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- Publisher
- American Society for Biochemistry and Molecular Biology
- Copyright
- Copyright © 2010 Elsevier Inc.
- ISSN
- 0021-9258
- eISSN
- 1083-351X
- DOI
- 10.1074/jbc.m109.095240
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- See Article on Publisher Site
Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 33, pp. 25332–25344, August 13, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Inhibition of NFB and Pancreatic Cancer Cell and Tumor Growth by Curcumin Is Dependent on Specificity Protein □ S Down-regulation Received for publication, December 15, 2009, and in revised form, May 10, 2010 Published, JBC Papers in Press, June 9, 2010, DOI 10.1074/jbc.M109.095240 ‡1 ‡1 § ‡,2 Indira Jutooru , Gayathri Chadalapaka , Ping Lei , and Stephen Safe From the Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843 and the Institute of Biosciences and Technology, Texas A&M Health Sciences Center, Houston, Texas 77030 Curcumin activates diverse anticancer activities that lead to specific gene polymorphisms (4, 5). In addition to heritable inhibition of cancer cell and tumor growth, induction of apopto- mutations, several acquired gene mutations have been identi- sis, and antiangiogenic responses. In this study, we observed fied in sporadic pancreatic tumors (6, 7). The K-Ras oncogene is that curcumin inhibits Panc28 and L3.6pL pancreatic cancer primarily mutated in codon 12 in 90% of pancreatic tumors cell and tumor growth in nude mice bearing L3.6pL cells as and the mutation results in a constitutively active form of ras xenografts. In addition, curcumin decreased expression of p50 that can lead to increased cell proliferation. Mutations in the and p65 proteins and NFB-dependent transactivation and also cyclin-dependent kinase inhibitor p16, the tumor suppressor decreased Sp1, Sp3, and Sp4 transcription factors that are over- gene p53, and SMAD4, a downstream target of transforming expressed in pancreatic cancer cells. Because both Sp transcrip- growth factor also exhibit high mutation frequencies in pan- tion factors and NFB regulate several common genes such as creatic tumors. cyclin D1, survivin, and vascular endothelial growth factor that Because pancreatic cancers are frequently detected at an contribute to the cancer phenotype, we also investigated inter- advanced stage, treatments have provided very limited actions between Sp and NFB transcription factors. Results of improvements in tumor regression and overall survival times Sp1, Sp3, and Sp4 knockdown by RNA interference demonstrate after diagnosis (8, 9). 5-Fluorouracil alone or in combination that both p50 and p65 are Sp-regulated genes and that inhibition with other drugs has been extensively used for treatment of of constitutive or tumor necrosis factor-induced NFB by cur- advanced pancreatic cancer, and gemcitabine, a deoxycytidine cumin is dependent on down-regulation of Sp1, Sp3, and Sp4 analog (or antimetabolite), has partially replaced 5-fluorouracil proteins by this compound. Curcumin also decreased mito- as a treatment for pancreatic cancer. Gemcitabine provides chondrial membrane potential and induced reactive oxygen increased clinical benefits in terms of response rate, time to species in pancreatic cancer cells, and this pathway is required progression, and median survival and several other drugs for for down-regulation of Sp proteins in these cells, demonstrating treatment of pancreatic cancer are also being investigated (10– that the mitochondriotoxic effects of curcumin are important 13). Curcumin (diferuloylmethane) is a polyphenolic phyto- for its anticancer activities. chemical that exhibits a broad spectrum of anticancer activities against multiple tumor types (14–16), including pancreatic cancer (17–21). Curcumin decreased survival and induced apo- ptosis in pancreatic cancer cells and, in the same cells, curcu- Pancreatic ductal adenocarcinoma is a major cause of can- cer-related deaths in developed countries and, in 2009, it is min also decreased pro-survival nuclear factor B (NFB) DNA binding in a gel mobility shift assay (17). Treatment of athymic estimated that in excess of 34,000 new cases will be diagnosed in nude mice with orthotopically implanted tumors with 1 g/kg of the United States (1). Pancreatic ductal adenocarcinoma is a curcumin daily did not inhibit tumor volume but in combina- highly aggressive disease that invariably evades early diagnosis (2, 3). The mean survival time for patients with metastatic dis- tion studies, curcumin enhanced the activity of gemcitabine as an inhibitor of pancreatic tumor growth (19). Curcumin also ease is only 3–6 months, and only 20–30% of pancreatic cancer decreased several NFB-regulated genes in tumors and these cases are alive after 12 months. Several factors are associated include cyclin D1,c-myc, bcl-2, cyclooxygenase-2 (COX-2), with increased risk for pancreatic cancer and these include chronic pancreatitis, prior gastric surgery, smoking, diabetes, and vascular endothelial growth factor (VEGF) (19). Recent studies in this laboratory demonstrated that the anti- exposure to certain classes of organic solvents, radiation, and cancer activity of curcumin in bladder cancer cells and tumors was associated with repression of specificity protein (Sp) tran- * This work was supported, in whole or in part, by National Institutes of Health Grants R01CA108718 and R01CA136571 and a grant from Texas A&M AgriLife Research. □ S 3 The on-line version of this article (available at http://www.jbc.org) contains The abbreviations used are: COX-2, cyclooxygenase 2; Sp, specificity pro- supplemental Figs. S1–S6. tein; ROS, reactive oxygen species; DMEM, Dulbecco’s modified Eagle’s Both authors contributed equally to this work. medium; PARP, poly(ADP-ribose) polymerase; FACS, fluorescence-acti- To whom correspondence should be addressed: Dept. of Veterinary Physi- vated cell sorter; DMSO, dimethyl sulfoxide; VEGF, vascular endothelial ology and Pharmacology, Texas A&M University, 4466 TAMU, Veteterinary growth factor; FBS, fetal bovine serum; TNF, tumor necrosis factor ; DTT, Research Bldg., 410 College Station, TX 77843-4466. Tel.: 979-845-5988; dithiothreitol; siRNA, small interfering RNA; MMP, mitochondrial mem- Fax: 979-862-4929; E-mail: [email protected]. brane potential. This is an Open Access article under the CC BY license. 