Fisetin, a natural flavonoid, targets chemoresistant human pancreatic cancer AsPC-1 cells through DR3-mediated inhibition of NF-κB

Fisetin, a natural flavonoid, targets chemoresistant human pancreatic cancer AsPC-1 cells through DR3-mediated inhibition of NF-κB
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  Fisetin, a natural flavonoid, targets chemoresistant humanpancreatic cancer AsPC-1 cells through DR3 mediated inhibitionof NF- κ B Imtiyaz Murtaza 1 , Vaqar Mustafa Adhami , Bilal Bin Hafeez , Mohammad Saleem , and HasanMukhtar  1 Department of Dermatology, University of Wisconsin-Madison, WI 53706 USA  Abstract Death receptors of the tumor necrosis factor (TNF) receptor super family have been implicated inconstitutive activation of Nuclear Factor kappa B (NF- κ  B) in pancreatic cancer (PaC) cells. In thisstudy we demonstrate that fisetin, a natural flavonoid, induces apoptosis and inhibits invasion of chemoresistant PaC AsPC-1 cells through suppression of DR3 mediated NF- κ  B activation. Fisetintreatment resulted in dose-dependent inhibition of PaC cell growth and cell proliferation withconcomitant induction of apoptosis. A cDNA array analysis revealed that fisetin modulatesexpression of more than 20 genes at transcription level with maximum decrease observed in DR3expression and a parallel increase observed in the expression levels of I κ  B α , an NF- κ  B inhibitor.Down-regulation of DR3 in PaC cells was found to down regulate activated pNF- κ  B/p65, pIkB α / β kinases (pIKK’s), MMP9 and XIAP that mostly impart chemoresistance in PaC. Immunoblottingand EMSA analysis showed a marked decrease in pNF- κ  B and NF- κ  B DNA binding activityrespectively with modest decrease in NF- κ  B promoter activity and significant decrease in MMP9 promoter activity with fisetin treatment. Importantly, consistent with these findings, we further found that transient down-regulation of DR3 by RNA interference significantly augmented fisetininduced changes in cell proliferation, cell invasion and apoptosis paralleled with decrease in pNF- κ  B, pIKK  α / β , MMP9, XIAP and NF- κ  B DNA binding activity. Blocking of DR3 receptor with anextra cellular domain blocking antibody demonstrated similar effects. These data provide evidencethat fisetin could provide a biological rationale for treatment of pancreatic cancer or as an adjuvantwith conventional therapeutic regimens. Keywords Pancreatic cancer; Fisetin; apoptosis; Invasion; DR3 INTRODUCTION Pancreatic cancer (PaC) represents one of the most aggressive tumor types with extremely poor prognosis.1 Constitutively activated nuclear factor kappa B (NF- κ  B) has beenassociated with a variety of aggressive tumor types, including pancreatic cancer.2-4Constitutive activation of NF- κ  B and its regulated genes strongly enhance invasive properties as well as impart chemoresistance to PaC cells.5 Members of TNF superfamilyincluding TNFR1, DR4, DR5, DR6, Fas and their ligands have been reported to play an 1 Correspondence to: Hasan Mukhtar , PhD. Helfaer Professor of Cancer Research, Director and Vice Chair of Research,Department of Dermatology, University of Wisconsin, 1300 University Avenue, MSC-25B, Madison, Wisconsin, 53706,, Phone: 608-263-3927, Fax: 608-263-5223; Imtiyaz Murtaza,  PhD. Address: Sher-e-Kashmir University of Agricultural Sciences & Technology, Shalimar Campus, Srinagar, 191121, J&K India  NIH Public Access Author Manuscript  Int J Cancer  . Author manuscript; available in PMC 2010 November 15. Published in final edited form as: Int J Cancer  . 2009 November 15; 125(10): 2465–2473. doi:10.1002/ijc.24628. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    important role in many cellular activities including proliferation, migration, differentiation,apoptosis, angiogenesis and inflammation and are highly expressed in number of  pathological conditions.6 The signaling pathways induced by these receptors are similar and rely on oligomerization of the receptor by ligand binding, recruitment of death domain proteins, such as TRADD, FAD, or TRAF2, RIP1, through homophilic interaction of their death domains, and subsequent activation of the caspases apoptotic cascade or thetranscription factor NF- κ  B.4 Members of TNF superfamily ligands have been shown toinduce NF- κ  B activation via binding to their cognate receptors.7 NF- κ  B regulates theexpression of genes that regulate transformation, tumor promotion, tumor invasion,angiogenesis, and metastasis.8DR3, a member of the TNF family and a receptor