Cancer Letters 353 (2014) 32–40

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Cancer Letters

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Rottlerin suppresses growth of human pancreatic tumors in nude mice,and pancreatic cancer cells isolated from KrasG12D mice

http://dx.doi.org/10.1016/j.canlet.2014.06.0210304-3835/� 2014 Elsevier Ireland Ltd. All rights reserved.

⇑ Corresponding authors.E-mail addresses: rsrivastava.lab@gmail.com (R.K. Srivastava), sharmila.

shankar@va.gov (S. Shankar).

Minzhao Huang a, Su-Ni Tang a, Ghanshyam Upadhyay a, Justin L. Marsh b, Christopher P. Jackman b,Rakesh K. Srivastava a,⇑, Sharmila Shankar c,⇑a Department of Pharmacology, Toxicology and Therapeutics, and Medicine, The University of Kansas Cancer Center, The University of Kansas Medical Center, 3901 RainbowBoulevard, Kansas City, KS 66160, USAb Department of Biochemistry, University of Texas Health Science Center at Tyler, Tyler, TX 75708, USAc Kansas City VA Medical Center, 4801 Linwood Boulevard, Kansas City, MO 64128, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 February 2014Received in revised form 12 May 2014Accepted 9 June 2014

Keywords:RottlerinPancreatic cancerCancer preventionAktSonic HedgehogNotch

The purpose of the study was to examine the molecular mechanisms by which rottlerin inhibited growthof human pancreatic tumors in Balb C nude mice, and pancreatic cancer cells isolated from KrasG12D mice.AsPC-1 cells were injected subcutaneously into Balb c nude mice, and tumor-bearing mice were treatedwith rottlerin. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively.The expression of components of Akt, Notch, and Sonic Hedgehog (Shh) pathways were measured by theimmunohistochemistry, Western blot analysis, and/or q-RT-PCR. The effects of rottlerin on pancreaticcancer cells isolated from KrasG12D mice were also examined. Rottlerin-treated mice showed a significantinhibition in tumor growth which was associated with suppression of cell proliferation, activation ofcapase-3 and cleavage of PARP. Rottlerin inhibited the expression of Bcl-2, cyclin D1, CDK2 and CDK6,and induced the expression of Bax in tumor tissues compared to untreated control. Rottlerin inhibitedthe markers of angiogenesis (Cox-2, VEGF, VEGFR, and IL-8), and metastasis (MMP-2 and MMP-9), thusblocking production of tumorigenic mediators in tumor microenvironment. Rottlerin also inhibited epi-thelial–mesenchymal transition by up-regulating E-cadherin and inhibiting the expression of Slug andSnail. Furthermore, rottlerin treatment of xenografted tumors or pancreatic cancer cells isolated fromKrasG12D mice showed a significant inhibition in Akt, Shh and Notch pathways compared to controlgroups. These data suggest that rottlerin can inhibit pancreatic cancer growth by suppressing multiplesignaling pathways which are constitutively active in pancreatic cancer. Taken together, our data showthat the rottlerin induces apoptosis and inhibits pancreatic cancer growth by targeting Akt, Notch andShh signaling pathways, and provide a new therapeutic approach with translational potential forhumans.

� 2014 Elsevier Ireland Ltd. All rights reserved.

Introduction

Rottlerin is a polyphenolic compound derived from Mallotusphilipinensis (Euphorbiaceae) [1]. Rottlerin is widely used as aninhibitor of the PKCd [1], but PKCd independent mechanisms ofaction have also been proposed [2,3]. Several mechanisms of rott-lerin have been proposed which include depolarization of themitochondrial membrane potential [4], drop in ATP levels, activa-tion of 50-AMP-activated protein kinase (AMPK), and generation

of mitochondrial reactive oxygen species (ROS) [2,5,6]. We haverecently demonstrated that rottlerin can induce autophagy in pan-creatic, breast and prostate cancer stem cells by regulating AMPK[3,7,8]. Rottlerin has been shown to demonstrate anticancer activ-ity in various tumors [3,9,10]. In spite of these findings, the molec-ular mechanisms by which it inhibits tumor growth, angiogenesisand metastasis is not well-understood.

Pancreatic cancer is one of the most aggressive human cancers,with more than 200,000 deaths worldwide every year [11]. It is acomplex disease which is regulated by several factors such asmutations in p53, p16, and SMAD [12–14]. Most importantly, themutations in Kras gene is found in codon 12 of more than 95%pancreatic cancer patients; this mutation causes constitutive

M. Huang et al. / Cancer Letters 353 (2014) 32–40 33

activation of Kras which is responsible for enhancing cell growthand metastasis [14]. Despite recent efforts, conventional treatmentapproaches, such as surgery and traditional chemotherapy, haveonly slightly improved patient outcomes. Therefore, more effectiveand well-tolerated therapies are required to reverse the currentpoor prognosis of pancreatic cancer.

