress and

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cell death.

AMPK plays a crucial role in the activation of autophagy [36, 79] and regulates the key initiator of

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autophagy, ULK1 [36]. In a previous investigation from our laboratories, it was demonstrated that

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incubation of cells with Dp44mT can induce ULK1 activation in an AMPK-dependent manner,

lP

suggesting up-regulation of autophagy via the AMPK pathway [9]. Studies utilizing the vesicular acidification inhibitor, Baf A1, have demonstrated that Dp44mT increases autophagic flux, while also

na

reducing the degradation of autophagosomes [8]. In the current investigation, Dp44mT was

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demonstrated to be capable of initiating autophagy after silencing AMPK, although the rate of

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autophagic initiation was slightly hindered.

Autophagy is a process through which cells recycle cytoplasmic contents, including proteins and organelles via the lysosome during cellular stress [26-28]. Due to this, autophagy can act as a protective mechanism for cells under stress conditions [26-28]. Studies have demonstrated that targeting genes or proteins involved in the regulation of autophagy either through genetic silencing or chemical inhibitors, can at least in some cases, result in the tumor cells becoming responsive to anti-cancer therapies [8083]. The increased anti-proliferative and apoptotic activity of Dp44mT observed herein in absence of AMPK can be exploited therapeutically. For instance, AMPK inhibitors could potentially be used in 30

Journal Pre-proof combination with Dp44mT to enhance its anti-cancer efficacy. Currently, while several agents are known to inhibit AMPK, such as the poorly selective compound C, and more recently, the more potent pyrimidine derivative, SBI-0206965 [84], future research in this area will be a promising avenue to develop novel anti-cancer therapeutics.

Previous studies have also demonstrated that AMPK activation leads to the up-regulation of p53, and

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p53-dependent apoptosis [3, 85]. Bhutia et. al, demonstrated that proteins associated with autophagy,

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such as ATG5 and Beclin-1, can act as a switch between protective autophagy and apoptosis [86]. In this investigation, increased Beclin-1 expression was observed after AMPK silencing via a mechanism

-p

which remains unclear. Moreover, increased levels of the apoptotic markers, cleaved caspase 3 and

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cleaved PARP, were demonstrated in AMPK silenced cells in the presence and absence of Dp44mT.

lP

This latter finding suggests at some stage in the cellular response process to Dp44mT there was a shift from autophagy to apoptosis. Such a switch has also been observed for other cytotoxic agents, such as

5. Conclusions

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pathway [87, 88].

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cadmium or tamoxifen, with tamoxifen possibly initiating this switch through the AMPK/mTOR

Collectively, Dp44mT activates the AMPK pathway, which results in an increased pHMGCR/HMGCR ratio due to a decrease in HMGCR protein levels that was inhibitory to cholesterol synthesis that stabilizes lysosomal membranes. The decrease in cholesterol observed after treatment with Dp44mT could potentiate LMP and tumor cell death. Nonetheless, silencing AMPK resulted in increased anti-proliferative activity of Dp44mT, suggesting AMPK mediates a protective effect against the activity of this anti-tumor agent, potentially through AMPK’s ability to mediate metabolic plasticity. In fact, AMPK silencing resulted in increased expression of markers of autophagy and 31

Journal Pre-proof apoptosis and decreased proliferation in the presence of Dp44mT. These findings suggest that through its metabolic effects, AMPK suppresses autophagy and apoptosis, which inhibits Dp44mT cytotoxicity.

Acknowledgments This project was supported by a Project Grant from the National Health and Medical Research Council

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of Australia (NHMRC) [1060482] to D.R.R. Additionally, D.R.R. appreciates NHMRC Senior Principal Research Fellowship support [1062607 and 1159596]. P.J.J. is grateful for a Cancer Institute

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New South Wales (CINSW) Career Development Fellowship [CDF171147]. S.S. would also like to

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thank Cancer Australia and Cure Cancer Australia for a Young Investigator PdCCRs grant [1125107]

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and AMP Foundation for the AMP Tomorrow Grant. S.S. would also like to thank Mr Guy Boncardo

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for the Boncardo Pancreatic Cancer Fellowship.

