7
Short-chain C6 ceramide sensitizes AT406-induced anti-pancreatic cancer cell activity Xiaoguang Zhao a, * , Baoyou Sun b , Jingjing Zhang c , Ruishen Zhang d , Qing Zhang e a Department of Neurosurgery, CPLA Bethune International Peace Hospital, Shijiazhuang, Hebei, China b Department of General Surgery, Shandong Provincial Hospital Afliated to Shandong University, Jinan, China c Department of Thoracic Surgery III, The Chest Hospital of Hebei Province, Shijiazhuang, Hebei, China d Hospital of China Railway Electric Bureau Group First Engineering Company, Shijiazhuang, Hebei, China e Department of Neurology, The Chest Hospital of Hebei Province, Shijiazhuang, Hebei, China article info Article history: Received 16 August 2016 Accepted 20 August 2016 Available online 22 August 2016 Keywords: Pancreatic cancer IAPs AT406 C6 ceramide Bcl-2 abstract Our previous study has shown that AT406, a rst-in-class small molecular antagonist of IAPs (inhibitor of apoptosis proteins), inhibits pancreatic cancer cell proliferation in vitro and in vivo. The aim of this research is to increase AT406's sensitivity by adding short-chain C6 ceramide. We show that co- treatment of C6 ceramide dramatically potentiated AT406-induced caspase/apoptosis activation and cytotoxicity in established (Panc-1 and Mia-PaCa-2 lines) and primary human pancreatic cancer cells. Reversely, caspase inhibitors largely attenuated C6 ceramide plus AT406-induced above cancer cell death. Molecularly, C6 ceramide downregulated Bcl-2 to increase AT406's sensitivity in pancreatic cancer cells. Intriguingly, C6 ceramide-mediated AT406 sensitization was nullied with Bcl-2 shRNA knockdown or pretreatment of the Bcl-2 inhibitor ABT-737. In vivo, liposomal C6 ceramide plus AT406 co- administration dramatically inhibited Panc-1 xenograft tumor growth in severe combined immunode- cient (SCID) mice. The combined anti-tumor activity was signicantly more potent than either single treatment. Expressions of IAPs (cIAP1/XIAP) and Bcl-2 were downregulated in Panc-1 xenografts with the co-administration. Together, we demonstrate that C6 ceramide sensitizes AT406-mediated anti- pancreatic cancer cell activity possibly via downregulating Bcl-2. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction The prognosis of pancreatic cancer has been poor, and the long- term survival of the patients is still dismissal [1e3]. Gemcitabine and it-based combination treatments are currently being utilized for chemotherapy of the pancreatic cancer patients [4]. Yet, the response has not been satisfactory [1e3]. The research eld is focusing on developing alternative anti-pancreatic cancer agents [3,5,6]. It has also been our focus for many years [7]. The inhibitor of apoptosis proteins (IAPs), including X-linked IAP (XIAP) and cellular IAP1/2 (cIAP1/cIAP2), are a family of key apoptosis-inhibitory proteins [8]. Of which, XIAP directly inhibits several key pro-apoptotic caspases, including caspase-3 and -7, and -9 [8,9]. cIAP1 and cIAP2 are able of inactivating tumor necrosis factor associated factor 2 (TRAF2) and TNF receptor-1/-2, thus inhibiting caspase-8 [8e10]. Previous studies have shown that IAPs play a pivotal role in apoptosis resistance in multiple cancer cells [8e10]. IAPs are often over-expressed in pancreatic cancers, which are critical for apoptosis inhibition and chemo-resistance [11]. IAPs inhibition, on the other hand, would provoke pancreatic cancer cell apoptosis [7,11]. Recent research efforts have developed a novel, potent and orally bio-available antagonist of IAPs, named AT406 [12]. This rst- in-class small molecular IAP antagonist directly binds to and in- hibits IAP family members, such as XIAP, cIAP1 and cIAP2 [12]. Our recent study has shown that AT406 induced cell death and apoptosis in established and primary human pancreatic cancer cells [7]. In vivo, oral administration of AT406 was found to efciently inhibit Panc-1 pancreatic tumor growth in severe combined immunodecient (SCID) mice [7]. The aim of this current research is to sensitize AT406's anti-cancer activity via supplementing short- chain C6 ceramide. Ceramides, enriched in cell membranes, are a family of lipid * Corresponding author. Department of Neurosurgery, CPLA Bethune Interna- tional Peace Hospital, No. 398 Zhongshan West Road, Shijiazhuang, Hebei, 050082, China. E-mail address: [email protected] (X. Zhao). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc http://dx.doi.org/10.1016/j.bbrc.2016.08.121 0006-291X/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Biochemical and Biophysical Research Communications 479 (2016) 166e172

