Curcumin and synthetic analogs induce reactive oxygen species and decreases specificity protein (Sp) transcription factors by targeting microRNAs
© Gandhy et al.; licensee BioMed Central Ltd. 2012
Received: 13 April 2012
Accepted: 23 November 2012
Published: 30 November 2012
Curcumin inhibits growth of several cancer cell lines, and studies in this laboratory in bladder and pancreatic cancer cells show that curcumin downregulates specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and pro-oncogenic Sp-regulated genes. In this study, we investigated the anticancer activity of curcumin and several synthetic cyclohexanone and piperidine analogs in colon cancer cells.
The effects of curcumin and synthetic analogs on colon cancer cell proliferation and apoptosis were determined using standardized assays. The changes in Sp proteins and Sp-regulated gene products were analysed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a), miR-20a, miR-17-5p and ZBTB10 and ZBTB4 mRNA expression.
The IC50 (half-maximal) values for growth inhibition (24 hr) of colon cancer cells by curcumin and synthetic cyclohexanone and piperidine analogs of curcumin varied from 10 μM for curcumin to 0.7 μM for the most active synthetic piperidine analog RL197, which was used along with curcumin as model agents in this study. Curcumin and RL197 inhibited RKO and SW480 colon cancer cell growth and induced apoptosis, and this was accompanied by downregulation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and Sp-regulated genes including the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET), survivin, bcl-2, cyclin D1 and NFκB (p65 and p50). Curcumin and RL197 also induced reactive oxygen species (ROS), and cotreatment with the antioxidant glutathione significantly attenuated curcumin- and RL197-induced growth inhibition and downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes. The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR)-27a, miR-20a and miR-17-5p that regulate these repressors.
These results identify a new and highly potent curcumin derivative and demonstrate that in cells where curcumin and RL197 induce ROS, an important underlying mechanism of action involves perturbation of miR-ZBTB10/ZBTB4, resulting in the induction of these repressors which downregulate Sp transcription factors and Sp-regulated genes.
KeywordsCurcumin ROS induction Sp transcription factors MicroRNAs
Traditional medicines have been extensively used for treatment of multiple diseases including cancer, and many widely used anticancer drugs are derived from natural sources [1, 2]. Curcumin is a major aromatic constituent of turmeric (Curcuma longa) and has been widely investigated for its anticancer activities in multiple cancer cell lines and in vivo tumor models [3, 4]. Curcumin has been used in clinical trials for pancreatic cancer, and it is anticipated that curcumin or a suitable derivative will eventually play a clinical role in cancer chemotherapy as a “stand alone” drug or in combination therapies [5–9]. A major problem associated with the use of curcumin is its low bioavailability and this has resulted in efforts to improve formulations for delivery of curcumin and also to develop curcumin analogs that are more potent and more bioavailable [5, 10–14].
The focus on curcumin as an anticancer agent is due, in part, to its broad spectrum of activities. Curcumin inhibits cancer cell and tumor growth, decreases survival, and inhibits angiogenesis and inflammation. Many, but not all of these responses, are observed in different cancer cell lines, and several pathways and genes responsible for these effects have been reported [3, 4]. For example, curcumin-induced growth arrest and apoptosis in various HCT-116-derived colon cancer cells was due to induction of various caspases and inhibition of β-catenin signaling pathways . Other studies in colon cancer cells report similar responses and also show downregulation of cyclin D1, bcl-2, VEGF and p65 (NFκB) and other pro-oncogenic factors [15–19].
Studies in this laboratory have shown that curcumin inhibits bladder and pancreatic cancer cell and tumor growth and that the anticancer activity is due, in part, to downregulation of specificity protein (Sp) transcription factors Sp1, Sp3, Sp4 and Sp-regulated genes [20, 21]. Sp transcription factors are overexpressed in multiple cancer cell lines and tumors [20–26] and represent an example of non-oncogene addiction by cancer cells [27, 28], and this is primarily due to the pro-oncogenic activity of Sp-regulated genes. Results of drug (including curcumin) treatment and Sp knockdown by RNA interference have identified Sp-regulated genes that are important for cell proliferation [cyclin D1, epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET)], survival (bcl-2, survivin), angiogenesis [vascular endothelial growth factor (VEGF) and its receptors (VEGR)], and inflammation (p65 and p50) [20–22, 29–32]. In this study, we investigated the anticancer activities of curcumin and several synthetic analogs using colon cancer cells as a model. Our major objectives were to compare the relative potencies of curcumin with the synthetic analogs, to determine their effects on Sp transcription factors and Sp-regulated genes, and the mechanisms responsible for downregulation of Sp transcription factors.
