Clofibrate Induces Heme Oxygenase 1 Expression through a PPARα-Independent Mechanism in Human Cancer Cells Open Access

Clofibrate, a member of the fibrate family, is a well-established activator for a nuclear receptor, the peroxisome proliferator-activating receptor alpha (PPARα), which regulates the expression of genes involved in fatty acid beta-oxidation and is a major regulator of lipid homeostasis [1]. In recent years, in addition to its antilipidemic activity, fibrates were found to induce apoptosis of cancer cells and suppress tumor growth in various experimental model systems [2,3,4]. Given that several fibrate derivatives including clofibrate have been used in humans as hypolipidemic agents, with their pharmacokinetics being well-documented and tolerable side effects clearly identified, there is a great potential to develop this group of compounds into anticancer agents in the near future. Therefore, further characterization of clofibrate's biological activity in cancer cells and its interaction with cancer related molecules will facilitate the development of this compound as well as other fibrates into clinical oncology practice. In this context, we have recently reported that clofibrate acts synergistically with clioquinol, a metal binding compound, to suppress cancer cell viability that is mediated by PPARα signaling [5]. Furthermore, we have shown that clofibrate suppresses hypoxia-induced HIF-1α expression and signaling in breast and ovarian cancer cells, thereby exerting antiangiogenic activity [6]. More interestingly, clofibrate and docosahexaenoic acid, both being PPARα ligands, were found to suppress superoxide dismutase 1 (SOD1) gene expression [7]. SOD1 is a primary antioxidant enzyme, and suppression of its expression leads to enhanced oxidative stress in human cancer cells [8].

HO-1 is a cytoprotective enzyme that catalyzes the degradation of heme to biliverdin, with the concurrent release of iron and carbon monoxide (CO) [9]. Induction of HO-1 expression is a recognized approach to protect cells from oxidative damage and other disease conditions [10]. In cancer cells, however, HO-1 was known to promote cell growth and render tumor cells chemo-resistance [11,12]. For this reason, inhibiting HO-1 activity is considered a viable approach to more effectively kill cancer cells and to enhance the cytotoxicity of chemotherapeutics [11,12,13]. Note that activation of PPARα has been shown to induce HO-1 expression in human vascular cells via the PPAR responsive elements (PPRE) present in the HO-1 gene promoter [14]. However, there have been no reports on PPARα-mediated HO-1 expression in cancer cells. Considering the potential of clofibrate and other PPARα ligands as anticancer agents and the role of HO-1 in cancer cell viability and chemo-resistance, the present study examined the effects of clofibrate on HO-1 expression and characterized the mechanisms of clofibrate-induced HO-1 expression in a human ovarian cancer cell model system, the A2780 cell line. We report here that clofibrate induces HO-1 expression in A2780 cells. To our surprise, the induction of HO-1 by clofibrate is mediated through a PPARα-independent mechanism, and the Nrf2 signaling pathway is indispensible for this induction.

The pGL3/4.5-HO-1 luciferase reporter construct containing the 4.5 kilo base fragment of the human HO-1 gene promoter was a kind gift from Dr. Anupam Agarwal (University of Alabama at Birmingham, Birmingham, AL). The HO-1 3'-UTR and the glyceraldehydes-3-P-dehydrogenase (GAPDH) 3'-UTR reporter constructs were purchased from SwitchGear Genomics (Menlo Park, CA). The antibodies for Nrf2 (sc-722), and PPARα (sc-9000) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The GAPDH antibody (Cat. 20035) was from ProMab Biotechnologies, Inc. (Albany, CA). The HO-1 antibody (SPA-896) was from Stress-gen (Ann Arbor, MI). The Dual-Luciferase Reporter kit was from Promega (Madison, WI). The QuikChange Site-Directed Mutagenesis Kit was from Stratagene (La Jolla, CA). The Carboxy-H₂DCFDA probe was from Molecular Probes Inc. (Eugene, OR). The β-actin antibody (A5441), Clofibrate, Fenofibrate, Troglitazone, GW6471, GW9662, N-Acetyl-L-cysteine (NAC) and other chemical agents were analytic grade and purchased from Sigma-Aldrich (St. Louis, MO).

The human ovarian carcinoma cell line A2780 was a kind gift from Dr. Stephen Howell (University of California, San Diego, CA). The human prostate cancer line DU145 was obtained from the American Type Culture Collection (Manassas, VA, USA). A2780 cells were cultivated in RPMI 1640 medium, and DU145 cells in DMEM medium, both being supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cells were routinely grown under a humid environment at 37°C, 5% CO₂, and passaged twice a week.