Biochemical and Biophysical Research Communications · 2017-01-05 · Introduction The prognosis of ... In brief, equal amounts of cytosol protein extracts per sample were resolved

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lable at ScienceDirect

Biochemical and Biophysical Research Communications 479 (2016) 166e172

Contents lists avai

Biochemical and Biophysical Research Communications

journal homepage: www.elsevier .com/locate/ybbrc

Short-chain C6 ceramide sensitizes AT406-induced anti-pancreaticcancer cell activity

Xiaoguang Zhao a, *, Baoyou Sun b, Jingjing Zhang c, Ruishen Zhang d, Qing Zhang e

a Department of Neurosurgery, CPLA Bethune International Peace Hospital, Shijiazhuang, Hebei, Chinab Department of General Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Chinac Department of Thoracic Surgery III, The Chest Hospital of Hebei Province, Shijiazhuang, Hebei, Chinad Hospital of China Railway Electric Bureau Group First Engineering Company, Shijiazhuang, Hebei, Chinae Department of Neurology, The Chest Hospital of Hebei Province, Shijiazhuang, Hebei, China

a r t i c l e i n f o

Article history:Received 16 August 2016Accepted 20 August 2016Available online 22 August 2016

Keywords:Pancreatic cancerIAPsAT406C6 ceramideBcl-2

* Corresponding author. Department of Neurosurtional Peace Hospital, No. 398 Zhongshan West Road,China.

E-mail address: [email protected] (X.

http://dx.doi.org/10.1016/j.bbrc.2016.08.1210006-291X/© 2016 The Authors. Published by Elsevier

a b s t r a c t

Our previous study has shown that AT406, a first-in-class small molecular antagonist of IAPs (inhibitor ofapoptosis proteins), inhibits pancreatic cancer cell proliferation in vitro and in vivo. The aim of thisresearch is to increase AT406's sensitivity by adding short-chain C6 ceramide. We show that co-treatment of C6 ceramide dramatically potentiated AT406-induced caspase/apoptosis activation andcytotoxicity in established (Panc-1 and Mia-PaCa-2 lines) and primary human pancreatic cancer cells.Reversely, caspase inhibitors largely attenuated C6 ceramide plus AT406-induced above cancer celldeath. Molecularly, C6 ceramide downregulated Bcl-2 to increase AT406's sensitivity in pancreatic cancercells. Intriguingly, C6 ceramide-mediated AT406 sensitization was nullified with Bcl-2 shRNA knockdownor pretreatment of the Bcl-2 inhibitor ABT-737. In vivo, liposomal C6 ceramide plus AT406 co-administration dramatically inhibited Panc-1 xenograft tumor growth in severe combined immunode-ficient (SCID) mice. The combined anti-tumor activity was significantly more potent than either singletreatment. Expressions of IAPs (cIAP1/XIAP) and Bcl-2 were downregulated in Panc-1 xenografts with theco-administration. Together, we demonstrate that C6 ceramide sensitizes AT406-mediated anti-pancreatic cancer cell activity possibly via downregulating Bcl-2.© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

The prognosis of pancreatic cancer has been poor, and the long-term survival of the patients is still dismissal [1e3]. Gemcitabineand it-based combination treatments are currently being utilizedfor chemotherapy of the pancreatic cancer patients [4]. Yet, theresponse has not been satisfactory [1e3]. The research field isfocusing on developing alternative anti-pancreatic cancer agents[3,5,6]. It has also been our focus for many years [7].