Both curcumin and the most active synthetic analog RL197 inhibited colon cancer cell growth with an IC50 (growth inhibition) of 10 and 0.7 μM, respectively. Both compounds induced reactive oxygen species (ROS) and downregulated Sp1, Sp3, Sp4 and Sp-regulated genes, and these responses were attenuated by the antioxidant glutathione (GSH). The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR)-27a, miR-20a and miR-17-5p that regulate the repressors.
Cell lines, reagents and antibodies
RKO and SW480 human colon carcinoma cell lines and CCD-18Co colon fibroblasts were obtained from American Type Culture Collection (Manassas, VA). Cells were initially grown and multiple aliquots were frozen and stored at -80°C for future use. Cells were purchased more than 6 months ago and were not further tested or authenticated by the authors. Cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) with phenol red supplemented with 10% FBS, and 10 mL/L of 100X antibiotic/antimycotic solution (Sigma-Aldrich Co., St. Louis, MO). Cells were cultured in 150-cm2 plates in an air/CO2 (95:5) atmosphere at 37°C. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) except c-MET and survivin (Cell Signaling Technology, Danvers, MA), NFκB-p50 and NFκB-p65 (Abcam Inc., Cambridge, MA), Sp1 (Millipore, Billerica, MA), and FAS (Sigma-Aldrich Co., St. Louis, MO). Glutathione, 98% (γ-L-glutamyl-L-cysteinyl-glycine, GSH) and lactacystin (proteasome inhibitor) were purchased from Sigma-Aldrich. Carboxy-H2DCFDA was purchased from Invitrogen (Carlsbad, CA). Curcumin (98% pure) was purchased from Indofine Chemical Company, Inc. (Hillsborough, NJ), and curcumin analogs were synthesized as described  and RL197 synthesis is outlined below.
Melting points were determined on a Mettler Toledo FP62 melting block and were uncorrected. High resolution mass spectrometry was recorded using a VG70-250S double focusing magnetic sector mass spectrometer. NMR spectra, at 25°C, were recorded at 500 MHz for 1H and 125 MHz for 13C on Varian INOVA-500 spectrometer. Chemical shifts are given in ppm on the δ scale referenced to the solvent peaks CHCl3 at 7.26 and CDCl3 at 77.00. 1-Boc-4-piperidone, and 2,5-dimethoxybenzaldehyde were purchased from the Sigma-Aldrich Company. (3E,5E)-3,5-Bis(2,5-dimethoxybenzylidene)-1-t-butoxycarbonylpiperidin-4-one (RL197). To a mixture of 1-Boc-4-piperidone (0.70 g, 3.5 mmol) and 2,5-dimethoxybenzaldehyde (1.20 g, 7.4 mmol) in methanol (50 mL) was added sodium methoxide (5M, 0.75 ml) and the mixture was stirred for 18 hr at room temperature. The resulting precipitate was removed by filtration, then washed with cold methanol and purified by recrystallisation from ethanol to give RL197 as a yellow solid (1.20 g, 69%); mp 167.7°C. Found: C, 67.79; H, 6.79; N, 2.73. C28H33NO7 requires: C, 67.86; H, 6.71; N, 2.83. 1H-NMR (CDCl3) δ: 1.26 (s, 9H), 3.79 (s, 6H), 3.82 (s, 6H), 4.59 (bs, 4H), 6.80 (bs, 2H), 6.85 (d, J = 9Hz, 2H), 6.89 (dd, J = 2, 9 Hz, 2H), 7.95 (bs, 2H); 13C-NMR (CDCl3) δ: 187.76, 154.35, 153.05, 152.74, 133.30 (br), 124.83, 116.13, 115.46 (br), 111.80, 80.20, 56.04, 55.84, 45.05, 28.05: (HRMS (+ve ESI) calc for C28H33NaNO7: 518.2149 m/z [MNa+], found: 518.2115 m/z.