for TNF, is a protein on the surface of cells that has been reported to be capable of inducing NF- κ  B activation when over expressed in mammalian cells.9-10 Recent studies suggest that DR3-blocking agents might be moreeffective at specifically treating autoimmune disease since mice engineered to lack DR3were resistant to those diseases suggesting that blocking DR3 in mice, and possibly inhumans, is a potential therapy for these diseases.11 While earlier studies mostly confined expression of DR3 to lymphoid cells, however recent studies have reported its expression innon-lymphoid tissues and pancreatic cancer cells.12-13Fisetin (3,7,3 ′ ,4 ′ -tetrahydroxyflavone) a naturally occurring flavonoid found in many fruitsand vegetables exhibits a wide variety of functions including antioxidant, neurotropic,antiangiogenic and antiproliferative effects.14-19 Fisetin was found to suppress TNF, NF- κ  B, NF- κ  B-dependent reporter gene expression and activity.17 In this study we show thatfisetin mediates its apoptotic, anti-proliferative and anti-invasive effects in chemoresistantPaC cells by modulating DR3 receptor mediated down regulation of NF- κ  B signaling pathway. MATERIAL & METHODS Reagents and Antibodies Fisetin (3, 3 ′ , 4 ′ , 7-Tetrahydroxyflavone) 99% pure was purchased from Sigma (St. Louis,MO). Antibodies against XIAP, DR3, pNF- κ  B/p65, NF- κ  B/p65, I κ  B α , pI κ  B α , IKK  α / β  and MMP9 were obtained from Cell Signaling (Beverly, MA), anti-PARP/116 from Upstate(Lake Placid, NY) and anti-PARP/85 from Promega (Madison, WI). Horseradish peroxidase(HRP) conjugated secondary antibodies were obtained from Amersham (Arlington Height,IL). DR3-shRNA and scrambled-shRNA were purchased from Qiagen (Valencia, CA). BCAProtein assay kit was obtained from Pierce (Rockford, IL). Novex precast Tris-glycine gelswere obtained from Invitrogen (Carlsbad, CA). Annexin-V-Fluos staining kit was purchased from Roche (Indianapolis, IN). Cell culture and treatment Human PaC cell line AsPC-1 was obtained from ATCC (Manassas, VA) and grown inappropriate media supplemented with 10% FBS and 1% Penicillin-Streptomycin under standard cell culture conditions. A stock solution of fisetin (25 mM) was prepared bydissolving in dimethyl sulfoxide (DMSO, 0.1% v/v). The cells (60-70% confluent) weretreated with fisetin (0-80 μ M) for 24 and 48 h in complete growth medium and cell viabilitywas performed and lysates prepared for Western blotting or stored at − 80 °C for later use. Cell viability assay The effect of fisetin on cell viability was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazoliumbromide) assay as described earlier.16 Murtaza et al.Page 2  Int J Cancer  . Author manuscript; available in PMC 2010 November 15. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t     Apoptosis assay The annexin-V-Fluos staining kit was used for the detection of apoptotic cells according tovendor’s protocol and described earlier.16 The AsPC-1 cells were grown to 70% confluenceand treated with fisetin (10-60 μ M) for 48 h. [ 3 H]-Thymidine incorporation assay AsPC-1 cells (60% confluent) grown in 24 well culture plates were subjected to fisetintreatment for 48 h, the last 16 h of which were in the presence of [ 3 H]thymidine (0.5 μ Ci/ml). Cells were then washed twice with PBS at room temperature and then with ice-cold 5%trichloroacetic acid (TCA). The cells were next incubated with TCA solution on ice for 30min and subsequently the acid-insoluble fraction was dissolved in 1 ml of 0.5M NaOH.Incorporated [ 3 H]thymidine was quantified by liquid scintillation counting. Microarray analysis Twenty four hours post incubation with fisetin, treated and untreated cells were harvested and RNA was isolated by using RNAeasy kit (Qiagen, Valencia, CA). Next, 4 μ g of RNAwas enzymatically converted into cDNA. The cDNA was then biotinylated with the TrueLabeling-AMP™ 2.0 Kit as per vendor’s protocol (Super Array, Frederick, MD). Thelabeled probes were hybridized on the arrays (Human Cancer Pathway Finder, Superarray,Frederick, MD) imprinted with 113 genes representative of the six biological pathwaysinvolved in transformation and tumsrcenesis. Hybridization was followed by detection withthe chemiluminescent reagents and x-ray film development. Data was acquired and analyzed  by using GE superarray software (Superarray, Frederick, MD). DR3-shRNA transfection Transfections were performed by using nucleofection kit (Amaxa, Walkersville, MD).Briefly, 10 6  cells were transfected with 50 nM of shRNA