The inhibitor of apoptosis proteins (IAPs), including X-linked IAP(XIAP) and cellular IAP1/2 (cIAP1/cIAP2), are a family of keyapoptosis-inhibitory proteins [8]. Of which, XIAP directly inhibitsseveral key pro-apoptotic caspases, including caspase-3 and -7, and-9 [8,9]. cIAP1 and cIAP2 are able of inactivating tumor necrosis

gery, CPLA Bethune Interna-Shijiazhuang, Hebei, 050082,

Zhao).

Inc. This is an open access article u

factor associated factor 2 (TRAF2) and TNF receptor-1/-2, thusinhibiting caspase-8 [8e10]. Previous studies have shown that IAPsplay a pivotal role in apoptosis resistance in multiple cancer cells[8e10]. IAPs are often over-expressed in pancreatic cancers, whichare critical for apoptosis inhibition and chemo-resistance [11]. IAPsinhibition, on the other hand, would provoke pancreatic cancer cellapoptosis [7,11].

Recent research efforts have developed a novel, potent andorally bio-available antagonist of IAPs, named AT406 [12]. This first-in-class small molecular IAP antagonist directly binds to and in-hibits IAP family members, such as XIAP, cIAP1 and cIAP2 [12]. Ourrecent study has shown that AT406 induced cell death andapoptosis in established and primary human pancreatic cancer cells[7]. In vivo, oral administration of AT406 was found to efficientlyinhibit Panc-1 pancreatic tumor growth in severe combinedimmunodeficient (SCID) mice [7]. The aim of this current researchis to sensitize AT406's anti-cancer activity via supplementing short-chain C6 ceramide.

Ceramides, enriched in cell membranes, are a family of lipid

nder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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A.

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*C6-0C6-10 μM

C6-0C6-10 μM

Fig. 1. C6 ceramide dramatically potentiates AT406-induced anti-pancreatic cancercell activity in vitro. Panc-1 cells (AeC), Mia-PaCa-2 cells (D), the primary humanpancreatic cancer cells (“Primary Pan Can”, E) or pancreatic epithelial HPDE6c7 cells(F) were treated with applied concentrations of AT406 (“AT”, 10e1000 nM) or plus C6ceramide (“C6”) for designated periods of time, cell survival was tested by CellTiter-Gloluminescent assay (A and D-F) or the clonogenic assay (B); Cell proliferation was testedby BrdU incorporation assay (C). “Ctrl” stands for untreated control cells (Same for allfigures). “Toge” stands for AT406 plus C6 ceramide treatment (Same for all Figures). Allvalues were expressed as mean ± SD (Same for all figures). For each assay, n ¼ 5.Experiments in this figure were repeated three times, and similar results were ob-tained. *p < 0.05 vs. group of “Ctrl”. #p < 0.05 vs. AT406 only treatment.

X. Zhao et al. / Biochemical and Biophysical Research Communications 479 (2016) 166e172 167

signaling molecules which are important in regulating key cellularfunctions, including cell growth, differentiation, apoptosis, andautophagy [13,14]. The short-chain cell permeable ceramides (C2,C4, C6 and C8) have demonstrated promising anti-cancer activity,alone or in combination with conventional anti-cancer agents [14].Existing evidences have shown that short-chain C6 ceramide coulddramatically augment the anti-tumor activity by doxorubicin,paclitaxel (Taxol), histone deacetylase inhibitor (HDACi) andvincristine [15e17]. In this report, we demonstrate that C6 cer-amide could also sensitize AT406's activity against humanpancreatic cancer cells.

2. Materials and methods

2.1. Reagents, chemicals and antibodies

AT406was purchased from Selleck (Shanghai, China). LiposomalC6 ceramide and free C6 ceramide were provided by Dr. XiwenFan's Lab [18] The caspase-3 specific inhibitor Ac-DEVD-CHO, thecaspase-9 inhibitor Ac-LEHD-CHO and the pan caspase inhibitorAc-VAD-CHO were from Calbiochem (La Jolla, CA). All the anti-bodies utilized in the study were obtained from Cell Signaling Tech(Shanghai, China). Cell culture reagents were obtained from Gibco(Shanghai, China).