Cell proliferation assay and annexin V staining
RKO and SW480 cancer cells were seeded in DMEM High Glucose with 10% FBS on 12-well plates and allowed to attach for 24 hr. The medium was then changed to DMEM High Glucose containing 2.5% charcoal-stripped FBS and cells were treated with either the vehicle (DMSO) or the indicated compounds for 24 hr. Cells were trypsinized and counted using a Coulter Z1 particle counter. For Annexin V staining, cells were seeded in 6-well plates, allowed to attach overnight, and treated with curcumin or RL197 as indicated. Annexin V and propidium iodide staining was determined using the Vybrant apoptosis assay kit #2 (Molecular Probes, Grand Island, NY) and images were captured at 20X magnification using IN cell analyzer 6000 (GE Healthcare Biosciences, Piscataway, NJ).
RKO and SW480 cancer cells were seeded in DMEM High Glucose with 10% FBS on 6-well plates and allowed to attach for 24 hr. The medium was then changed to DMEM High Glucose containing 2.5% charcoal-stripped FBS and treated with either the vehicle (DMSO) or the indicated compounds and analyzed by western blots as described [20, 21].
Cellular ROS levels were evaluated with the cell permeant probe carboxy-H2DCFDA (5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate) from Invitrogen. Following treatment, cells seeded on 6-well plates were loaded with 10 mM of carboxy-H2DCFDA for 1 hr, washed once with serum-free medium, and analyzed for ROS levels using BD Accuri C6 Flow Cytometer using the FL1 channel. Analysis of data was determined with BD Accuri CFlow software (set at 480 nm and 525 nm excitation and emission wavelengths, respectively). Each experiment was carried out in triplicate and results are expressed as means ± S.E. for each treatment group.
Measurement of mitochondrial membrane potential (MMP)
MMP was measured with Mitochondrial Membrane Potential Detection Kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol using the JC-1 dye; mitochondrial membrane potential was measured using BD Accuri C6 Flow Cytometer and data were analyzed using the BD Accuri CFlow software. J-aggregates are detected as red fluorescence and J-monomers are detected as green fluorescence. Each experiment was determined in triplicate, and results are expressed as means ± S.E. for each treatment group.
Quantitative real time PCR of mRNA and miRNAs
Sp1 (Forward): 5'-TCA CCT GCG GGC ACA CTT-3'
Sp1 (Reverse): 5'-CCG AAC GTG TGA AGC GTT-3'
TBP (Forward): 5'-TGCACAGGAGCCAAGAGTGAA-3'
TBP (Reverse): 5'-CACATCACAGCTCCCCACCA-3'
ZBTB10 (Forward): 5'-GCTGGATAGTAGTTATGTTGC-3'
ZBTB10 (Reverse): 5'-CTGAGTGGTTTGATGGACAGA-3'
ZBTB4 (Forward): 5'-ACCTGTGCAGGAATTTCCAC-3'
ZBTB4 (Reverse): 5'-GAGCGGCCAAGTTACTGAAG-3'
Primers for Sp3 and Sp4 were purchased from Qiagen.
Statistical significance of differences between the treatment groups was determined using the Student’s t test, and levels of probability were noted. IC50 values were calculated using linear regression analysis and expressed in micromolar (μM) concentrations at 95% confidence intervals.
Curcumin inhibits colon cancer cell growth and downregulates Sp transcription factors and Sp-regulated genes
Curcumin and RL197 activate ROS in colon cancer cells
Curcumin and RL197 disrupt miR27a:ZBTB10 and miR-20a/17-5p:ZBTB4 interactions
Non-oncogene addiction by cancer cells is now recognized as an important pathway for maintaining the cancer cell phenotype [27, 28] and Sp transcription factors are an example of non-oncogenes that fulfill this function. Sp1, Sp3 and Sp4 are overexpressed in multiple cancer cell lines and tumor types and Sp-regulated genes and oncogenes play an important role in cancer cell proliferation, survival, angiogenesis and inflammation [20–26, 29–38]. These transcription factors are ideal for development of mechanism-based drugs since Sp1 expression decreases with age [39–41], and results of animal studies show that Sp1, Sp3 and Sp4 are highly expressed in tumor but not in non-tumor tissues [21, 23]. The high expression of Sp transcription factors is due, in part, to miR-dependent repression of the Sp repressors ZBTB10 and ZBTB4, which competitively bind GC-rich gene promoters and deactivate transcription [25, 26]. MiR-27a and miRs-20a/17-5p, which are overexpressed in many tumors interact with and suppress ZBTB10 or ZBTB4, respectively, and overexpression of ZBTB10 and ZBTB4 or transfection of cells with miR-27a and miR-20a/17-5p antagomirs also decrease expression of Sp transcription factors [25, 26].