25332 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 33 •AUGUST 13, 2010 Effects of Curcumin on NFB Are Sp-dependent scription factors Sp1, Sp3, and Sp4, which was accompanied by Coulter Z1 particle counter. Each experiment was done in trip- decreased expression of Sp-regulated survival, angiogenic and licate and results are expressed as mean S.E. for each treat- growth promoting genes (22). In this study, we show that cur- ment group. cumin also decreased expression of Sp proteins and Sp-depen- Transfection and Luciferase Assay—The pancreatic cancer dent gene products in pancreatic cancer cells and mouse cells (1 10 per well) were plated in 12-well plates in DMEM/ tumors (xenograft). Moreover, in pancreatic cancer cells, the Ham’s F-12 medium supplemented with 2.5% charcoal- p65 and p50 subunits of NFB are also Sp-regulated genes and stripped FBS. After 24 h, various amounts of DNA (i.e. 0.4 gof inhibition of constitutive and induced NFB expression by cur- PGL2-Luc, 0.4 g of PGL2-Luc, 0.04 gof -galactosidase, and cumin is also due, in part, to down-regulation of Sp transcrip- 0.4 g of pSp1 (4)-Luc, 0.4 g of pSp3-Luc, 0.4 g of VEGF tion factors. Moreover, the mechanism of Sp down-regulation (2068)-Luc, 0.4 g of pSurvivin (269)-Luc) were transfected by curcumin is due to the mitochondriotoxicity of this com- using Lipofectamine reagent according to the manufacturer’s pound and the subsequent induction of reactive oxygen species protocol. Five h post-transfection, the transfection mixture was (ROS). replaced with complete medium containing either vehicle (DMSO) or the indicated compound in DMSO. After 22 h, cells EXPERIMENTAL PROCEDURES were then lysed with 100 lof1 reporter lysis buffer, and cell Cell Lines—The Panc28 cell line was a generous gift from Dr. extracts (30 ml) were used for luciferase and -galactosidase Paul Chiao and L3.6pL cells were kindly provided by Dr. Isaiah assays. A Lumicount luminometer was used to quantitate lucif- Fidler (University of Texas M.D. Anderson Cancer Center, erase and -galactosidase activities, and the luciferase activities Houston, TX). Panc1 and PC3 cells were obtained from ATCC were normalized to -galactosidase activity. (Manassas, VA) and RKO cells were kindly provided by Dr. Western Blots—Pancreatic cancer cells were seeded in Stanley Hamilton (M.D. Anderson Cancer, Houston, TX). DMEM/Ham’s F-12 medium containing 2.5% charcoal- Antibodies and Reagents—Both pancreatic cancer cell lines stripped FBS and after 24 h, cells were treated with either vehi- were maintained in Dulbecco’s modified Eagle’s medium cle (DMSO) or the indicated compounds. Cells were collected (DMEM)/F-12 supplemented with 5% FBS, 0.22% sodium using high-salt buffer (50 mmol/liter of HEPES, 0.5 mol/liter of bicarbonate, and 10 ml/liter of 100 antibiotic/antimycotic NaCl, 1.5 mmol/liter of MgCl , 1 mmol/liter of EGTA, 10% mixture solution (Sigma). Cells were grown in 150-cm culture glycerol, and 1% Triton X-100) and 10 l/ml of Protease Inhib- plates in an air/CO (95:5) atmosphere at 37 °C. Cyclin D1, Sp3, itor Mixture (Sigma). Protein lysates were incubated for 3 min Sp4, VEGF, GKLF4, c-jun, and p50 antibodies were purchased at 100 °C before electrophoresis, and then separated on 10% from Santa Cruz Biotechnology (Santa Cruz, CA). Cleaved SDS-PAGE at 120 V for 3–4 h. Proteins were transferred onto PARP and COX-2 antibody were purchased from Cell Signaling polyvinylidene difluoride membranes by wet electroblotting in Technology (Danvers, MA) and Sp1 antibody was purchased a buffer containing 25 mmol/liter of Tris, 192 mmol/liter of from Millipore (Billerica, MA). Survivin antibody was pur- glycine, and 20% methanol for 1.5 h at 180 mA. Membranes chased from R&D Systems (Minneapolis, MN). NFB-p65 anti- were blocked for 30 min with 5% TBST-Blotto (10 mmol/liter of body was from Abcam (Cambridge, MA). Monoclonal -actin Tris-HCl, 150 mmol/liter of NaCl (pH 8.0), 0.05% Triton X-100, antibody was purchased from Sigma. Horseradish peroxidase and 5% nonfat dry milk) and incubated in fresh 5% TBST-Blotto substrate for Western blot analysis was obtained from Milli- with 1:500 primary antibody overnight with gentle shaking at pore. Dithiothreitol and -L-glutamyl-L-cysteinyl-glycine (GSH) 4 °C. After washing with TBST for 10 min, the polyvinylidene were obtained from Sigma. TNF was purchased from R&D difluoride membrane was incubated with secondary antibody Systems. Curcumin (98% pure) was purchased from Indofine (1:5000) in 5% TBST-Blotto for2hby gentle shaking. The Chemical Company, Inc. (Hillsborough, NJ). Lipofectamine membrane was washed with TBST for 10 min, incubated with 6 and Lipofectamine 2000 was purchased from Invitrogen. Lucif- ml of chemiluminescence substrate for 1 min, and exposed to erase reagent was from Promega (Madison, WI). -Galactosid- Kodak image station 4000 mm Pro (Carestreamhealth, Wood- ase reagent was obtained from Tropix (Bedford, MA). The bridge, CT). VEGF and survivin promoter constructs were provided by Drs. Electrophoretic Mobility Shift Assay—Cells were rinsed in Gerhard Siemeister and Gunter Finkenzeller (Institute of cold phosphate-buffered saline buffer and harvested in reporter Molecular Medicine, Tumor Biology Center, Freiburg, Ger- lysis buffer (Promega). After a 15-min incubation on ice and many) and Dr. M. Zhou (Emory University, Atlanta, GA) 10-min centrifugation at 16,000 g, 4 °C, the pellet was resus- respectively. Sp1 and Sp3 promoter constructs were kindly pended in 1 reporter lysis buffer (Promega) supplemented provided by Drs. Carlos Cuidad and Veronique Noe with 0.5 mol/liter of KCl and incubated on ice for 30 min. The (University of Barcelona, Barcelona, Spain). NFB promoter supernatant containing nuclear proteins was collected after construct was purchased from Stratagene (Cedar Creek, centrifugation for 10 min at 16,000 g at 4 °C, and quantified TX). for protein concentrations by the Bradford method. The NFB Cell Proliferation Assay—Pancreatic cancer cells (1 10 per probe was prepared by annealing the two complementary well) were plated in 12-well plates and allowed to attach for polynucleotides, the NFB sense strand probe was 5-AGT 24 h. The medium was then changed to DMEM/Ham’s F-12 TGA GGG GAC TTT CCC AGG C-3. The annealed probe was medium containing 2.5% charcoal-stripped FBS, and vehicle 5-end labeled using T4 polynucleotide kinase (Invitrogen) and (DMSO), GSH, DTT, and/or curcumin were added. Cells were [- P]ATP (PerkinElmer Life Sciences). The labeled probe was then trypsinized and counted at the indicated times using a purified with the Chroma Spin TE-10 column (BD Biosciences, AUGUST 13, 2010• VOLUME 285 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 25333 Effects of Curcumin on NFB Are Sp-dependent San Jose, CA). The electrophoretic mobility shift assay reaction turer’s protocol using JC-1 dye and mitochondrial membrane was carried out in the reporter lysis buffer supplemented with potential shift was measured using FACS Calibur flow cytome- 0.1 mol/liter of KCl. Each reaction contained 2 g of nuclear ter using CellQuest acquisition software (BD Biosciences). J-ag- protein, 1 g of poly(dI-dC) (Roche Molecular Biochemicals) gregates are detected as red fluorescence and J-monomers are with or without unlabeled competitor oligonucleotides, and 10 detected as green fluorescence. fmol of labeled probe; the mixture was incubated for 15 min on Immunohistochemistry—Tissue sections were deparaf- ice. Protein-DNA complexes were resolved by 5% native PAGE finized in xylene and treated with a graded series of alcohol and at 160 V at room temperature for 1.5 h and visualized after rehydrated in phosphate-buffered saline. Antigen