2.2. Cell culture

Cultures of established human pancreatic cancer cell lines(Panc-1 and Mia-PaCa-2) and the pancreatic epithelial cell lineHPDE6c7 were described previously [7].

2.3. Primary culture of human pancreatic cancer cells

As described [7], the fresh pancreatic cancer specimens frominformed-consent patients were washed and minced. Resolvingcancer tissues were then digested [7]. Afterwards, the single-cellsuspensions were pelleted and washed. Primary cancer cells werethen cultured in the medium described [19]. The protocol wasapproved by the Institutional Review Board and the Ethics Com-mittee of authors' institutions. All studies were conducted inaccordance with the principles expressed in the Declaration ofHelsinki.

2.4. Cell viability assay

The CellTiter-Glo luminescent cell viability assay kit was appliedto evaluate cell viability according to the manufacturer's in-structions (Promega, Shanghai, China). The detailed protocol wasdescribed in our previous study [7]. Luminescence OD, indicator ofcell viability, was recorded by a Fluorescence/Multi-DetectionMicroplate Reader (Synergy 2, BioTek, Winooski, VT) [7].

2.5. Clonogenic assay

As described [7], cells were seeded at 3000/well onto 6-wellplates. Following the applied treatments, cells were then allowedto grow for additional 10 days. The colonies were then fixed andmanually counted.

2.6. Cell proliferation assay

The BrdU assay kit (Cell Signaling Technology, Shanghai, China)was applied to test cell proliferation following applied treatment[7].

2.7. Caspase activity assay

Following treatment of cells, cytosolic proteins (25 mg pertreatment) [7] were incubated with the caspase assay buffer [7]with corresponding caspase-3/-9 substrate [7]. The released AFCwas measured via a multi-detection microplate reader with exci-tation of 400 nm [7].

2.8. TUNEL staining assay

Cell apoptosis was detected by terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) In Situ Cell DeathDetection Kit (Roche, Shanghai, China) as described [7]. TUNEL ratio(TUNEL/DAPI � 100%) was recorded under a fluorescence micro-scope (Zeiss, 1: 100 magnification). For each condition, a total of ten

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X. Zhao et al. / Biochemical and Biophysical Research Communications 479 (2016) 166e172168

random views containing at least 500 cells were included to countTUNEL ratio.

2.9. Annexin V assay of apoptosis

The FITC-conjugated Annexin V (Bender, Burlingame, CA)staining assay was performed to quantify cell apoptosis aftertreatment [7].

2.10. Western blot assay

The detailed protocol for Western blot assay was describedpreviously [19]. In brief, equal amounts of cytosol protein extractsper sample were resolved by SDS-PAGE and analyzed by Westernblot. The antibody-antigen binding was visualized via the Super-Signal West Pico ECL substrates (Pierce). Band intensity (totalgray) was quantified via the Image J software.

A. B. C.

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G. H.Primary PaPrimary Pan Can

Fig. 2. C6 ceramide potentiates AT406-induced pancreatic cancer cell apoptosis. Panc-1 cthe pancreatic epithelial HPDE6c7 cells (I) were treated with applied concentration of AT40activation and cell apoptosis were tested by listed assays (A-E, G-I); For E and F, Panc-1 ce(“DEVD”, 40 mM), the caspase-9 inhibitor Ac-LEHD-CHO (“LEHD”, 40 mM) or the pan caspassay, n ¼ 5. Experiments in this figure were repeated three times, and similar results were o#p < 0.05 vs. group of “DMSO (0.1%)” (E and F).