directed against DR3. Controlcells were transected with scrambled shRNA (50 nM). After overnight incubation,transfected cells were treated with fisetin (20-40 μ M) and harvested after 24 h post fisetintreatment. Cell lysates were processed for immunoblot analysis. In addition, in a parallel setof experiments, annexin-V assay and cell invasion assay were performed. Western Blot Analysis Cell lysates were prepared in cold lysis buffer [(0.05 mmol/L Tris-HCl, 0.15 mmol/L NaCl,1 mole/L EGTA, 1 mol/L EDTA, 20 mmol/L NaF, 100 mmol/L Na 3 VO4, 0.5% NP-40, 1%Triton X-100, 1 mol/L phenyl methylsulfonyl flouride (pH 7.4) with freshly added proteaseinhibitor cocktail (Calbiochem, La Jolla, CA). The lysate was collected and protein contentmeasured by BCA method. For Western blotting, 40 μ g protein was resolved over 12% Tris-glycine polyacrylamide gels under non-reduced conditions, transferred onto nitrocellulosemembranes and subsequently incubated in blocking buffer (5% nonfat dry milk/1% Tween20; in 20mmol/L TBS, pH 7.6) for 2 hours. The blots were incubated with appropriate primary antibody, washed and incubated with appropriate secondary HRP-conjugated antibody. The blots were detected with chemiluminescence and autoradiography, usingXAR-5 film (Kodak, Rochester, NY). Equal loading of protein was confirmed by strippingthe blots and reprobing with β -actin. Wound closure assay Cells were plated in 24 well plates. When the cells were ~90% confluent, a wound wasinduced on the monolayer cells by scraping a gap using a micropipette tip and then fisetinwas added immediately after the wound induction. The speed of wound closure was Murtaza et al.Page 3  Int J Cancer  . Author manuscript; available in PMC 2010 November 15. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    compared between fisetin treated and untreated control group. Photographs were taken 24 hafter wound incision. Chemoinvasion assay We used a chemoinvasion kit (Millipore, Danvers, MA) for cell invasion assay. Confluentcells were adjusted to 10 6  cells per 100 μ l of cell suspension, resuspended in serum-freemedia with appropriate concentration of fisetin, and applied onto the matrigel-filled transwell chamber. After 14 h incubation, noninvasive cells were scraped off with a cottonswab and invasive cells were fixed and stained for 10 minutes with crystal violet (0.5% in20% methanol) and washed with water. Gelatin zymography Cells were seeded at a density of 10 6  and grown in medium containing 10% FBS. After 24h, appropriate concentrations of fisetin were added and cells were allowed to grow for additional 24 h. Supernatant was collected and cleared by centrifugation, and cells weredetached from the wells with trypsin and counted. Supernatants (normalized to cellnumbers) were then loaded onto zymogram gels. After electrophoresis, the gel wasincubated with renaturing buffer for 30 min at room temperature and incubated overnight at37 °C with zymogram developing buffer. The gel was stained with 0.02% Coomassie brilliant blue in 20% acetic acid. Molecular weights of the gelatinolytic bands wereestimated using molecular weight markers. Transcriptional activity of NF- κ B and MMP9 The human MMP-9 promoter luciferase plasmid (pGL2-MMP-9-luc )  was received as a kind gift. Empty pGL2 was procured from Upstate Laboratories (Lake Placid, NY). All plasmidswere transformed in agar media and extracted by using Maxiprep kit (Qiagen, Valencia,CA). Cells plated at a density of 5 × 10 4  cells/well were transfected with the plasmids(200ng/well) for 24 h.  Renilla  luciferase (20 ng/well, pRL-TK; Promega, Madison, WI) wasused as an internal control. In addition, for controls, the same amount of empty vectors, weretransfected in cells. After 12 h post-transfection, cells were treated with fisetin (5-10 μ M)and incubated for 24 h. The cells were then harvested and transcriptional activity wasmeasured in terms of luciferase activity by using dual-luciferase reporter assay system(Promega, Madison, WI). Relative luciferase activity was calculated with the values fromvector alone group with or without Fisetin treated group. Nuclear extract preparation and electrophoresis mobility shift assays (EMSA) EMSA for NF- κ  B was performed using lightshift™ chemiluminiscent EMSA kit (Pierce,Rockford, IL) as per manufacturer’s protocol and described earlier [20]. Effect of fisetin on cell surface expression of DR3 For analysis of cell surface expression of DR3, fisetin treated cells were harvested and suspended in Dulbecco’s PBS containing 