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian tar-get of rapamycin (mTOR) pathway is implicated in the pathogene-sis of pancreatic ductal adenocarcinoma (PDAC) [15,16]. A recentstudy used compounded mice where Pten conditional knockoutmice [Pten(lox/lox)] were crossed with conditionally activatedKrasG12D mice [17]. These compound heterozygous mutant micedemonstrated aggressive development of all stages of pancreaticcancer including acinar-to-ductal metaplasia (ADM), pancreaticintraepithelial neoplasia (mPanIN), and PDAC within 1 year [17].Most importantly, all compounded mice (KrasG12D with activatedPten homozygous deletion) developed pancreatic cancer within3 weeks. Similarly, we have recently demonstrated that resveratrolcan inhibit pancreatic carcinogenesis in KrasG12D mice [18]. Thesestudies highlight the significance of PI3K/AKT pathway and acti-vated KrasG12D in pancreatic carcinogenesis. Here, we sought toexamine the anti-proliferative effects of rottlerin on pancreaticcancer cells isolated from KrasG12D mice.

The Notch signaling pathway is evolutionary conserved andplays critical roles in neurogenesis, myogenesis, vasculogenesis,and hematopoiesis [19]. Accumulating evidence demonstratesthe clinical significance of Notch signaling in growth, differentia-tion, and apoptosis in several cancers including pancreatic cancer[20]. In animal studies, Notch1-signaling pathway regulates pan-creatic embryogenesis by promoting self-renewal and exocrinelineage development of pancreatic progenitor cells [21]. Further-more, a recent study has demonstrated that activated Kras in aci-nar cells can induce PanINs, and activation of Notch pathway canfurther synergize with Kras in pancreatic carcinogenesis [22]. Theinvolvement of Notch pathway in pancreatic cancer was furtherconfirmed due to fact that higher nuclear expression of Notch1, -3 and -4, Hes-1, and Hey-1 was demonstrated in locally advancedand metastatic pancreatic tumors compared to resectable cancers[23]. The role of Notch in modulating the antitumor activity of rott-lerin has not been demonstrated.

Sonic Hedgehog (Shh) is a member of the hedgehog (Hh) family[24]. Hh pathway plays a significant role in pancreatic cancer pro-gression in both sporadically or in genetically predisposed individ-uals [25]. Hh signaling is required for embryogenesis and isimportant in postnatal tissue renewal and in malignancy. Shhbinds to Patched (Ptch) receptors, which leads to loss in Ptchactivity followed by phosphorylation and posttranscriptionalstabilization of Smoothened (Smo) [26]. Finally, Gli family oftranscription factors are activated resulting in gene induction[27]. We have recently demonstrated that inhibition of Smo orGli by small molecules inhibited human pancreatic cancer stemcell characteristics and tumor growth [28,29]. It is not knownwhether rottlerin can inhibit pancreatic tumor growth by sup-pressing Shh pathway.

The purpose of this study was to examine the molecular mech-anisms by which rottlerin inhibits human pancreatic tumorgrowth, angiogenesis, and metastasis in Balb C nude mice. In addi-tion, the molecular mechanisms by which rottlerin inhibitedgrowth of pancreatic cancer cells isolated from KrasG12D mice wereexamined because KrasG12D mice closely mimics pancreatic cancerdevelopment in humans. Our data demonstrated that rottlerininhibited tumor growth, angiogenesis and metastasis of AsPC-1tumors in nude mice, and mouse pancreatic cancer cell growththrough the inhibition of Akt, Shh and Notch signaling pathways,and can therefore be developed for the prevention and/or treat-ment of pancreatic cancer.

Materials and methods

Reagents

Antibodies (phospho-Akt, Akt, Notch1, Notch3, Hes1, Gli1, Gli2, cyclin D1, CDK-2, CDK-6, PCNA, Ki67, caspase-3, PARP, Bcl-2, Bax, Cox-2, VEGF, VEGFR, MMP-2,MMP-9, E-Cadherin, Snail, Slug and b-actin) for Western blot and immunohisto-chemistry were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Rott-lerin was obtained from LKT Laboratories, Inc. (St. Paul, MN). TUNEL assay kit waspurchased from Roche Applied Sciences (Indianapolis, IN). Matrigel was purchasedfrom Becton Dickinson (Bedford, MA).