2.11. Bcl-2 shRNA knockdown

Briefly, cells were seeded onto the polybrene (Sigma)-coated 6-well plate with 50% confluence, and were infected with the lenti-viral Bcl-2 shRNA (“-1/-2”) [7] for 24 h. Cells were then subjected topuromycin selection. Bcl-2 downregulation in stable cells wasconfirmed by Western blot assay [7]. Control cells were infectedwith same amount of scramble nonsense control shRNA lentivirus(Santa Cruz).

2.12. Mouse xenograft model and immunohistochemistry (IHC)staining

Animal studies were approved by the IACUC and Ethics Com-mittee of all authors' institutions. Panc-1 cells (1 � 107 per mouse)were injected s.c. into the right flanks of male severe combinedimmunodeficient (SCID) mice (Animal Research Centre, Shanghai,China). When established tumors were around 0.1 cm3 in volume,

D.

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1.2

Ctrl C6 AT Toge500 nM10 μM 48hrs

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*#

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Ctrl C6 AT Toge500 nM10 μM 48hrs

rs0

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Panc-1 cells

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DVEDLEHDVAD

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AT Toge500 nM 48hrs

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I.n Can HPDE6c7 cells

ells (AeF), the primary human pancreatic cancer cells (“Primary Pan Can”, G and H) or6 (“AT”, 500 nM) or plus C6 ceramide (“C6”, 10 mM) for designated time, caspase-3/-9lls were also pre-treated (for 1 h) with the caspase-3 specific inhibitor Ac-DEVD-CHOase inhibitor Ac-VAD-CHO (“VAD”, 40 mM), cell viability was also tested (F). For eachbtained. *p < 0.05 vs. group of “Ctrl”. #p < 0.05 vs. AT406 only treatment (A-D, G and H).

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Tubulin

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RNA

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Fig. 3. C6 ceramide downregulates Bcl-2 to increase AT406's sensitivity inpancreatic cancer cells. Panc-1 cells (A) or the primary human pancreatic cancer cells(“Primary Pan Can”, B) were treated with AT406 (“AT”, 500 nM) or plus C6 ceramide(“C6”, 10 mM) for 24 h, expression of listed proteins was tested by Western blot assay.Panc-1 cells (C) or the primary human pancreatic cancer cells (D), pretreated with/outABT-737 (“ABT”, 10 mM, 1 h), were stimulated with AT406 (“AT”, 500 nM) or plus C6ceramide (“C6”, 10 mM) for 72 h, cell viability was tested by the CellTiter-Glo assay.Stable Panc-1 cells with scramble control shRNA (“C-shRNA”) or Bcl-2 shRNA (“-1/-2”)were treated with AT406 (500 nM) or plus C6 ceramide (“C6”, 10 mM) for 72 h, Bcl-2expression (E) and cell viability (F) were tested. Relative expression of listed pro-teins (vs. Tubulin) was quantified (A, B and E, lower panels). Experiments in this figurewere repeated three times, and similar results were obtained. *p < 0.05 vs. group of“Ctrl”.#p < 0.05 vs. AT406 only treatment.

X. Zhao et al. / Biochemical and Biophysical Research Communications 479 (2016) 166e172 169

mice were randomized into four groups (with ten mice per group):vehicle (Saline, oral gavage), AT406 (5 mg/kg, oral gavage, at Day-1,2, 3, 8,15 and 22) [7] and/or liposomal C6 ceramide (25mg/kg, i.v. atDay-1, 2, 3, 8, 15 and 22) [20]. Tumor volume was calculated usingthe described formula [7]. For IHC study, Panc-1 xenografts werefixed; Tissue paraffin sections (4-mm) were blocked and incubatedwith primary antibody (Bcl-2, 1:50). The HRP-conjugated second-ary antibody (1: 50) was then added, followed by 3,30-dia-minobenzidine color development.

2.13. Statistical analysis

Statistical analysis was carried out via the SPSS 18.0 software(Chicago, IL). Results were compared via one-way analysis of vari-ance (ANOVA) followed by Turkey's test. All values were expressedas mean ± standard deviation (SD). A p value of less than 0.05 wasconsidered statistically significant [7].