1% FBS and 0.1% sodium azide. The cells were preincubated with 10% goat serum for 20 min and washed, and then monoclonal rabbit IgGanti-DR3 antibodies were added. Following 1 h incubation at 4 °C, cells were washed and incubated for an additional 1 h in FITC-conjugated goat anti-rabbit IgG antibody. The cellswere analyzed using a FACS Calibur flow cytometer and Cell Quest acquisition and analysis programs (BD Biosciences, San Jose, CA). Effect of blocking of DR3 extracellular domain with antibody A DR3 specific antibody was used at a concentration of 5 μ g/ml to further ascertain the roleof DR3 in induction of apoptosis and invasion in AsPC-1 cells. AsPC-1 cells were treated  Murtaza et al.Page 4  Int J Cancer  . Author manuscript; available in PMC 2010 November 15. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    with either a DR3 antibody, 20 μ M fisetin or a combination of both. Cells were analyzed for apoptosis induction, invasion and DR3 expression as detailed above. Statistical analyses Student’s t test for independent analysis was applied to evaluate differences between thetreated and untreated groups with respect to the expression of various proteins. A p-value of <0.05 was considered to be statistically significant. RESULTS Effect of fisetin on cell growth and viability Recently, it has been shown that fisetin caused significant growth-inhibitory effects ondifferent cancer cells in a time and dose-dependent manner [14-19]. To evaluate the effect of fisetin on the growth of human PaC cells we selected AsPC-1 cells. The choice of these cellswas based on the fact that these cells demonstrate resistance to conventionalchemotherapeutic regimens. Treatment of AsPC-1 cells with fisetin resulted in a dose-dependent growth inhibition with an IC 50  of 38 μ M at 48 h (Figure 1A). These resultssuggested that the cell line AsPC-1 that is highly resistant to currently availablechemotherapeutic drugs remarkably showed sensitivity to fisetin treatment. Effect of fisetin on AsPC-1 cell proliferation It is well known that proliferating cells exhibit increased [ 3 H]-thymidine incorporation intoDNA which arises from increased growth factor expression and activity in cancer cells. Next, we determined the effect of fisetin treatment (20-40 μ M) on the rate of proliferation of AsPC-1 cells by measuring the rate of uptake of thymidine by the dividing cells. Weobserved that treatment of cells with fisetin significantly decreased the thymidineincorporation in a dose-dependent manner, further validating the anti-proliferative efficacyof fisetin against human PaC AsPC-1 cells (Figure 1B). Effect of fisetin on apoptosis induction in AsPC-1 cells To investigate whether fisetin induced apoptosis in AsPC-1 cells we used the annexin-Vstaining and PARP cleavage as a measure of apoptosis. Annexin-V specially binds to phosphatidylserine and has been employed for determination of apoptotic cells. AsPC-1cells treated with fisetin (10-60 μ M; 48 h) showed dose-dependent induction of apoptosis(Figure 1C). Additionally, PARP cleavage as detected by immunoblot analysis showed thatthe full size PARP (116 KD) protein was cleaved to yield an 85 KD fragment after treatmentwith fisetin (Figure 1D). Gene expression profile of fisetin treated AsPC-1 cells  Next, we analyzed the effect of fisetin treatment of AsPC-1 cells on a subset of 113 geneswith known relevance to cancer cell transformation and tumsrcenesis (Figure 2). Compared to vehicle treated cells, fisetin treatment caused significant modulation in the expression of many genes that were either unregulated or downregulated ranging between 1.5- and 7-fold (supplementary table 1). The highly upregulated genes were mostly related to cell cyclecontrol, invasion, and metastasis (p21, p16, I κ  B α , NME4, KISS1) and downregulated genesreported to be antiapoptotic, such as BCL2L1 and genes having their role in cell invasion, proliferation and metastasis like NF- κ  B, MMP9, EGFR, HER-2. Interestingly, death domain containing TNFR superfamily members TNFR1 and TNFRSF25 (DR3) receptors werehighly down regulated on fisetin treatment, with DR3 showing the maximum modulationamong all the selected genes. These data support the hypothesis that fisetin has a broader effect on cell survival, proliferation, invasion and metastasis of PaC cells. Murtaza et al.Page 5  Int J Cancer  . Author manuscript; available in PMC 2010 November 15. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  
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