Cell proliferation

Mouse pancreatic cancer cells (1 � 104) were incubated with rottlerin (0–1 lM)in 1 ml of RPMI 1640 medium in 6-well plate for 48 h. At the end of incubation per-iod, number of live cells were counted using cell counter (Invitrogen).

Antitumor activity of rottlerin

We have purchased Balb C nude mice (4–6 weeks old) from the National CancerInstitute, Frederick, MD. To study the possible effects of rottlerin on tumor growth,AsPC-1 cells (2 � 106 cells mixed with Matrigel, 50:50 ratio) were injected subcuta-neously into the flanks of Balb/c nu/nu mice (4–6 weeks old, n = 7). Rottlerin (0 or20 mg/kg) was administered to tumor-bearing mice through gavage (5 days perweek, once daily) for 6 weeks. At the end of the experiment, all the mice from con-trol and rottlerin-treated groups (seven mice in each group) were euthanized.Tumors from each mouse were isolated and divided into two groups, the first groupfor the Western blot analysis and the second group for immunohistochemistry. Forthe Western blot analysis, we have pooled the tumor tissues from seven mice due tosmall size of tumors in rottlerin-treated group.

Generation of KrasG12D mice

We have obtained LSL K-rasG12D and Pdx-1-Cre mice from the National CancerInstitute (Frederick, MD). The breeding and characterization of these mouse strainswere performed as previously described [18]. In brief, LSL K-rasG12D mice wereintercrossed with the Pdx-1-Cre mice to obtain KrasG12D (Pdx1-Cre; LSL-KrasG12D)mice. The pancreatic cancer cells were isolated from 10-months old KrasG12D mice(n = 7) [18]. Mouse pancreatic cancer cells were treated with rottlerin in vitro toexamine its effects on cell growth and colony formation, and signaling pathways.The experiments were repeated three times.

Western blot analysis

The Western blot analysis was performed as we described earlier [30]. In brief,whole tissues were snap frozen in liquid nitrogen and crushed immediately usingan electronic pestle into ice-cold lysis buffer. Whole cell lysates were extractedfrom tumor tissues using RIPA lysis buffer containing 1� protease inhibitor cocktail.Equal amounts of protein (40 lg) from each group were resolved on 12% SDS–PAGE.Proteins from the gel were transferred on polyvinylidene difluoride membranesusing semi-dry system (Bio-Rad). Membranes were blocked in buffer 5% nonfatdry milk in 1� Tris Buffer Saline (TBS) and incubated overnight with primary anti-bodies (1:1000 dilution) at 4 �C. After blocking, membranes were washed threetimes with TBS-T for 10, 5, and 5 min each, and were incubated with secondaryantibodies conjugated with horseradish peroxidase at 1:5000 dilution in TBS for1 h at room temperature. Membranes were again washed three times in TBS-Tand developed using enhanced chemiluminescence substrate (Super Signal WestPico substrate, Pierce). Protein bands were visualized on X-ray film.

Immunohistochemistry and TUNEL assay

Immunohistochemistry (IHC) of tumor tissues collected from control and rott-lerin-treated mice was performed as we described elsewhere [30]. In brief, IHC wascarried out on formalin-fixed and paraffin-embedded 5-lM sections of tumor sam-ples using primary antibody (1:100 dilution) overnight at 4 �C. Slides were devel-oped as per manufacturer’s instruction (Thermo Fisher Scientific). As a negativecontrol, isotype-matched IgG were used. Apoptosis was measured by terminaldeoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay(Roche Applied Sciences).

Statistical analysis

Differences between groups were analyzed by ANOVA, followed by Bonferoni’smultiple comparison tests. Tumor volumes of mice (n = 7) were compared using theKruskal–Wallis test using PRISM software (Graph-Pad Software). Levels of statisti-cal significance were set at P less than 0.05. Experiments were performed in tripli-cates, and values were presented as mean ± SD.

34 M. Huang et al. / Cancer Letters 353 (2014) 32–40

Results

Rottlerin inhibits the growth of AsPC-1 xenografts in Balb C nude mice

In order to examine the tumorigenic potential of rottlerin, wefirst examined the effects of rottlerin on growth of AsPC-1 xeno-grafted tumors in Balb C nude mice. AsPC-1 cells were injectedsubcutaneously into the flanks of Balb C nude mice. After tumorformation, mice were treated with rottlerin (0 or 20 mg/kg bodyweight) through gavage (Monday to Friday, once daily) for6 weeks. Rottlerin inhibited AsPC-1 pancreatic tumor growth inBalb C nude mice (Fig. 1A). Furthermore, rottlerin had no effecton the body weight of AsPC-1 tumor-bearing mice, although micegained weight during the treatment in both the groups. We did notobserve any toxicity in the liver, spleen and intestine of mice trea-ted with rottlerin, suggesting it is safe and can be used for thetreatment and prevention of pancreatic cancer.