3. Results

3.1. C6 ceramide dramatically potentiates AT406-induced anti-pancreatic cancer cell activity in vitro

This study focused on the potential effect of short-chain C6ceramide on AT406's activity in pancreatic cancer cells. CulturedPanc-1 pancreatic cells were treated with AT406 or plus C6 cer-amide. In line with our previous findings [7], we showed thatAT406 dose-dependently inhibited Panc-1 cell survival (Fig. 1A).Yet, AT406's activity alone was moderate, with the IC-50 over500 nM (Fig. 1A). Significantly, co-treatment with C6 ceramide (at10 or 25 mM) remarkably potentiated AT406's sensitivity, leading toa dramatic viability reduction in Panc-1 cells (Fig. 1A). The IC-50 ofAT406 decreased to less than 50 nM when combined with C6 cer-amide (10 or 25 mM) (Fig. 1A). Notably, treatment with C6 ceramidealone (at 10 or 25 mM) only induced minor viability reduction inPanc-1 cells (Fig. 1A). Yet, a low concentration of C6 ceramide(1 mM) showed no such effects (Fig. 1A).

The clonogenicity assay results in Fig. 1B demonstrated thatAT406 dose-dependently decreased the number of viable Panc-1colonies, which was again facilitated with co-treatment of C6 cer-amide (10 mM) (Fig. 1B). To test Panc-1 cell proliferation, BrdUincorporation assay [7] was applied. Results showed that the anti-proliferative activity by AT406 was largely enhanced when com-bined with C6 ceramide (10 mM) (Fig. 1C). Treatment C6 ceramide(10 mM) alone only slightly inhibited Panc-1 cell colony formation(Fig. 1B) and proliferation (Fig. 1C).

We also tested the effect of C6 ceramide on AT406 in otherpancreatic cancer cells. CellTiter-Glo assay results showed that C6ceramide (10 mM) largely potentiated AT406 (500 nM)-induceddeath of Mia-PaCa-2 cells (Fig. 1D) and primary human pancreaticcancer cells (Fig. 1E). On the other hand, the same AT406 plus C6ceramide regimen was non-cytotoxic to HPDE6c7 cells, which arenormal pancreatic epithelial cells (Fig. 1F). Collectively, these re-sults demonstrate that co-treatment of C6 ceramide dramaticallysensitizes AT406-induced anti-pancreatic cancer cell activity invitro.

3.2. C6 ceramide potentiates AT406-induced pancreatic cancer cellapoptosis

Our previous study has shown that AT406 provoked apoptoticcell death in pancreatic cancer cells [7], we thus studied the po-tential effect of C6 ceramide on the process. Treatment with AT406(500 nM) again induced activation of caspase-3 (Fig. 2A) andcaspase-9 (Fig. 2B) in Panc-1 cells. Further, the TUNEL percentage

(Fig. 2C) and the Annexin V intensity OD (Fig. 2D) were bothincreased following AT406 (500 nM) treatment. Remarkably, suchpro-apoptotic activity by AT406 was significantly augmented withco-treatment of C6 ceramide (Fig. 2AeD). The latter alone onlyinduced minor cell apoptosis (Fig. 2AeD). Importantly, as shown inFig. 2E and F, the caspase-3 specific inhibitor Ac-DEVD-CHO, thecaspase-9 inhibitor Ac-LEHD-CHO or the pan caspase inhibitor Ac-VAD-CHO all largely attenuated AT406 plus C6 ceramide-inducedPanc-1 cell apoptosis (Fig. 2E) and death (Fig. 2F). These results

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X. Zhao et al. / Biochemical and Biophysical Research Communications 479 (2016) 166e172170

suggested that C6 ceramide potentiated AT406-induced apoptoticdeath of pancreatic cancer cells. Similarly in primary humanpancreatic cancer cells (Fig. 2G and H), C6 ceramide significantlyfacilitated AT406-induced cell apoptosis. Notably, the same co-treatment failed to induce significant apoptosis in the non-cancerous HPDE6c7 epithelial cells (Fig. 2I). Collectively, these re-sults suggest that C6 ceramide potentiates AT406-inducedpancreatic cancer cell apoptosis.