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Journal Pre-proof Figure Legends Figure 1. (A, B) Line drawings of Dp44mT and the Cu-Dp44mT complex (i.e., [Cu(Dp44mT)Cu]). (C, D) Dp44mT activates AMPK in PANC-1 pancreatic cancer cells after incubations of 24-, 48- and 72-h/37°C. (C) PANC-1 cells were incubated for 24-72 h/37oC with control medium only, or this medium containing Dp44mT (1.25- or 2.5-µM). Western blotting was then performed to investigate p-AMPK and AMPK levels. (D)

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Densitometric analysis (arbitrary units) of the results in (C). Results are shown as mean ± SEM (3 experiments): *p < 0.05, **p < 0.01; ***p < 0.001 compared to the respective

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control cells (i.e., 0 µM) at each time point.

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Figure 2. Dp44mT activates AMPK in AsPC-1 and MIAPaCa-2 pancreatic cancer cells

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after incubations of 24-, 48- and 72-h. (A) AsPC-1 cells were incubated for 24, 48- or 72h/37oC with control medium only, or this medium containing Dp44mT (2.5 µM). Western

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blotting was then performed to investigate p-AMPK and total AMPK levels. (B) Densitometric analysis (arbitrary units) of (i) p-AMPK, (ii) total AMPK, and (iii) the p-

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AMPK/AMPK ratio in AsPC-1 cells. (C) MIAPaCa-2 cells were incubated for 24-, 48- or 72-

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h/37oC with control medium only, or this medium containing Dp44mT (2.5 µM). Western blotting was then performed to investigate p-AMPK and total AMPK levels. (D) Densitometric analysis (arbitrary units) of: (i) p-AMPK, (ii) total AMPK, and (iii) the pAMPK/AMPK ratio in MIAPaCa-2 cells. Results are shown as mean ± SEM (3 experiments): *p < 0.05, **p < 0.01; ***p < 0.001 compared to respective control cells. #p < 0.05; ###p < 0.001 compared to respective treatment at 24 h.

Figure 3. AMPK increases the anti-proliferative activity of Dp44mT after 24 h in PANC-1, AsPC-1 and MIAPaCa-2 cells. (A) PANC-1 cells were pre-incubated for 48 44

Journal Pre-proof h/37ºC with either a control siRNA, or a selective siRNA against AMPK. Cells were then subsequently incubated with control medium only, or this medium containing Dp44mT (0-20 µM) for: (i) 24 h/37ºC or (ii) 72 h/37ºC. Cellular proliferation was then examined using MTT analysis. (B) AsPC-1 cells were pre-incubated for 48 h/37ºC with either a control siRNA, or a selective AMPK siRNA. Cells were then subsequently incubated with Dp44mT (0-20 µM) for: (i) 24 h/37ºC, or (ii) 48 h/37ºC. Cellular proliferation was then assessed using MTT analysis. (C) MIAPaCa-2 cells were pre-incubated for 72 h/37ºC with either control siRNA,

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or a selective siRNA against AMPK. Cells were then subsequently incubated with Dp44mT (0-20 µM) for (i) 24 h/37ºC or (ii) 72 h/37ºC. Cellular proliferation was assessed using MTT

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analysis. Results are shown as mean ± SEM (3 experiments). *p < 0.05, **p < 0.01; ***p <

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0.001 compared to the respective control cells (i.e., 0 µM Dp44mT); #p < 0.05, ##p < 0.01

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compared to the NC siRNA at the same respective Dp44mT concentration.