Rottlerin inhibits tumor cell proliferation, and induces apoptosisthrough activation of caspase-3 and cleavage of poly(ADP-ribose)polymerase (PARP)

We next examined the effects of rottlerin on cell proliferation intumor tissues derived from control and rottlerin treated mice using

Fig. 1. Rottlerin inhibits the growth of AsPC-1 tumors xenografted in Balb C nude mice. (Aimplanted into the flanks of Balb C nude mice. Tumor-bearing mice were treated with rott6 weeks. Tumor volume and body weight of mice were recorded weekly. Data represent tExpression of PCNA, Ki67, caspase-3, PARP, Bcl-2 and Bax in tumor tissues. ImmunohistocPARP, Bcl-2 and Bax in tumor tissues isolated from control and rottlerin-treated mice. Eacproliferation and apoptosis. Western blot analysis was performed to measure the expressused as a loading control. (D) Quantification of TUNEL positive cells. Apoptosis was measfrom control, P < 0.05.

anti-PCNA or anti-Ki67 antibody (Fig. 1B and C). PCNA and Ki67 arethe markers of cell proliferation. Rottlerin inhibited cell prolifera-tion in tumor tissues obtained from AsPC-1 xenografts comparedto control mice, as measured by immunohistochemistry (IHC)and the Western blot (WB) analysis.

Caspase activation and cleavage of its substrate PARP are thehall marks of apoptosis [31]. We next examined whether rottlerininduced tumor cell apoptosis through activation of caspase-3 andcleavage of PARP (Fig. 1B and C). Caspase-3 activation was mea-sured by IHC and Western blot analysis using active anti-cas-pase-3 antibody. Rottlerin induced caspase-3 activation. PARP isa substrate of caspase-3 [31]. Rottlerin treatment resulted in cleav-age of PARP. Activation of caspase-3 by rottlerin correlated withcleavage of PARP in tumor tissues. Tumors from rottlerin-treatedgroup demonstrated significantly higher percentage of TUNEL-positive cells than those of control group (Fig. 1D). Overall, thesedata suggest that rottlerin inhibited cell proliferation and inducedapoptosis in pancreatic tumor tissues through inhibition of PCNA,Ki67, and activation of caspase-3 and cleavage of PARP.

Rottlerin regulates Bcl-2 family members in tumor tissues

The Bcl-2 family members consist of anti- and pro-apoptoticproteins [32]. Most anticancer drugs exert their effects through

) AsPC-1 cells (2 � 106 cells mixed with Matrigel, 50:50 ratio) were subcutaneouslylerin (0 or 20 mg/kg body weight) through gavage (Monday to Friday, once daily) forhe mean (n = 7) ± S.D. ⁄=significantly different from respective controls, P < 0.05. (B)hemistry was performed to measure the expression of PCNA, Ki67, active caspase-3,h picture is a representative of 5–7 images. (C) Effects of rottlerin on markers of cellion of PCNA, Ki67, caspase-3, PARP, Bcl-2 and Bax in tumor tissues. The b-actin was

ured by TUNEL assay. Data represent the mean (n = 7) ± S.D. ⁄=significantly different

M. Huang et al. / Cancer Letters 353 (2014) 32–40 35

modulation of these proteins. We therefore measured the effects ofrottlerin on the expression of anti-apoptotic Bcl-2, and pro-apopto-tic Bax in tumor tissues (Fig. 1B and C). Rottlerin inhibited theexpression of Bcl-2 and induced the expression of pro-apoptoticprotein Bax in tumor tissues. These data suggest that rottlerincan inhibit pancreatic tumor growth by regulating Bcl-2 familymembers.

Rottlerin regulates cell cycle proteins in human pancreatic tumortissues

Cell cycle arrest is the major event to block tumor progressionand metastasis. Since rottlerin inhibited cell proliferation andinduced apoptosis, we next sought to examine its effects on cellcycle regulatory proteins. Rottlerin inhibited the expression ofcyclin D1, CDK-2 and CDK-6 (Fig. 2). The inhibition of cyclin D1,CDK-2 and CDK-6 observed after rottlerin treatment of mice mayhave important implications in cell cycle progression, and cancertherapy.