3.3. C6 ceramide downregulates Bcl-2 to increase AT406'ssensitivity in pancreatic cancer cells

Our recent study has implied the Bcl-2, a well-known anti-apoptosis protein [21], could be a major resistance factor of AT406in pancreatic cancer cells [7]. We found that Bcl-2 inhibition orshRNA knockdown potentiated AT406-induced pancreatic cancercell death [7]. We therefore examined the potential effect of C6ceramide (or plus AT406) on Bcl-2 expression in pancreatic cancercells. In line with our previous findings [7], Fig. 3A results showedthat AT406 treatment in Panc-1 cells downregulated key IAPs

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Fig. 4. Liposomal C6 ceramide sensitizes AT406-induced anti-pancreatic cancer activityDay-1, 2, 3, 8, 15 and 22 with vehicle control (“Saline”), AT406 (5 mg/kg body weight, gavavolumes (A), tumor daily growth (in mm3 per day, B) and mice body weights (in grams, C) weinitial AT406 plus “Lipo C6” co-administration, two tumors per set) was tested by Westernliposomal C6 ceramide co-administration. *p < 0.05 vs. group of “Saline”. #p < 0.05 vs. gro

(cIAP1 and XIAP), but didn't affect Bcl-2 expression. Significantly,co-treatment of C6 ceramide also induced Bcl-2 degradation,therefore leading to a profound cytochrome C release (Fig. 3A, alsosee quantification data). Similar results were also obtained in theprimary cancer cells, where AT406 plus C6 ceramide co-treatmentinduced downregulation of Bcl-2 and IAPs (cIAP1 and XIAP),causing a dramatic cytochrome C release (Fig. 3B).

The above results suggest that C6 ceramide-mediated AT406sensitization could be due to its ability of downregulating Bcl-2. Tosupport this hypothesis, Panc-1 cells and primary cancer cells werepre-treated with Bcl-2 inhibitor ABT-737 [22]. Consistent with ourprevious finding [7], ABT-737 potentiated AT406's cytotoxicity inthe cancer cells (Fig. 3C and D). Significantly, C6 ceramide wasunable to further enhance AT406's cytotoxicity when ABT-737 waspresent (Fig. 3C and D). Similarly, in Bcl-2-silenced Panc-1 cells (bytargeted shRNA-1/-2) [7] (Fig. 3E), AT406's cytotoxicity wasaugmented (Fig. 3F). Yet, C6 ceramide-mediated AT406 sensitiza-tion was again nullified with Bcl-2 knockdown (Fig. 3F). Together,these results suggest that C6 ceramide-induced Bcl-2 down-regulation could be reason of AT406 sensitization in pancreatic

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in vivo. Panc-1 tumor bearing SCID mice (n ¼ 10 of each group) were administrated atge) and/or plus liposomal C6 ceramide (“Lipo C6”, 25 mg/kg body weight, i.v.), tumorre recorded; Expression of listed proteins in the xenografted tumor tissues (Day-3 afterblot assay (D) and IHC staining assay (E, for Bcl-2). “Combine” stands for AT406 plusup of AT406 only. Bar ¼ 100 mm (E).

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cancer cells.

3.4. Liposomal C6 ceramide sensitizes AT406-induced anti-pancreatic cancer activity in vivo

At last, by using the Panc-1 xenograft SCID mice model [7], wetested the potential effect of C6 ceramide on AT406-induced anti-tumor activity in vivo. Since free C6 ceramide is not soluble whengiven in vivo, we hereby applied the liposomal C6 ceramide (a giftfromDr. Fan [18]). As shown in Fig. 4A, oral administration of AT406(5 mg/kg, gavage) inhibited Panc-1 xenograft growth in SCID mice.Significantly, co-administration of the liposomal C6 ceramide(“Lipo C6”, i.v., at 25 mg/kg) dramatically potentiated AT406's anti-tumor activity, leading to profound inhibition on Panc-1 xenograftgrowth (Fig. 4A). The anti-tumor activity by the combination wasapparently superior than either single treatment (Fig. 4A). Treat-ment with liposomal C6 ceramide alone also exerted moderateinhibition on Panc-1 xenograft growth (Fig. 4A). Daily tumorgrowth (mm3 per day)was again lowest in AT406 plus liposomal C6co-administration mice (Fig. 4B). AT406 or liposomal C6 alone lessefficiently inhibited tumor growth (Fig. 4B).