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Figure 4. AMPK silencing leads to decreased susceptibility of PANC-1 cells towards Dp44mT-induced LMP. (A) PANC-1 cells were pre-incubated for 72 h/37ºC with either NC

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siRNA, or a selective siRNA against AMPK. Cells were then subsequently incubated with

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Cu-Dp44mT (2.5 µM) for 0-2.5 h h/37ºC. Acridine orange staining was then performed. (B) Red/green acridine orange fluorescence ratio from the cells in (A) above. An average of 5 cells/ image/condition using 3 replicates/experiment, repeated 3 times (approximately 45 cells in total) were used for quantification. Scale bar: 10 μm. Immunofluorescence photographs are representative from 3 experiments. Results are shown as mean ± SEM (3 experiments): *p < 0.05 compared to the relative NC siRNA-treated cells after a 2.5 h/37ºC incubation.

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Journal Pre-proof Figure 5. The cholesterol-depleting agent, methyl- cyclodextrin (MCD), re-sensitizes AMPK silenced PANC-1 cancer cells towards Dp44mT-mediated LMP. (A) PANC-1 cells were pre-incubated for 72 h/37 ºC with either NC siRNA, or a selective siRNA against AMPK. Cells were then subsequently preincubated for 1 h/37 ºC in the presence or absence of MCD (10 mM) and then incubated with either Cu-Dp44mT (2.5 µM), Cu-Dp44mT (2.5 µM) + MCD (10 mM), or MCD (5 mM) for 2.5 h h/37 ºC. Acridine orange staining was

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then performed. (B) Red/green acridine orange fluorescence ratio from the cells in (A) above. Scale bar: 10 μm. An average of 5 cells/ image/condition using 3 replicates/experiment, repeated 3 times (approximately 45 cells in total) were used for quantification.

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Immunofluorescence photographs are representative from 3 experiments. Results are shown

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as mean ± SEM (3 experiments): *p < 0.05 or **p < 0.01 compared to the respective control

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cells. # p < 0.05 comparison of Cu-Dp44mT to Cu-Dp44mT + MCD.

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Figure 6. High glucose increases cellular cholesterol levels and decreases the susceptibility of PANC-1 cancer cells towards Dp44mT-mediated LMP. (A) PANC-1

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cells were cultured in either standard control glucose (25 mM) media, or high glucose (50

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mM) media for 2 weeks, followed by the determination of cellular cholesterol levels. (B) PANC-1 cells were cultured in either standard control glucose (25 mM) media, or high glucose (50 mM) media for 2 weeks. Cells were then subsequently incubated with CuDp44mT (2.5 µM) for 2.5 h h/37ºC. Acridine orange staining was then performed. (C) Red/green acridine orange fluorescence ratio from the cells in (B) above. Scale bar: 10 μm. An average of 5 cells/image/condition using 3 replicates/experiment, repeated 3 times (approximately 45 cells in total) were used for quantification. Immunofluorescence photographs are representative from 3 experiments. Results are shown as mean ± SEM (3 experiments): *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the respective control 46

Journal Pre-proof cells. ##p < 0.01 compared to the corresponding Cu-Dp44mT treatment under standard control glucose (25 mM) conditions as indicated.

Figure 7. Dp44mT inhibits cholesterol levels via its effect on increasing the inhibitory pHMGCR/HMGCR ratio in PANC-1 cells by decreasing total HMGCR protein levels. (A) PANC-1 cells were pre-incubated for 48 h/37ºC with either the NC siRNA, or a selective siRNA against AMPK. Cells were then subsequently incubated with Dp44mT (5 µM) for 24

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h/37ºC. Western blot analysis was then performed to study the effect on the levels of AMPK, p-AMPK (Thr172), p-HMGCR (Ser872), or total HMGCR. (B) Densitometric analysis

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(arbitrary units) of the results in (A). (C) PANC-1 cells were pre-incubated for 48 h/37ºC

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with either the NC siRNA, or a selective siRNA against AMPK. Cells were then subsequently incubated with Dp44mT (5 µM) for 24 h/37ºC, followed by the determination of cellular

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cholesterol levels. Results are shown as mean ± SEM (3 experiments): *p < 0.05, **p < 0.01,

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***p < 0.001 compared to the NC siRNA control. #p < 0.05, compared to the corresponding

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treatment in NC siRNA-treated cells as indicated.