Rottlerin inhibits angiogenesis

Increased mitogenic signaling and angiogenesis, frequentlyfacilitated by VEGF overexpression, drive cancer growth [33]. Sinceangiogenesis plays a major role in tumor growth [33], we sought tomeasure the effects of rottlerin on angiogenesis by measuring theexpression of VEGF and its receptor VEGFR in pancreatic tumor tis-sues. Treatment of mice with rottlerin resulted in significant inhi-bition in both VEGF and VEGFR in tumor cells (Fig. 3A and B). Thesedata suggest that rottlerin may inhibit angiogenesis by suppressingthe expression of VEGF/VEGFR.

It is well known that cyclooxygenase-2 (Cox-2) play a signifi-cant role in tumorigenesis, and promotes vascular permeabilityand mediates the proliferation of endothelial cells [34]. We there-fore examined whether rottlerin inhibits the expression of Cox-2 inpancreatic cancer. Rottlerin inhibited the expression of Cox-2 inpancreatic tumor tissues (Fig. 3A and B). These data suggest that

Fig. 2. Effects of rottlerin on the expression of cell cycle-related proteins in tumor tiperformed as described in material and methods. Each picture is a representative of 5–7 iD1, CDK-2 and CDK-6. The b-actin was used as a loading control.

rottlerin can inhibit pancreatic tumor growth by suppressingCox-2.

Various cytokines regulate cancer cell growth and angiogenesis[35]. Specially, proinflammatory cytokines such as IL-8 have beenshown to enhance angiogenesis and remodel the tumor microenvi-ronment [35,36]. We therefore measured the expression of IL-8 inpancreatic tumor tissues. Treatment of AsPC-1 tumor-bearing micewith rottlerin resulted in suppression of IL-8 (Fig. 3A and B). Thesedata suggest that IL-8 may mediate antitumor activity of rottlerin.

We next confirmed the effects of rottlerin on angiogenesis bycounting the number of blood vessels. H&E staining, anti-CD31antibody and anti-vWF-antibody were used to count blood vesselsin tumor tissues (Fig. 3C). Rottlerin inhibited number of blood ves-sels in tumor tissues, suggesting it may inhibit angiogenesis.

We have demonstrated that the number of circulating vascularendothelial growth factor receptor 2 (VEGFR2)-positive endothelialcells may serve as an indicator of tumor angiogenesis [37]. Wetherefore counted the numbers of VEGFR2-positive endothelialcells in the blood derived from mice treated with rottlerin(Fig. 3D). Rottlerin-treated mice demonstrated significantly lessnumber of circulating VEGFR2-positive endothelial cells comparedto that of control mice. Overall, these data suggest that rottlerincan inhibit angiogenesis by suppressing the expression of VEGF/VEGFR, Cox-2, and IL-8.

Rottlerin inhibits epithelial to mesenchymal-transition in pancreatictumor tissues

Epithelial tumors metastasize via epithelial-to-mesenchymaltransition (EMT) [38]. During EMT, expressions of Slug and Snailtranscription factors are induced, as a result the expression ofE-cadherin is inhibited and the expression of N-cadherin is up-reg-ulated [39]. Furthermore, the expressions of MMPs, which digestthe extracellular matrix and basement membrane, are enhancedduring EMT [40]. We therefore measured the effects of rottlerinon the expression of E-cadherin, Slug, Snail, MMP-2 and MMP-9.Treatment of mice with rottlerin induced the expression of

ssues. (A) Expression of cyclin D1, CDK2 and CDK6. Immunohistochemistry wasmages. (B) Western blot analysis was performed to measure the expression of cyclin

Fig. 3. Effects of rottlerin on markers of angiogenesis. (A) Immunohistochemistry was performed to examine the expression of VEGF, VEGFR, Cox-2, and IL-8 in tumor tissuesisolated from control and rottlerin-treated mice. Each picture is a representative of 5–7 images. (B) Western blot analysis was performed to measure the expression of VEGF,VEGFR, Cox-2, and IL-8. The b-actin was used as a loading control. (C) Number of blood vessels. Tumor tissue sections were stained with H&E, anti-CD31 antibody and anti-von Willebrand Factor (vWF) antibody and the numbers of blood vessels were counted. Each column represents the mean (n = 10) ± SEM. ⁄=significantly different fromrespective control, P < 0.05. (D) Blood VEGFR2-positive cells. VEGF receptor 2 (VEGFR2)-positive circulating endothelial cells collected from rottlerin-treated and control mice.The blood cells from peripheral blood attached to the slide were stained with anti-VEGFR2 antibody, and the numbers of VEGFR2 positive cells were counted. Each columnrepresents the mean (n = 10) ± SEM. ⁄=significantly different from respective control, P < 0.05.