Several drugs that target Sp transcription factors have been identified and these include the non-steroidal anti-inflammatory drugs (NSAIDs) tolfenamic acid, COX-2 inhibitors and the nitro-NSAID GT-094, and several natural products including betulinic acid (BA), celastrol and the synthetic triterpenoids methyl 2-cyano-3,12-dioxooleana-a-dien-28-oate (CDDO-Me) and methyl 2-cyano-3,4-dioxo-18β-olean-1,12-dien-30-oate (CDODA-Me) [20–26, 29–38]. In bladder cancer cells, curcumin induced proteasome-dependent downregulation of Sp1, Sp3 and Sp4 , whereas in pancreatic cancer cells this response was ROS-dependent and reversed by cotreatment with antioxidants such as GSH . Both curcumin and the potent RL197 analog induced ROS (Figure 5A) and decreased expression of Sp1, Sp3, Sp4 and Sp-regulated proteins (Figures 2 and 3) in colon cancer cells, and cotreatment with GSH inhibited these responses and also partially reversed the growth inhibitory effects of these compounds (Figure 5D). Similar effects have previously been observed for BA and GT-094 in colon cancer cells and induction of ROS is also a critical element for their cytotoxicity [34, 35]. However, the identities of individual ROS species induced by curcumin and RL197 have not been determined and are currently being investigated.
Increased ROS contributes to tumor formation due to several factors including oxidative DNA damage; however, several anticancer drugs also induce ROS and this plays an important role in their cancer chemotherapeutic activity [42, 43]. Induction of ROS by CDDO-Me, BA/GT-094 and celastrol in pancreatic, colon and bladder cancer cells, respectively, results in downregulation of miR-27a and induction of ZBTB10 [29, 34, 35]. Moreover, celastrol also downregulates miR-20a and other miR paralogs with the same seed sequence in bladder cancer cells and this is accompanied by induction of ZBTB4. This study demonstrates that like celastrol, curcumin- and RL197-induced ROS in RKO cells also decreases miR-dependent regulation of ZBTB10 and ZBTB4 (Figure 6). Previous studies show that curcumin induces ROS in some cancer cell lines [44–48], and results of this study suggest that curcumin and RL197 induce ROS in RKO cells and ROS-mediated disruption of miR-ZBTB interactions results in downregulation of Sp transcription factors and Sp-regulated gene products. These results for curcumin and RL197 in colon cancer cells are consistent with previous studies with other ROS inducers which act as anticancer agents through the common pathway illustrated in Figure 6D.
Previous reports show that the many anticancer agents such as curcumin and RL197 target Sp transcription factors and Sp-regulated genes [20–26, 30–38] and thereby inhibit non-oncogene addiction by cancer cells and tumors. The mechanisms of Sp downregulation are both drug- and cell context-dependent, and this study demonstrates the important role of ROS in disrupting miR-mediated suppression of ZBTB10 and ZBTB4. Current studies are focused on the critical trans-acting factors that are induced or inhibited by ROS and are required to decrease miR-27a and miR-20a/17-5p expression and possibly other miRs that form part of the miR-23a~miR-27a~miR-24-2 and miR-17-92 clusters, respectively.
SUG carried out the majority of the in vitro studies, analyzed and summarized the results, and helped draft this manuscript. KK co-supervised research and carried out PCR studies on miR expression. LL and RJR synthesized compounds and helped with drafting of this manuscript. SS conceived of this project, wrote the manuscript, and supervised research. All authors read and approved the final manuscript.
Hepatocyte growth factor receptor
Epidermal growth factor receptor
Fatty acid amide hydrolase
Reactive oxygen species
Small inhibitory RNA
Vascular endothelial growth factor
Vascular endothelial growth factor receptor.
National Institutes of Health (R01-CA136571) and Texas AgriLife.The authors would like to thank Gayathri Chadalapaka and Ping Lei for their help.
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