retrieval was exposing it to ImageTek-H autoradiography X-Ray film. done using 10 mM sodium citrate (pH 6.0–6.2) and endogenous siRNA Interference Assays—siRNAs for Sp1, Sp3, Sp4, p65, peroxidase was blocked by the 3% hydrogen peroxide in meth- p50, and LMN were purchased from Sigma. The siRNA com- anol for 6 min. Slides were then incubated with blocking serum plexes used in this study are indicated as follows: LMN, (Vecstatin ABC Elite kit, Vector Laboratories, Burlingame, CA) SASI_Hs02_00367643; Sp1, SASI_Hs02_00363664; Sp3, for 45 min. Samples were then incubated overnight with Sp1, 5-GCG GCA GGU GGA GCC UUC ACU TT; Sp4, 5-GCA Sp3, Sp4, VEGF, cyclin D1, survivin, p50, and p65 antibodies at GUG ACA CAU UAG UGA GCT T; p65 (REL1096), 5-GAT 4 °C. Sections were then washed in phosphate-buffered saline/ TGA GGA GAA ACG TAA ATT; and p50 (REL 1911), 5-GTC Tween and then incubated with biotinylated secondary anti- ACT CTA ACG TAT GCA ATT. body followed by streptavidin. The brown staining specific for The Panc28 and L3.6pL pancreatic cancer cell lines were antibody binding was developed by exposing the avidin and seeded (6 10 per well) in 12-well plates in DMEM/Ham’s biotinylated peroxidase complex to diaminobenzidine reagent F-12 medium supplemented with 2.5% charcoal-stripped FBS (Vector Laboratories) and sections were then counterstained without antibiotic and left to attach for 1 day. The triple Sp with hematoxylin (Vector Laboratories). siRNA knockdown (iSp1, iSp3, and iSp4 complex) along with Quantitative Real Time PCR of mRNA—cDNA was prepared iLamin as control was performed using Lipofectamine 2000 from Panc28 and L3.6pL cell lines using Superscript II reverse transfection reagent as per the manufacturer’s instructions. transcriptase (Invitrogen) according to the manufacturer’s pro- Xenograft Study—Female athymic nude mice, age 4 to 6 tocol. Each PCR was carried out in triplicate in a 20-l volume weeks, were purchased from Harlan. L3.6pL cells (3 10 )ina using SYBR GreenER (Invitrogen) at 95 °C for 10 min, then 40 1:1 ratio of Matrigel (BD Biosciences) were injected into the cycles of 95 °C for 15 s and 60 °C for 1 min in the Applied either side of the flank area of nude mice. Seven days after Biosystems 7500 Fast Real-time PCR System. The following tumor cell inoculation, mice were divided into two groups of 10 primers were used: p50 (forward), 5-ACCCTGACCTTGCC- animals each. The first group received 100 l of vehicle (corn TATTTG-3 and (reverse). 5-AGCTCTTTTTCCCGATC- oil) by intraperitoneal injection, and the second group of ani- TCC-3; p65 (forward), 5-CGGGATGGCTTCTATGAGG-3 mals received 100 mg/kg/day injection of curcumin in corn oil and (reverse) 5-CTCCAGGTCCCGCTTCTT-3. The primers every 2nd day for 18 days (9 doses) by intraperitoneal injection. for TBP, Sp1, Sp3, and Sp4 genes have previously been The mice were weighed, and tumor areas were measured described (23). throughout the study. After 20 days, the animals were sacri- Fluorescence-activated Cell Sorting Assays—Both Panc28 ficed; final body and tumor weights were determined and and L3.6pL pancreatic cancer cells were treated with vehicle, plotted. curcumin, or siRNA for iLamin or Sp1/3/4. Cells were analyzed GSH Estimation—GSH-Glo Glutathione assay kit (Promega) on a FACSCalibur flow cytometer using CellQuest acquisition was used to estimate GSH levels according to the manufactur- software (BD Biosciences). Propidium iodide fluorescence was er’s protocol using a 96-well cell culture plate and luminescence collected through a 585/42-nm band pass filter, and list mode measured using a Lumicount luminometer. data were acquired on a minimum of 20,000 single cells defined ROS Estimation—Cellular ROS levels were evaluated with by a dot plot of propidium iodide width versus propidium the cell permeant probe CM-H DCFDA (5-(and-6)-chlorom- iodide area. Data analysis was performed using Modfit LT soft- ethyl-27-dichlorodihydrofluorescein diacetate acetyl ester) ware (Verity Software House, Topsham, ME). from Invitrogen. Following 20–24 h treatment, cells plated on a Statistical Analysis—Statistical significance of differences 96-well cell culture plate were loaded with 10 M CM- between the treatment groups was determined by an analysis of H DCFDA for 30 min, washed once with serum-free medium, variance and Student’s t test, and levels of probability were and analyzed for ROS levels using the BioTek Synergy 4 plate noted. IC values were calculated using linear regression anal- reader (Winooski, VT) set at 480 and 525 nm excitation and ysis and expressed in micromolar at 95% confidence intervals. emission wavelengths, respectively. Following reading of ROS, RESULTS cultures were then treated with Janus green and cell counts were determined with the plate reader set to an absorbance of Curcumin Inhibits Constitutive NFB—Fig. 1 illustrates the 610 nm, and ROS intensities were then corrected accordingly. concentration-dependent inhibition of Panc28 and L3.6pL Each experiment was done in triplicate and results are pancreatic cancer cell proliferation after treatment with 10–50 expressed as mean S.E. for each treatment group. M curcumin for 24 h, and IC values for this response were Measurement of Mitochondrial Membrane Potential 34.0 and 28.8 M, respectively. After prolonged treatment of (MMP)—MMP was measured with Mitochondrial Membrane these cells for 96 and 144 h, IC values were 12.4 and 11.2 M Potential Detection Kit (Stratagene) according to the manufac- and 11.8 and 9.9 M in Panc28 and L3.6pL cells, respectively, 25334 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 33 •AUGUST 13, 2010 Effects of Curcumin on NFB Are Sp-dependent Panc28 and L3.6pL cells represent gemcitabine-resistant and non-re- sistant cell lines, respectively. The effects of curcumin on distribution of cells in G /G ,S,andG /M phases of o 1 2 the cell cycle were determined 0–24 h after treatment with 50 M curcumin (supplemental Fig. S1B). In Panc28 cells, curcumin decreased G /M and increased the percentage of cells in S phase over time and this effect was also observed in L3.6pL cells after treatment for 24 h. Curcumin-depen- dent inhibition of DNA replication as evidenced by inhibition of G /G 0 1 to S phase progression was not observed. Interactions between cur- cumin and NFB signaling have been extensively reported (14–16). Fig. 1B illustrates Western blots of nuclear extracts from DMSO- and curcumin (35 and 50 M)-treated L3.6pL and Panc28 cells; 35 M decreased p65 and p50 protein lev- els in L3.6pL but not Panc28 cells, whereas 50 M curcumin decreased expression of both proteins in both cell lines. It has also been reported that curcumin decreases NFB binding to its cognate response ele- ment in gel mobility shift assays (17), and Fig. 1C summarizes bind- ing nuclear extracts from L3.6pL cells treated with DMSO (solvent) or curcumin (25 and 50 M)toan oligonucleotide containing a con- sensus NFB site. The free probe in the absence of nuclear extract (lane 1) did not form a retarded band; however, nuclear extracts from sol- vent-treated cells formed an NFB retarded band (lane 2, indicated by an arrow). The retarded band inten- sity was decreased when nuclear FIGURE 1. Curcumin inhibits pancreatic cancer cell growth, decreases expression of p65, p50 proteins, NFB-DNA binding, and transactivation of the NFB promoter. A, inhibition of Panc28 and L3.6pL cell extracts from cells treated with 25 growth. Cells were treated with DMSO (solvent control) or 10, 25, 35, or 50 mol/liter of curcumin, and effects or 50 M curcumin were used (lanes on cell growth were determined after treatment for 24 h as described under “Experimental Procedures.” 