36 M. Huang et al. / Cancer Letters 353 (2014) 32–40

E-cadherin and inhibited the expression of Slug, Snail, MMP-2 andMMP-9 in tumor tissues (Fig. 4A and B). Our data demonstrate thatrottlerin can inhibit/reverse tumor metastasis by inducing theexpression of E-cadherin and inhibiting its associated transcriptionfactors, and MMPs. Overall, our data demonstrate that rottlerinmay inhibit metastasis in pancreatic cancer.

Rottlerin inhibits Notch, Akt and Sonic Hedgehog pathways inpancreatic cancer

Since Notch pathway is constitutively active in tumor tissues[41,42], we sought to measure the expression of Notch1, Notch3and their down-stream target gene Hes1. Rottlerin inhibited theexpression of Notch1, Notch3 and Hes1 (Fig. 5A and B). These datasuggest that rottlerin can regulate pancreatic cancer growth bysuppressing Notch pathway.

The serine/threonine kinase Akt/PKB is a major signaling path-way integrating metabolic, survival, growth, and cell cycle regula-tory signals, and it is frequently and aberrantly activated in laterstages of pancreatic ductal adenocarcinoma (PDAC) [43]. SincePI3K/Akt pathway has been shown to regulate pancreatic carcino-genesis, we measured the expression of Akt in tumor tissues(Fig. 5). Rottlerin inhibited the expression of phospho-Akt in tumortissues (Fig. 5A and B). Rottlerin had no effects on the expression oftotal Akt (Fig. 5B). Overall, these data suggest that rottlerin caninhibit Akt pathway which regulates tumor cell proliferation andcarcinogenesis in pancreatic cancer.

Activated Hh signaling pathway plays significant role in pancre-atic tumorigenesis [24]. We next sought to examine the effects ofrottlerin on the expression of Gli1 and Gli2 transcription factors.Since Gli binding sites are present in the promoters of Gli1/Gli2,they regulate their own transcription. Rottlerin inhibited theexpression of Gli1 and Gli2 (Fig. 5C and D). These data suggest thatrottlerin can inhibit pancreatic tumor growth by suppressing Shhpathway.

Rottlerin inhibits growth of pancreatic cancer cells isolated fromKrasG12D mice

Akt, Notch and Shh signaling pathways are well-establishedsurvival pathways in human pancreatic cancer. Constitutive acti-vation of each of these pathways has been shown to increasecancer cell proliferation and drug resistance. The activation ofeach of these pathways has been demonstrated in KrasG12D mice.Kras is a master regulator of pancreatic cancer as it is mutatedin more than 95% human PDAC [14]. We therefore examinedthe effects of rottlerin on cancer cell growth (proliferation andcolony formation) in mouse pancreatic cancer cells isolated from10-months old KrasG12D mice. Rottlerin inhibited cell prolifera-tion and colony formation in mouse pancreatic cancer cellsin vitro (Fig. 6A).

We next determined whether Akt, Notch and Shh signalingpathways were down-regulated by rottlerin in pancreatic cancercells isolated from KrasG12D mice in vitro. Rottlerin inhibited the

Fig. 4. Effects of rottlerin on markers of epithelial–mesenchymal transition. (A) Immunohistochemistry was performed to examine the expression of E-cadherin, Slug, Snail,MMP-2 and MMP-9 in tumor tissues isolated from control and rottlerin-treated mice. Each picture is a representative of 5–7 images. (B) Western blot analysis was performedto measure the expression of E-cadherin, Slug, Snail, MMP-2 and MMP-9. The b-actin was used as a loading control.

M. Huang et al. / Cancer Letters 353 (2014) 32–40 37

expression of phospho-Akt in mouse pancreatic cancer cells iso-lated from KrasG12D mice (Fig. 6B). However, it had no effect ontotal Akt expression. Rottlerin inhibited the expression of Notch1,Notch3 and their down-stream target Hes1 in mouse pancreaticcancer cells isolated from KrasG12D mice (Fig. 6C). Similarly, rott-lerin inhibited the expression of Gli1 and Gli2 and their down-stream target cyclin D1 in mouse pancreatic cancer cells(Fig. 6D). These data indicate that rottlerin suppresses mouse pan-creatic cancer growth by inhibiting Notch, Shh and Akt signalingpathways.

Discussion

Pancreatic cancer is one of the most aggressive and devastatingmalignancies. We have demonstrated, for the first time, that rott-lerin inhibited AsPC-1 xenografted tumor growth which was asso-ciated with suppression of Akt, Notch and Shh pathways. Thesepathways have been shown to play major roles in pancreatic carci-nogenesis. Furthermore, rottlerin inhibited the growth of pancre-atic cancer cells isolated from KrasG12D mice through suppressionof Akt, Notch and Shh pathways. Rottlerin was originally discov-ered as PKCd inhibitor. We and others have recently demonstratedthat rottlerin can exert its effects through PKCd independent mech-anisms as well [2,3]. The anti-tumor activity of rottlerin wasexerted through inhibition of cell proliferation, and induction ofapoptosis. Rottlerin also inhibited markers of angiogenesis andmetastasis, suggesting that it can be used as anticancer agent.