Notably, the mice body weights were not affected by the AT406and/or liposomal C6 treatment (Fig. 4C). We also failed to observeany signs of toxicities in the tested animals. Therefore, the SCIDmice appeared well-tolerated to the single or the combinationtreatment. When analyzing the apoptosis molecule in Panc-1 tu-mor masses, we showed expressions of cIAP1, XIAP and Bcl-2 wereall downregulated in AT406 plus liposomal C6-treated Panc-1 xe-nografts (Day-3 after administration, Fig. 4D). Notably, our previousstudies found that Bcl-2 was not affected by AT406 single treatment[7]. Expression of cleaved-caspase-3, the indicator of cell apoptosis,was significantly increased in the combination-treated tumors(Fig. 4D). IHC staining assay further confirmed Bcl-2 down-regulation in Panc-1 tumors with the co-administration (Fig. 4E).Therefore, the signaling results in Panc-1 tumor masses areconsistent with the in vitro findings.

4. Discussions

The results of our recent study [7] have indicated that Bcl-2 mayact as the primary resistance factor of AT406 [7]. We previouslyshowed that AT406 failed to affect expression of Bcl-2 in humanpancreatic cancer cells [7]. Yet, shRNA knockdown or pharmaco-logical inhibition (ABT-737) of Bcl-2 dramatically potentiatedAT406-induced lethality in humanpancreatic cancer cells [7]. In thepresent study, we demonstrated that C6 ceramide downregulatedBcl-2 in pancreatic cancer cells, which could be the reason for thesubsequent AT406 sensitization. Notably, ABT-737 or Bcl-2 knock-down almost nullified C6 ceramide-mediated AT406 sensitizationin pancreatic cancer cells. Our results of Bcl-2 downregulation byC6 ceramide were consistent with other studies. For example, Jiet al., have shown C6 ceramide, alone or together with Doxorubicin,significantly downregulated Bcl-2 in multiple cancer cell lines [17].It will be worthy testing the underlying mechanisms of Bcl-2downregulation by C6 ceramide.

Although exerting promising anti-cancer cell activity [23], C6ceramide and other short-chain ceramides have extremely poorsolubility, which significantly limited their applications in vivo[16,24e26]. Therefore, liposome-based nanotechnologies havebeen applied for package and the system delivery of these short-chain ceramides [16,24e26]. Existing evidences have suggestedthat liposome package of C6 ceramide could offer its rapid tissuedistributionwhen given systematically (i.v.) [24]. Further, liposomalC6 ceramide showed no apparent adverse effects [18,20,24].Significantly, liposomal C6 ceramide demonstrated selective

toxicity only to cancerous cells, yet sparing normal non-cancerouscells [16,18,20,25,26]. Here, we showed that co-administration ofliposomal C6 ceramide in SCID mice largely augmented AT406-induced anti-cancer activity in vivo. These results suggest thatliposomal C6 ceramide could act as an important adjuvant forAT406 to kill pancreatic cancer cells.

Pancreatic cancer is still one of the most aggressive humanmalignancy with an extremely poor prognosis [27]. Many of thepancreatic cancer cells are strikingly resistant to current conven-tional gemcitabine-based treatments, which have failed to signifi-cantly improve patients' survival [27]. It is therefore extremelyimportant to develop novel therapeutic agents against this devas-tating disease [27]. The results of the current report indicate thatAT406 plus C6 ceramide may be worthy further testing in otherpancreatic cancer models.

Competing interests

The authors declare that they have no competing interests.

Transparency document

Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.bbrc.2016.08.121.

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