3 and 4). The intensity of the NFB- B, effects of curcumin on p65 and p50 subunits of NFB in Panc28 and L3.6pL cells. Cells were treated with DMSO (0), 35 or 50 mol/liter of curcumin for 24 h, and p65 and p50 protein levels in nuclear extracts were DNA complex (lane 2) was decreased determined as described under “Experimental Procedures.” -Actin served as loading control. C, gel mobility after competition with 100-fold shift assay. Panc28 and L3.6pL cells were treated with DMSO or 25 or 50 mol/liter of curcumin for 24 h, and nuclear lysates were incubated with P-labeled GC-rich oligonucleotide alone or in the presence of other excess of unlabeled wild-type (lane factors. Retarded bands were analyzed by electrophoretic mobility shift assay as described under “Experimen- 5) but not mutant (lane 6)NFB oli- tal Procedures.” D, decrease in transactivation of NFB promoter. Panc28 and L3.6pL cells were transfected gonucleotide. In addition, the inten- with the NFB-luc construct, then treated with DMSO or 25 and 50 M curcumin, and luciferase activity was determined as described under “Experimental Procedures.” Results are expressed as mean S.E. for three sity of the NFB-DNA complex was replicate determinations for each treatment group, and significant (p 0.05) compared with solvent (DMSO) also decreased after incubation with control indicated by an asterisk. EMSA, electrophoretic mobility shift assay. p50/p65 (combined) antibodies due and concentrations of curcumin required for growth inhibition to immunodepletion (lane 7); however, we did not observe a decreased with increasing treatment times as observed for supershift complex with these antibodies. We also investigated many anticancer drugs. Supplemental Fig. S1A shows that the effects of curcumin on NFB-dependent transactivation in AUGUST 13, 2010• VOLUME 285 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 25335 Effects of Curcumin on NFB Are Sp-dependent pancreatic cancer cells transfected with pNFB-luc, a construct con- taining 5 tandem NFB response elements linked to a luciferase reporter gene. The results show that curcumin decreased luciferase activity (Fig. 1D) and this was con- sistent with results in Fig. 1, A–C, showing that curcumin repressed constitutive NFB primarily through down-regulation of p50 and p65 proteins. Curcumin Decreases Sp Tran- scription Factors and Sp-dependent Responses—Recent studies in this laboratory showed that curcumin decreased expression of Sp tran- scription factors Sp1, Sp3, and Sp4 in bladder cancer cells (22). We also observed that these proteins were overexpressed in L3.6pL and Panc28 (Fig. 2A) cells, and curcu- min induced a concentration- dependent decrease of these pro- teins in both cell lines. In bladder cancer cells, curcumin-induced Sp down-regulation was blocked by proteasome inhibitors; however, results in Fig. 2B show that the pro- teasome inhibitor MG132 did not alter the effects of curcumin on Sp1, Sp3, and Sp4 protein expression in pancreatic cancer cells. Curcumin also decreased Sp3 and Sp4 mRNA in both cell lines and decreased Sp1 mRNA in L3.6pL but not in Panc28 cells (supplemental Fig. S2). Curcu- min also decreased expression of several Sp-dependent genes in Panc28 and L3.6pL cells and these included VEGF, VEGFR1, cyclin D1, and survivin (Fig. 2C). This was accompanied by increased PARP cleavage, a marker of apoptosis. Using Panc28 cells as a model, cur- cumin decreased luciferase activity in cells transfected with constructs FIGURE 2. Curcumin activates proteosome-independent down-regulation of Sp proteins, decreases cell containing GC-rich promoter inserts growth, angiogenic and apoptotic proteins, and their promoters. A, decreased Sp proteins. Panc28 and from the Sp1 (pSp1For4), Sp3 L3.6pL cells were treated with DMSO or 10, 25, 35, and 50 mol/liter of curcumin for 24 h and whole cell lysates were analyzed by Western blot analysis as described under “Experimental Procedures.” B, curcumin causes (pSp3For5), VEGF (pVEGF), and proteasome-independent Sp degradation. Cells were treated with DMSO or 50 mol/liter of curcumin in the survivin (pSurvivin) genes linked to presence or absence of proteasome inhibitor MG132, and the effects on Sp protein degradation were deter- a luciferase reporter gene (Fig. 2D). mined after treatment for 24 h by Western blot as described under “Experimental Procedures.” C, curcumin decreases expression of Sp-dependent gene products. Panc28 and L3.6pL cells were treated with DMSO or 10, Thus, curcumin decreased expres- 25, 35, or 50 mol/liter of curcumin for 24 h, and whole cell lysates were analyzed by Western blot analysis as sion of Sp1, Sp3, Sp4, and several Sp- described under “Experimental Procedures.” -Actin served as a loading control. D, curcumin decreases trans- dependent gene products in pancre- activation in cells transfected with Sp1, Sp3, VEGF, and survivin promoter constructs. Cells were treated with DMSO (solvent control) or 25 or 40mol/liter of curcumin, and the effects on transactivation of promoters were atic cancer cells. Previous studies determined after treatment for 24 h as described under “Experimental Procedures.” Results are expressed as showed that curcumin decreased mean S.E. for three replicate determinations for each treatment group, and significant (p 0.05) decreases in luciferase activity compared with the solvent (DMSO) control are indicated (*). growth and expression of Sp1, Sp3, 25336 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 33 •AUGUST 13, 2010 Effects of Curcumin on NFB Are Sp-dependent cells transfected with a mixture (iSp) containing small inhibitory RNAs for Sp1 (iSp1), Sp3 (iSp3), and Sp4 (iSp4) as previously described (22). Fig. 3A illustrates that after transfection of these cells with iSp, there was a decrease in p65 and p50 expression and Sp proteins were also down-regulated. Similarly, transfection of cells with siRNA for p65 plus p50 (combined) (ip65–50) decreased expression of p65 and p50 proteins (Fig. 3B); however, Sp1, Sp3, and Sp4 protein expres- sion was unchanged (data not shown). These results demonstrate that Sp transcription factors regu- late expression of p65 and p50. Results in Fig. 3C compare the effects of transfection of iSp and ip65–50 on expression of putative NFB- and Sp-regulated gene prod- ucts, cyclin D1, VEGF, and survivin (19, 22, 24–32) and iSp ip65–50 in decreasing expression of all three proteins. In these