Western blot was performed as described [15]. In brief, for cellular protein isolation, cells were lysed in buffer containing 50 mM Tris-HCl pH 7.8, 100 mM NaCl, 5 mM Na-EDTA, 0.1% SDS, 1 mM PMSF, 1% Triton-X-100, and 2.5% glycerol. The lysate was sonicated on ice for 3 strokes of 10 seconds each and centrifuged at 15,000×g for 15 minutes to remove insoluble material. For nuclear protein extraction, cells were detached by adding 2.5 ml wash buffer (for a 100-mm dish) containing 1 mM HEPES, pH 7.9, 150 μM MgCl₂, 1 mM KCl, 50 μM Dithiothreitol (DTT), 100 μM PMSF, 200 ng/ml Aprotinin, 1µg/ml Leupeptin, 200 ng/ml Pepstatin A, and 0.01% NP-40. The lysate was centrifuged at 4,550×g for 2 minutes. Pellets were re-suspended in 50 μl suspension buffer containing 25% glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml pepstatin A. The suspension was incubated on ice for 30 minutes and centrifuged for 20 minutes at 15,000×g to remove insoluble material. Thirty to forty micrograms of protein from each sample was separated on a 10% SDS-PAGE gel, transferred to a PVDF membrane, and blotted with antibodies against HO-1, Nrf2, PPARα, GAPDH, or β-actin.

A2780 cells were seeded in 100-mm cell culture dishes and reached 70-80% confluence after 24 hours of plating. The cells were then transfected with the luciferase reporter constructs including the pGL3/4.5-HO-1 and its mutants, the HO-1-3'-UTR and GAPDH-3'-UTR using the Fugene HD transfection reagent (Roche, Mannheim, Germany) as previously described [6]. The next day, cells were lifted and plated into 24-well plates at a density of 2×10⁵ per well. 48 hours after transfection, cells were treated with various reagents at indicated concentrations and durations. Cell lysates were prepared and luciferase activity was assayed using the Dual-Luciferase Reporter kit, as previously described [6]. The firefly luciferase activity was normalized to the amount of protein present in each sample. The data are expressed as percentages of luciferase activity detected in treated relative to untreated control cells.

siRNA for Nrf2, PPARα were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cells were co-transfected with 225 nM (final concentration) of Nrf2, PPARα, or scrambled non-specific siRNAs (as control) and 2 μg of PGL3/4.5-HO-1 into A2780 cells cultured in a 100-mm dish using the Fugene HD transfection reagent (Roche, Mannheim, Germany) following the manufacturer's protocols. The next day, cells were lifted and plated into 24-well plates (for luciferase activity assay) and 100-mm dishes (for Western blot analysis). Thirty six hours after the transfection, clofibrate was added to the medium at indicated concentrations and durations. The cells were lysed and assayed for luciferase activity. The knockdown of Nrf2 and PPARα was confirmed by Western blot analysis.

Total RNA was isolated from A2780 cells using the Trizol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. RNA samples were reverse-transcribed (RT) with the SuperScript II kit (Invitrogen, Carlsbad, CA) as previously described [8]. The real-time polymerase chain reactions was performed by using the following transcript specific primers: HO-1, forward: 5'-GCC TCC TCT CGA GCG TCC TCA-3', reverse: 5'-CTG GGG CAT GCT GTC GGG TTG-3'; GAPDH, forward: 5′-CTC CTG TTC GAC AGT CAG CCG C-3', reverse: 5′-ACG ACC AAA TCC GTT GAC TCC G -3′. The samples were initially denatured at 95ºC for 10 minutes prior to thermal cycling. The thermal cycle parameters were as follows: 95ºC for 15 seconds, 60ºC for 60 seconds, for a total of 40 cycles using ABI 7300 (Life Technologies, Carlsbad, CA) and RT² SYBR Green qPCR Mastermix (SABiosciences, Valencia, CA) following manufacture's protocol.

The PGL3/4.5-HO-1 construct was used to generate mutants with deletion of the two AREs [16,17] and two PPREs located within the promoter region [14]. This was achieved as we recently described [18].