The PI3K/Akt signaling pathway regulates cell proliferation andsurvival, and is frequently activated in PDAC. In our study using inhuman xenografts and mouse pancreatic cancer cells, rottlerininhibited the activation of Akt which was associated with suppres-sion of tumor and cell growth, respectively. In a recent study, theheterozygous loss of Pten in KrasG12D mutant mice acceleratedthe development of acinar-to-ductal metaplasia (ADM), mPanIN,and PDAC within 1 year [17], confirming the role of PI3K/AKTand activated Kras in pancreatic cancer progression. In anotherstudy, we have recently demonstrated that rottlerin inducesautophagy followed by apoptosis via inhibition of PI3K/Akt/mTORpathway and activation of caspase cascade in human pancreaticcancer stem cells [3].

Sonic Hedgehog pathway has been demonstrated to play a sig-nificant role in pancreatic carcinogenesis. Hedgehog signalingpathway is highly activated in tumor epithelia and surroundingstromal tissues derived from pancreatic adenocarcinoma patients.Tumor growth can be controlled in an autocrine or paracrine man-ner by Hh signaling pathway. Paracrine activity of the Hh signalingin tumor microenvironment can regulate tumor cell proliferation,metastasis and drug resistance. We have recently demonstratedthat the components of Shh pathway are highly expressed inhuman pancreatic cancer stem cells and pancreatic cancer celllines, and several chemopreventive agents inhibited pancreaticcancer growth [18,30,44,45]. Similarly in the present study, rott-lerin inhibited AsPC-1 tumor growth and mouse PDAC cell growthby suppressing Shh pathway. In another study, it has been shownthat inhibition of the Hh pathway decreased cell proliferation and

Fig. 5. Effects of rottlerin on Notch, Akt and Shh pathways. (A) Immunohistochemistry was performed to measure the expression of Notch1, Notch3, Hes1 and pAkt in tumortissues isolated from control and rottlerin-treated mice. Each picture is a representative of 5–7 images. (B) Western blot analysis was performed to measure the expression ofNotch1, Notch3, Hes1, p-Akt and Akt. The b-actin was used as a loading control. (C) Immunohistochemistry was performed to measure the expression of Gli1 and Gli2 intumor tissues isolated from control and rottlerin-treated mice. Each picture is a representative of 5–7 images. (D) Western blot analysis was performed to measure theexpression of Gli1 and Gli2. The b-actin was used as a loading control.

38 M. Huang et al. / Cancer Letters 353 (2014) 32–40

induced apoptosis through inhibition of the PI3K/Akt pathway[46]. We have also demonstrated that inhibition of the Shh path-way significantly inhibited EMT by suppressing the activation oftranscription factors Snail and Slug, which were correlated withreduced pancreatic cancer stem cell invasion [30,44,45,47].Overall, these data suggest that the Shh pathway may be a poten-tial therapeutic target in human pancreatic cancer.

Accumulating evidence suggests an important role for Cox-2 inthe pathogenesis of a wide range of malignancies. Cox-2 is upreg-ulated in human PDAC [34]. Cox-2 deletion in Pdx1+ pancreaticprogenitor cells significantly delayed the development of PDAC inmice with K-ras activation and Pten haploinsufficiency. Conversely,Cox-2 overexpression promoted early onset and progression ofPDAC in the K-ras mouse model. This study confirmed the role ofPTEN in determining lethal PDAC onset and overall survival ofmice. Cox-2 overexpression increased p-Akt levels in the precursorlesions of Pdx1(+); K-ras(G12D)(/+); Pten(lox)(/+) mice in theabsence of Pten LOH. In contrast, Cox-2 deletion in the same set-ting diminished p-Akt levels and delayed cancer progression. Thisstudy suggests an important cell intrinsic role for Cox-2 in tumorinitiation and progression through activation of the PI3K/Akt path-way [34]. In the present study, rottlerin inhibited both Cox-2 andp-Akt in pancreatic cancer, suggesting agents than can simulta-neously inhibit Cox-2 and Akt may be useful for the managementof pancreatic cancer.