studies, cells were transfected with siRNAs and whole cell lysates were analyzed by West- ern blots and, therefore, the observed extent of down-regulation was limited not only by the effec- tiveness of the siRNAs but also by transfection efficiencies. The effects of individual knockdown of Sp1 (iSp1), Sp3 (iSp3), and Sp4 (iSp4) on expression of p65 and p50 were also examined in Panc28 and L3.6pL cells (Fig. 3D). Transfection of Panc28 cells with iSp1, iSp3, or iSp4 decreased expression of p65 and FIGURE 3. Sp and NFB knockdown and effects on NFB subunits, angiogenic and survival proteins. Sp p50 proteins; however, the effects of (A) and NFB(B) knockdown by RNA interference. Panc28 and L3.6pL were transfected with iSp (A) or ip65/p50 combined knockdown (iSp) of all 3 (B), and effects on Sp proteins, and p65 and p50 subunits of NFB were determined by Western blot analysis as transcription factors was much described under “Experimental Procedures.” C, effects of Sp and NFB knockdown on expression of CD1, VEGF, and survivin proteins. Protein lysates from Panc28 and L3.6pL cells transfected with iSp or ip65/p50 were more effective than individual Sp analyzed for CD1, VEGF, and survivin proteins by Western blot analysis as described under “Experimental knockdown. The effects of iSp1, Procedures.” D, effects of Sp knockdown on p65, p50, cell proliferation, and PARP cleavage. Cells were trans- iSp3, and iSp4 on p65 and p50 pro- fected with various oligonucleotides, and cell numbers were counted or cell lysates were analyzed by Western blots as described under “Experimental Procedures.” E, effects of iSp on transactivation of NFB promoter. Cells tein expression in L3.6pL cells was were transfected iSp and NFB-luc, and luciferase activity was estimated as described under “Experimental highly variable. iSp1 and iSp4 were Procedures.” -Actin and Lamin served as a loading control and similar results were observed in duplicate experiments (A–C). Cell numbers (D) or luciferase activity (E) in transfection experiments were expressed as the most effective oligonucleotides mean S.E. for 3 replicate experiments and significant (p 0.05) decreases are indicated (*). for decreasing expression of p65 and p50, respectively, and iSp4 had and Sp4 in bladder cancer cells (22), and results in no effect on p65 expression. The combined knockdown of Sp1, supplemental Fig. S3 show that similar results were observed Sp3, and Sp4 (iSp) was the most effective treatment for decreas- after treatment of Panc1 pancreatic, PC3 prostate, and RKO ing p65 and p50 protein expression in L3.6pL cells. The differ- colon cancer cells. ential effect of Sp1, Sp3, and Sp4 on regulation of other genes Sp Transcription Factors Regulate NFB—The role of Sp has also previously been reported in different cancer cell lines transcription factors in regulating p65 and p50 proteins that (22, 33–35). In addition, Sp knockdown also inhibited Panc28 form the NFB complex was investigated in Panc28 and L3.6pL and L3.6pL cell growth and induced PARP cleavage, indicating AUGUST 13, 2010• VOLUME 285 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 25337 Effects of Curcumin on NFB Are Sp-dependent siRNAs for Sp1, Sp3, and Sp4 on luciferase activity in Panc28 and L3.6pL cells transfected with pNFB-luc, and luciferase activity was significantly decreased in all treatment groups (Fig. 3E). Results in Fig. 3, D and E, show that knock- down of individual Sp proteins is more effective as an inhibitor of NFB-dependent transactivation than their individual effects on decreasing levels of p65 or p50 pro- teins. This may be due, in part, to a role for Sp transcription factors in cooperatively activating NFB (36, 37) and this is being further inves- tigated. In contrast to the effects of Sp transcription factor knock- down on Sp-regulated proteins and NFB, the effects on distribu- tion of Panc28 and L3.6pL cells in G /G ,S,andG /M phases as 0 1 2 determined by FACS analysis were minimal (supplemental Fig. S4). As a control, we show that curcu- min differentially affects KLF4 but does not induce c-jun protein expression in Panc28 and L3.6pL cells (supplemental Fig. S5). Stressors such as TNF induce NFB-dependent responses through increased formation of the nuclear NFB complex and this was observed in L3.6pL cells, whereas minimal induction by TNF was observed in Panc28 cells. Using L3.6pL cells as a model, we show that 1 and 10 ng/ml of TNF increased nuclear p65/p50 levels and cotreatment with curcumin decreased TNF-induced nuclear accumulation of p65/p50 (Fig. 4A). Similar results were observed in a gel mobility shift assay (Fig. 4B). FIGURE 4. Role of Sp proteins in curcumin-dependent inhibition of TNF inducible responses in L3.6pL pancreatic cancer cells. A, curcumin decreases TNF-induced expression of p65 and p50 proteins. Cells were Nuclear extracts from cells treated treated with TNF in the presence or absence of 50 M curcumin, and nuclear lysates were examined for with solvent (control) or 1 and 10 expression of p65 and p50 proteins by Western blots as described under “Experimental Procedures.” B, curcu- min decreased TNF-induced NFB oligonucleotide-protein binding. L3.6pL cells were treated with DMSO or ng/ml of TNF formed an NFB- 50 mol/liter of curcumin in the presence or absence of TNF for 24 h, and nuclear extracts were incubated DNA retarded band using a P-la- with P-labeled NFB oligonucleotide alone or in the presence of other factors. Retarded bands were analyzed beled consensus NFB oligonucleo- by electrophoretic mobility shift assay as described under “Experimental Procedures.” Effects of curcumin (C) and iSp (D) on Sp/NFB-dependent protein expression are shown. L3.6pL cells were treated with 50 M curcu- tide and TNF increased retarded min (C) or transfected with iSp (D) in the presence or absence of TNF, and whole cell and nuclear lysates were band intensity, whereas band inten- analyzed for Sp1, Sp3, Sp4, p65, p50, CD1, COX-2, and VEGF proteins by Western blot analysis as described sities were decreased using extracts under “Experimental Procedures.” The gels were typical of results of at least two replicate determinations per treatment group. E, effects of curcumin on TNF-induced responses. L3.6pL cells were treated with curcumin from cells cotreated with TNF plus or TNF alone or in combination for up to 45 min, whole cell lysates were obtained and analyzed by Western curcumin. Retarded band intensi- blots as described under “Experimental Procedures.” ties decreased after cotreatment that agents such as curcumin that decrease Sp transcription with unlabeled wild-type but not with mutant NFB oligonu- factors also decrease Sp-dependent cell growth and survival cleotide, and after coincubation with p65 plus p50 antibodies pathways. We also investigated the effects of iSp and individual (combined). Fig. 4C shows that TNF had a minimal effect on 25338 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 33 •AUGUST 13, 2010 Effects of Curcumin on NFB Are Sp-dependent expression but enhanced levels of cyclin