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Figure 8. Dp44mT can increase autophagic initiation in PANC-1 cells, which is independent of AMPK and Beclin-1 expression. (A) PANC-1 cells were pre-incubated for 48 h/37ºC with either NC siRNA, or AMPK siRNA. Cells were then subsequently incubated with Baf A1 (100 nM) for 30 min/37ºC followed by Dp44mT (2.5 µM) for 24 h/37ºC. Western blot analysis was then performed to study the effect on the levels of LC3-I, LC3-II, or AMPK. (B) Densitometric analysis (arbitrary units) of the results in (A). (C) PANC-1 cells were pre-incubated for 72 h/37ºC with either NC siRNA or AMPK siRNA. Cells were then subsequently incubated with Dp44mT (2.5 µM) for 24 h/37ºC. Western blot analysis was then performed to study the effect on AMPK or Beclin-1 levels. (D) Densitometric analysis

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Journal Pre-proof (arbitrary units) of the results in (C). Results are shown as mean ± SEM (3 experiments *p < 0.05, **p < 0.01; ***p < 0.001 compared to relative NC siRNA control cells. #p < 0.05, ##p < 0.01; ###p < 0.001 compared to the respective treatment in cells without Baf A1. †p < 0.05; ††

p < 0.01; ††† p < 0.001 compared to the respective control as shown; n.s., p > 0.05 compared

to the control NC siRNA cells, as shown.

Figure 9. AMPK silencing increases the expression of Dp44mT-mediated apoptosis in

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PANC-1 cells. (A) PANC-1 cells were incubated with NC siRNA or AMPK siRNA for 48 h/37ºC followed by incubation for 24 h/37ºC with control medium only, or this medium

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containing Dp44mT (0-10 µM). Western blotting was then performed to investigate caspase

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3, cleaved caspase 3, PARP, cleaved PARP, or AMPK. (B) Densitometric analysis (arbitrary units) of the results in (A). Results are shown as mean ± SEM (3 experiments): *p < 0.05,

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**p < 0.01; ***p < 0.001 compared to NC siRNA control cells. #p < 0.05 comparing control

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and Dp44mT upon AMPK siRNA treatment as indicated.

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Figure 10. Schematic overview of the role of AMPK in the Dp44mT-mediated effects on

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lysosomal membrane permeabilization (LMP), autophagy and apoptosis in: (A) the absence of AMPK silencing and (B) presence of AMPK silencing. (A) The chelator Dp44mT, in the presence of AMPK, initiates LMP through the inhibition of cholesterol synthesis due to down-regulation of HMGCR expression. In fact, cholesterol is known to stabilize membranes [22], while decreased cholesterol levels sensitize tumor cells to LMP by Dp44mT. Dp44mT is also known to initiate autophagy via AMPK [4]. However, increased LMP prevents the fusion of the lysosome and the autophagosome, resulting in dysfunctional autophagy [8]. (B) In AMPK silenced cells, Dp44mT did not initiate LMP, and there was stabilization of lysosomes in the cell. However, silencing of AMPK increased the anti-

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Journal Pre-proof proliferative activity of Dp44mT potentially due to a loss of metabolic plasticity. The expression of markers of autophagy and apoptosis was also enhanced by AMPK silencing in

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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Highlights The anti-cancer agent, Dp44mT, increases the cellular p-AMPK/AMPK ratio. Dp44mT was more effective in inhibiting tumor cell proliferation after AMPK silencing. Dp44mT increases autophagic initiation after AMPK silencing. AMPK rescues Dp44mT-mediated cytotoxicity by inhibiting autophagy and apoptosis.

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