The Notch signaling pathway plays a critical role in cellulartransformation and tumorigenesis [20]. There is mountingevidence that this pathway is dysregulated in a variety of

malignancies including pancreatic cancer. Notch3-specific siRNAsuppressed Notch3 expression, and increased gemcitabine-induced, caspase-mediated apoptosis [48]. Furthermore, suppres-sion of Notch3 expression decreased PI3K/Akt activity andenhanced anti-proliferative effects of gemcitabine in BxPC-3 andPANC-1 cells. In the present study, rottlerin inhibited pancreaticcancer growth which was associated with inhibition of Notch1,Notch3 and their downstream target Hes1. Overall, these datademonstrate that Notch is a novel target for cancer therapeuticintervention and rottlerin can regulate pancreatic cancer growthby suppressing Notch pathway.

Angiogenesis (the growth of new vessels) plays a significantrole in tumor growth and metastasis. Vascular endothelial growthfactor (VEGF) is one of the main mitogen which regulates tumorgrowth, angiogenesis, and metastasis. In our study, rottlerin inhib-ited the expression of VEGF and its receptor, and number of bloodvessels in pancreatic tumor xenografts. We have also noticed a sig-nificant inhibition of circulating VEGFR2-positive endothelial cellsin xenografted mice treated with rottlerin. Thus, our studies dem-onstrate that rottlerin can inhibit angiogenesis by suppressingVEGF signaling pathway.

During EMT, secretion of several cytokines, chemokines andMMPs are increased [49]. These factors could play a significant rolein tumor progression, metastasis and in maintaining tumor micro-environment. Our data presented here, however, show that rott-lerin could modulate EMT and metastasis in pancreatic cancer byregulating the expression of MMPs. The IL-8/IL-8 receptor axishas been demonstrated to induce or maintain EMT and remodel

Fig. 6. Effects of rottlerin on cell proliferation and colony formation, and Akt, Notch and Shh pathways in pancreatic cancer cells isolated from KrasG12D mice. (A) Pancreaticcancer cells were isolated from 10-months old KrasG12D mice. Mouse pancreatic cancer cells were treated with rottlerin (0–1 lM) for 2 or 21 days to measure cell proliferationor colony formation, respectively. Data represent the mean (n = 7) ± SD. ⁄=significantly different from control, P < 0.05. Experiments were repeated three times. (B) Expressionof Akt in mouse pancreatic cancer cells. Western blot analysis was performed to measure the expression of phopho-Akt (pAkt) and Akt. The b-actin was used as a loadingcontrol. (C) Mouse pancreatic cancer cells were isolated from 10-months old KrasG12D mice, and treated with or without rottlerin (0.5 lM) for 36 h. At the end of incubationperiod, crude proteins were isolated to examine the expression of Notch1, Notch3 and Hes1 by the Western blot analysis. The b-actin was used as a loading control. (D) Mousepancreatic cancer cells were isolated from 10-months old KrasG12D mice, and treated with or without rottlerin (0.5 lM) for 36 h. At the end of incubation period, crudeproteins were isolated to examine the expression of Gli1, Gli2 and cyclin D1 by the Western blot analysis. The b-actin was used as a loading control.

M. Huang et al. / Cancer Letters 353 (2014) 32–40 39

the tumor microenvironment [35,36]. In the present study, rott-lerin inhibited the expression of IL-8 in tumor tissues, suggestingthat inhibition of IL-8 secreted by tumors could block tumor pro-gression by inhibiting or reversing EMT. Future work will need todetermine if rottlerin is capable of inhibiting metastatic processessimultaneously through multiple signaling pathways in pancreaticcancer.

In conclusion, the findings presented in this work are the first todemonstrate that rottlerin can inhibit pancreatic carcinogenesisthrough modulation of multiple signaling pathways. Specifically,we have shown that rottlerin inhibits AsPC-1 xenografted tumorgrowth by suppressing Akt, Notch and Shh pathways. Rottlerininhibits the production of pro-angiogenic IL-8 and VEGF/VEGFRas well as invasiveness-promoting MMP-2 and MMP-9, thus mod-ulating tumor microenvironment and inhibiting tumor growth.Furthermore, rottlerin inhibits growth of mouse pancreatic cellsobtained from KrasG12D mice by suppressing Akt, Notch and Shhpathways. These studies are important as they provide supportfor the potential role of rottlerin as a promising anticancer agent.Moreover, they offer a rational basis for the development of rott-lerin that may act either alone or in combination with presentlyavailable anticancer agents to enhance clinical activity and/orovercome cellular drug resistance.

Conflict of Interest

The authors have declared that there is no conflicts of interestsexist.

Authors’ contributions

MH, S-NT, GU, JLM and CJ performed the experiments, andanalyzed the data.

MH, S-NT and GU analyzed the data, and drafted and revised themanuscript.

SS and RKS designed the study, contributed reagents, andapproved the manuscript.

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