D1 and COX-2; however, in cells cotransfected with iSp, there was a significant decrease in Sp1, Sp3, and Sp4, and TNF-induced expression of COX-2 and cyclin D1 was also decreased. These results suggest that although TNF-depen- dent induction of cyclin D1 and COX-2 correlated with induction of p65/p50, these responses were blocked after down-regulation of Sp transcription factors. TNF and other stressors also rapidly induce several responses in cancer cell lines. Fig. 4E shows that after treat- ment of L3.6pL cells with TNF, there was increased expression of p65, p50, phosphorylated p65, IKK, and IKB within 5–10 min. However, in cells cotreated with curcumin plus TNF, all the stress-induced responses were in- hibited and Sp transcription factors were not decreased over this short incubation period. These Sp-inde- pendent effects of curcumin also con- tribute to the anticancer activity of this compound. Ongoing studies in this labora- tory indicate that drug-induced down-regulation of MMP and in- duction of ROS in cancer cell lines leads to decreased expression of Sp1, Sp3, and Sp4 and this is also observed after treatment with hydrogen peroxide (Fig. 7). Results in Fig. 5A show that after treatment of L3.6pL and Panc28 cells with 40M curcumin for 20 h, there was a signif- icant loss of MMP, respectively (also FIGURE 5. Effects of curcumin on MMP and ROS and related responses. Induction of changes in loss of MMP see supplemental Fig. S6). Moreover, (A) and ROS (B) by curcumin. Panc28 and L3.6pL cells were treated with DMSO or 25 or 40 mol/liter of curcumin for 24 h, in the presence or absence of antioxidant GSH, and mitochondrial membrane potential and in these same cells, cotreatment ROS were determined as described under “Experimental Procedures.” ROS mediated Sp degradation (C) cell with curcumin and the thiol antioxi- growth inhibition (D) in the presence or absence of antioxidants. Cells were treated with DMSO or 35 or 50 mol/liter of curcumin in the presence or absence of thiol antioxidants for 24 h, and cells were then counted or dants DTT and GSH, the curcumin- the whole cell lysates were analyzed by Western blots as described under “Experimental Procedures.” -Actin induced loss of MMP was signifi- served as a loading control. Results in A, B, and D are expressed as mean S.E. for three replicate determina- cantly inhibited. In addition, the tions for each treatment group, and significant (p 0.05). Curcumin-mediated decreases (*) or increases after cotreatment with antioxidants (**) compared with the solvent (DMSO) control are indicated. GSH levels in loss of MMP in L3.6pL and Panc28 Panc28 (4.33 M) and L3.6pL (2.64 M) cells were also determined as described under “Experimental cells treated with curcumin was Procedures.” accompanied by induction of ROS, expression of Sp1, Sp3, Sp4, and Sp-dependent gene products which was also significantly attenuated after cotreatment with VEGF; cyclin D1 was induced and COX-2 expression was the DTT or GSH (Fig. 5B). The role of antioxidants in protecting most highly induced by TNF. Curcumin alone (50 M)orin against curcumin-induced down-regulation of Sp1, Sp3, and combination with TNF resulted in decreased expression of all Sp4 was also investigated in Panc28 and L3.6pL cells. Treat- of these proteins. Results in Fig. 3A show that iSp inhibited ment with curcumin alone decreased expression of Sp1, Sp3, basal expression of p65 and p50, and this is confirmed in Fig. and Sp4 proteins in L3.6pL and Panc28 cells; however, cotreat- 4D, which also shows that iSp inhibited TNF-induced p65 and ment with the antioxidants GSH or DTT blocked down-regu- p50 expression in L3.6pL cells. TNF had minimal effects on Sp lation of these transcription factors (Fig. 5C). Curcumin-de- AUGUST 13, 2010• VOLUME 285 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 25339 Effects of Curcumin on NFB Are Sp-dependent respectively. In addition, there was some variability in protein levels at earlier time points; however, with few exceptions, the major effects were observed after 18–24 and 12–24 h in Panc28 and L3.6pL cells, respectively. Maximal effects were observed after 24 h in both cell lines. In parallel studies, ROS was induced in Panc28 and L3.6pL cells 12–24 and 18–24 h, respectively, after treatment with curcumin. These results demonstrate a time-depen- dent overlap in curcumin-induced ROS and the down-regulation of Sp1, Sp3, Sp4, p65, and p50. The direct effects of ROS on Sp proteins was investigated by treat- ing cells with hydrogen peroxide (Fig. 7). Hydrogen peroxide (50– 250 M) decreased expression of Sp1, Sp3, and Sp4 proteins in Panc28 and L3.6pL cells (Fig. 7A), and in cells cotreated with hydrogen peroxide in combination with gluta- thione, the down-regulation of Sp transcription factors was reversed by the antioxidant (Fig. 7B). This interaction was similar to the effects of glutathione on curcumin-induced down-regulation of Sp proteins illus- trated in Fig. 5C. Moreover, we also observed that, like curcumin, hydro- genperoxideinducedROSinthepan- creatic cancer cell lines (Fig. 7C). These results further support that induction of ROS by curcumin results in down-regulation of Sp transcrip- tion factors, and activation of this pathway contributes to the anticancer activity of curcumin. Curcumin Inhibits Tumor Growth and Down-regulates Sp Transcription FIGURE 6. Time course effects of curcumin on Sp1, Sp3, Sp4, p65 and p50, and ROS. Panc28 (A) and L3.6pL (B) cells were treated with DMSO (0 time) or 50M curcumin for different times over a 24-h period and whole cell lysates Factors—The in vivo antitumori- were analyzed by Western blots as described under “Experimental Procedures.” C, induction of ROS. The time course genic activity of curcumin was induction of ROS by curcumin in Panc28 and L3.6pL cells was determined as described under “Experimental Proce- dures.” Results are mean S.E. (3 replicates/group) and significant (p 0.05) induction of ROS is indicated (*). investigated in athymic nude bear- ing L3.6pL cells as xenografts. At a pendent inhibition of Panc28 and L3.6pL cell growth was also dose of 100 mg/kg/days, curcumin inhibited tumor weights reversed in cells cotreated with GSH or DTT and the effects of (Fig. 8A) and growth (Fig. 8B) over an 18-day treatment period. the antioxidants were more pronounced in Panc28 than L3.6pL We also examined Sp1, Sp3, and Sp4 protein expression in cells (Fig. 5D). tumors from the corn oil (control)- and curcumin-treated mice We also investigated the time course effects of curcumin on (Fig. 8, C and D). All three transcription factors were decreased Sp1, Sp3, Sp4, p65, and p50 protein levels in Panc28 and L3.6pL after treatment with curcumin; however, only Sp1 and Sp4 were (Fig. 6, A and B) and correlated these effects with induction of significantly (p 0.05) lower due to the inter-animal variability. ROS (Fig. 6C). Treatment of Panc28 cells with 50 M curcumin Immunohistochemical staining also showed that curcumin resulted in decreased levels of Sp1, Sp3, Sp4, p65, and p50 after decreased expression of nuclear Sp proteins, p65, p50, VEGF, 18, 8, 12, 8, and 6 h, respectively, whereas in L3.6pL cells survivin, and cyclin D1 (Fig. 8E). Thus, curcumin-dependent decreased levels were observed after 8, 10, 18, 8, and 18 h, down-regulation of Sp transcription factors correlated with the 25340 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 33 •AUGUST 13, 2010 Effects of Curcumin on NFB Are Sp-dependent transition, and inflammation, and this is accompanied by induction of genes such as cyclin D1, survivin, VEGF, bcl-2, and COX-2 that con- tribute to these responses (30–32). Curcumin has been extensively characterized as an anti-inflamma- tory and anticancer agent and these effects have been linked to modula- tion of several pathways and genes in different cancer cell lines (14– 16). In addition, curcumin has been extensively investigated as an inhib- itor of basal and induced NFB-de- pendent responses and this plays an important role in the remarkable anticancer activities of this com- pound (14–16). Studies in this laboratory have been focused on drugs such as tolfenamic acid, betulinic acid, and the synthetic triterpenoid methyl 2-cyano-3,11-dioxo-18- olean-1,12-dien-30-oate (CDODA- Me) that also inhibit tumor growth and this is due, in part, to down-reg- ulation of Sp1, Sp3, and Sp4 tran- scription factors (25–27, 38). These agents also decrease expression of several Sp-dependent genes includ- ing survivin, VEGF, and its recep- tors, cyclin D1, bcl-2, EGFR, and FIGURE 7. Hydrogen peroxide decreases Sp proteins and induces ROS. Effects of hydrogen peroxide alone several other genes. Initial studies (A) and in combination with GSH (B) are shown. Cells were treated with hydrogen peroxide or GSH alone or in combination for 24 h and whole cell lysates were analyzed by Western blots as described under “Experimental showed that Sp1 was highly Procedures.” C, induction of ROS. Panc28 and L3.6pL cells were treated with hydrogen peroxide for 18 or 24 h expressed in many pancreatic can- and ROS was determined as described under “Experimental Procedures.” Results are mean S.E. (3 replicates/ cer cell lines and was required for group) and significant (p 0.05) induction of ROS is indicated (*). VEGF expression (39), and it has subsequently been shown that Sp1, Sp3, and Sp4 are highly expressed in pancreatic and other can- growth inhibitory effects of this compound in both in vitro and cer cell lines (data not shown and see Refs. 23, 25–27, and 38). in vivo pancreatic cancer cells, suggesting that targeting these transcription factors play a role in the antitumorigenic activity Moreover, a recent report showed that Sp1 was a negative prog- of curcumin. nostic factor for pancreatic cancer patient survival (40). Many Sp-dependent genes are also co-regulated in some cells by DISCUSSION NFB and not surprisingly, there is also a striking similarity The nuclear NFB complex containing p65 (Rel A) and p50 between Sp- and NFB-dependent growth inhibitory, angio- (NFB1) or closely related proteins is a multifunctional nuclear genic and survival responses, and genes. Moreover, our recent transcription factor that regulates expression of multiple genes studies with curcumin in bladder cancer cells (22) showed that that promote inflammation and carcinogenesis (30–32). The this compound also decreased expression of Sp transcription inactive cytosolic NFB-IB complex is activated and pro- factors and Sp-dependent genes, and there was evidence in cessed through phosphorylation and proteasome-dependent 253JB-V cells that p65 was also an Sp-regulated gene. Curcu- degradation of IB and this results in enhanced accumulation min is currently in clinical trials for pancreatic cancer (21) and of nuclear NFB and modulation of NFB-dependent gene we used this tumor type as a model for investigating the effects expression. Upstream activators of nuclear NFB include vari- of this compound on Sp1, Sp3, Sp4, and NFB and also ous cellular stressors such as cytokines, apoptosis inducers, car- Sp-NFB interactions. cinogens and tumor promoters, ROS, endotoxins, and bacterial Curcumin inhibited Panc28 and L3.6pL cell proliferation and viral infections (30–32). Activation of NFB in a cancer cell (Fig. 1A) and decreased expression of both p65 and p50 and context results in the induction of cancer cell proliferation, sur- their DNA binding activity (Fig. 1, B and C) and luciferase activ- vival, angiogenesis and metastasis, epithelial to mesenchymal ity in cells transfected with an NFB-luc construct (Fig. 1D). AUGUST 13, 2010• VOLUME 285 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 25341 Effects of Curcumin on NFB Are Sp-dependent ment with curcumin was blocked by L3.6pL - Curcumin L3.6pL 1100 the proteasome inhibitor MG-132, whereas in pancreatic cancer cells, Control Curcumin MG-132 did not affect curcumin- (100 mg/kg/dose) 80 dependent repression of Sp1, Sp3, and Sp4 (Fig. 2B). Curcumin decreased pancreatic tumor growth in athymic nude mice bearing 04 8 12 16 20 24 28 Control 100 mg/kg/dose L3.6pL cells as xenografts and this No. of days was also accompanied by Sp down- regulation (Fig. 8) and parallels the Control Curcumin in vitro effects of curcumin in pan- Control Sp1 Curcumin creatic cancer cells (Fig. 2). Thus, curcumin decreases both Sp and Sp3 NFB transcription factors in pan- creatic cancer cells and this is Sp3 accompanied by decreased expres- sion of several genes that may be Sp4 regulated by both NFB and Sp transcription factors, depending on β -Actin the cell context. Sp1 Sp3 Sp4 Because curcumin decreased p65 p65 Sp1 Sp3 Sp4 and p50 proteins in Panc28 and L3.6pL cells (Fig. 1B), we hypothe- sized that this response may be dependent, in part, on down-regula- tion of Sp1, Sp3, and Sp4 in pancre- atic cancer cells (Fig. 2A). Direct evidence for the role of Sp transcrip- tion factors in regulating NFB was obtained by RNA interference in VEGF survivin CD1 p50 which cells were transfected with iSp, which contained siRNAs for Sp1, Sp3, and Sp4 (in combination). The results showed that knock- down of Sp transcription factors decreased expression of both p65 and p50 proteins (Fig. 3A); com- bined knockdown of p65 and p50 (ip65-p50) by RNA interference FIGURE 8. Curcumin inhibits pancreatic cancer xenograft tumor growth. Tumor weights (A) and volume (B) are shown. Athymic nude mice bearing L3.6pL xenografts were treated with corn oil or curcumin (100 mg/kg/ decreased expression of both pro- day), and tumor weights and volumes (mm ) were determined as described under “Experimental Procedures.” teins (Fig. 3B). Moreover, a compar- C, Western blot analysis of tumor lysates. Lysates from three mice in the treated and control groups were ison of the effects of iSp versus ip65- analyzed by Western blots as described under “Experimental Procedures.” -Actin served as loading control and for standardizing quantitative protein determinations. D, Sp proteins levels of control animals were set at p50 on several putative Sp- and 100%. Columns, means for three separate determinations; bars, S.E.; *, significantly (p 0.05) decreased protein NFB-regulated genes (cyclin D1, levels. E, immunohistochemical staining. Tumor slides from treated and untreated animals were stained as described under “Experimental Procedures.” VEGF, and survivin (Fig. 3C)) con- firmed that expression of these Thus, basal NFB, which is overexpressed in many cancer cell genes was primarily dependent on Sp transcription factors, and lines and tumors including pancreatic cancer (17), is also inhib- luciferase activity in cells transfected with NFB-luc was also ited in Panc28 and L3.6pL cells treated with curcumin and this decreased by Sp knockdown (Fig. 3E). However, comparison of is related, in part, to decreased expression of p65 and p50. In the effects of iSp versus ip65/p50 on down-regulation of cyclin parallel experiments, we also demonstrated that curcumin D1 (in L3.6pL cells) and VEGF in both cell lines (Fig. 3C) also decreased expression of Sp1, Sp3, and Sp4 (Fig. 2A) and several suggests that Sp transcription factors and NFB may coordi- Sp-dependent genes (Fig. 2C), and similar results were previ- nately regulate expression of these genes. Knockdown of indi- ously observed in bladder cancer cells (22), and results in vidual Sp proteins in Panc28 and L3.6pL cells differentially supplemental Fig. S3 show that curcumin inhibits growth and affected expression of p65 and p50 (Fig. 3D) and this was more down-regulates Sp1, Sp3, and Sp4 in Panc1, RKO, and PC3 pronounced in L3.6pL cells where iSp1 and iSp4 were the most cells. In bladder cancer cells, Sp down-regulation after treat- effective oligonucleotides for decreasing p65 and p50 proteins, 25342 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 33 •AUGUST 13, 2010 Tumor weight (%) Control Treated Control Treated Tumor Volume (mm ) O.D. β -Actin/control normalized Effects of Curcumin on NFB Are Sp-dependent respectively. Previous studies have reported similar differences ROS were observed primarily after prolonged treatment (8–12 in gene-specific regulation by Sp1, Sp3, and Sp4. For example, h) and were maximal after 24 h (Fig. 6). In addition, hydrogen Sp1 (but not Sp3 or Sp4) regulates estrogen receptor expres- peroxide also decreased expression of Sp1, Sp3, and Sp4 pro- sion in breast cancer cells (33), whereas all 3 transcription fac- teins in Panc28 and L3.6pL cells and cotreatment with glutathi- tors contribute to basal expression of VEGF receptor 2 in pan- one attenuated these effects (Fig. 7), and current studies are creatic cancer cells (34). Knockdown of Sp proteins also focused on analysis of the ROS species generated by treatment inhibited pancreatic cancer cell growth and induced PARP with curcumin and their individual role in repression of Sp cleavage (Fig. 3D). These responses were previously observed in proteins. Interaction of curcumin with pancreatic cancer cell L3.6pL and Panc1 cells and confirm that curcumin-induced mitochondria, induction of ROS, and the attenuation of cur- effects on Sp proteins contributes to the growth inhibitory and cumin-induced Sp down-regulation by antioxidants is also con- proapoptotic responses induced by this compound. sistent with a role for ROS in regulating expression of Sp1, Sp3, TNF induced levels of nuclear p65 and p50 proteins (Fig. 4, and Sp4. Previous studies showed that among several cancer A and B) and this resulted in induction of some NFB-depen- cell lines, their sensitivity to arsenic trioxide was dependent, in dent gene products such as COX-2 (Fig. 4C); both curcumin part, on constitutive glutathione levels (48), and the higher lev- and iSp inhibited not only basal (Fig. 3) but TNF-induced els of glutathione in Panc28 (4.33 M) versus L3.6pL (2.64 M) responses (Fig. 4) in pancreatic cancer cells. Thus, curcumin- cells may explain the increased resistance of the former cell line dependent inhibition of NFB is due, in part, to down-regula- to curcumin-mediated repression of Sp1, Sp3, and Sp4 proteins tion of Sp transcription factors and these results are consistent (Fig. 2A). In contrast, we also observed that antioxidants were with previous reports showing that the p65 and p50 promoters less effective in reversing curcumin-mediated inhibition of cell contain functional GC-rich Sp binding sites and both genes are proliferation in L3.6pL compared with Panc28 cells (Fig. 5D) regulated by Sp1 (41, 42). However, the role of Sp1, Sp3, and Sp4 and this is an example of cell context-dependent differences in in regulation of p65 and p50 will also be dependent on cell the contribution of the ROS-Sp degradation pathway to pan- context because we previously observed that knockdown of Sp creatic cancer cell growth inhibition. transcription factors in bladder cancer cells decreased p65 but Thus, like arsenic trioxide (43) and other mitochondriotoxic not p50 proteins (22). The temporal effects of curcumin on drugs, curcumin induces ROS in pancreatic cancer cells and decreased expression of Sp1, Sp3, Sp4, p65, and p50 are maxi- this results in down-regulation of Sp1, Sp3, and Sp4 proteins mal after 24 h but are also decreased to a lesser extent after and Sp-dependent gene products, which includes NFB. More- 8–12 h (Fig. 6, A and B). However, previous studies show that over, curcumin inhibited pancreatic tumor growth and this was curcumin can rapidly decrease TNF or stress-induced also accompanied by down-regulation of Sp1, Sp3, Sp4, and responses after short time periods, and TNF-induced activa- Sp-regulated genes (Fig. 8). These results highlight a novel tion of p65 and p50, phospho-p65, IKK, and IKB were all mechanism of action for curcumin that includes ROS-Sp and inhibited by curcumin within a 45-min time period (Fig. 4E). Sp-NFB interactions and further demonstrates that in pancre- Thus, curcumin-mediated short-term effects are independent atic cancer cells, Sp transcription factors are an important drug of decreased Sp protein levels and these rapid effects of curcu- target. The downstream targets of curcumin-induced ROS are min also contribute to the overall anticancer activity of this also being investigated and these include microRNAs such as compound. miR-27a that inhibit expression of the Sp repressor, ZBTB10 Ongoing studies in this laboratory have been investigating (38). Preliminary studies indicated that only minimal induction the mechanisms associated with drug-induced Sp down-regu- of ZBTB10 by curcumin is observed in pancreatic cancer cells lation in cancer cells (data not shown and see Ref. 38), and and a search for other curcumin-induced Sp repressor genes is recently we have shown that induction of ROS is a critical ele- ongoing. Current studies are also focused on development of ment for this response (43, 44). 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Journal
Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology
Published: Aug 13, 2010
Keywords: Mitochondrial Transport; Pancreas; Reactive Oxygen Species (ROS); RNA Interference (RNAi); Sp1; NFκB; Sp Transcription Factors; Curcumin; Pancreatic Cancer