ROS was detected using the Carboxy-H₂DCFDA Fluorescent Dye-Based probe. A2780 cells were plated in a 96-well plate with 5,000 cells per well. After reaching 80% confluence, cells were treated with clofibrate for 0.5 or 48 hours. Media was removed and the cells were incubated at 37°C with 4μM of Carboxy-H₂DCFDA in 1X HBSS for 45 minutes protected from light. After incubation, cells were washed twice with 1X HBSS and the fluorescent intensity was measured with a fluorescent plate reader at excitation 485 nm/emission 538 nM. The fluorescent reading was normalized by the cell viability, which was assayed using the MTS reagent (Promega, Madison, WI) as we described [5].

Statistical analysis was performed with Graphpad Prism software (San Diego, CA). One-way ANOVA with Dunnett's post-test was used to determine differences among control and experimental groups, with p < 0.05 or p < 0.01 as the level of statistical significance.

To determine the effects of clofibrate on HO-1 expression, the human ovarian cancer cell line A2780 was treated with 1 mM clofibrate for various times. Western blot analysis indicated that clofibrate induces HO-1 protein expression, with the induction being detected at 4 hours of treatment and most pronounced at 48 hours of treatment (Fig. 1A). Real-time PCR analysis indicated that clofibrate increases HO-1 mRNA expression with maximal level being detected at 16 hours of treatment and declined thereafter (Fig. 1B). Because clofibrate is a well-established ligand to PPARα, we analyzed nuclear PPARα expression in A2780 cells after clofibrate treatment. As expected, treatment with 1mM clofibrate for 6 hours enhanced nuclear PPARα protein contents. Interestingly, nuclear Nrf2 expression was also enhanced by clofibrate (Fig. 2). Since both Nrf2 and PPARα have been reported to mediate HO-1 gene transcription, this raises a question on which signaling pathways, the PPARα or the Nrf2, mediate HO-1 induction by clofibrate. The induction of HO-1 and an increase in nuclear Nrf2 and PPARα contents by clofibrate treatment were also evident in the human prostate cancer cell line DU145 although the induction seemed to be more rapid (Fig. 3). This suggests that clofibrate-induced HO-1 expression is not cell line dependent.

As endogenous HO-1 expression is barely detectable in A2780 cells [19], we used A2780 line as a model system to investigate the mechanisms of clofibrate-induced HO-1 gene expression in cancer cells. A human HO-1 promoter reporter gene construct [19] was applied to study transcription regulation of the HO-1 gene. A2780 cells were transfected with the HO-1 promoter reporter construct and treated with 1 mM clofibrate for 4, 21 and 48 hours. As shown in Figure 4A, the HO-1 promoter activity was significantly up-regulated at 21 hours and reached a peak at 48 hours of treatment (Fig. 4A). This induction of HO-1 promoter activity by clofibrate was also concentration-dependent (Fig. 4B). A luciferase reporter construct containing the 3' untranslated region of the HO-1 transcript was used to examine whether clofibrate targets the post-transcriptional regulation of the HO-1 gene. It turned out that clofibrate had no significant effect on the HO-1 3'UTR-driven reporter activity. These results indicate that the up-regulation of HO-1 by clofibrate was mainly through transcriptional regulation.

To determine which signaling pathway, the Nrf2 or the PPARα, is involved in clofibrate-induced HO-1 gene expression, we deleted the two antioxidant response elements (AREs) from the HO-1 promoter reporter construct (Fig. 5A). These AREs have been reported to be recognized by Nrf2, a well known transcription factor regulating expression of HO-1 as well as other antioxidant molecules in response to oxidative stress [16,17]. The two PPAR response elements (PPREs) [14] were also deleted from the HO-1 promoter report construct. These PPREs are known to be bound by PPARs and regulate expression of a variety of genes including the HO-1 gene [14]. As shown in Figure 5B, single deletion of ARE1 significantly reduced clofibrate-induced HO-1 promoter activity at 21 hours of treatment and double deletion of the AREs completely reversed the HO-1 promoter activity at 21 hours of treatment and dramatically attenuated the HO-1 promoter activity at 48 hours of treatment. On the other hand, deletion of PPREs had no significant effect on clofibrate-induced HO-1 gene promoter activity (Fig. 5C). These results suggest that it is the Nrf2 pathway but not the PPARα pathway that mediates clofibarte-induced HO-1 expression in our model system. siRNA knockdown was applied to further confirm the involvement of Nrf2 in clofibrate-induced HO-1 expression in cancer cells. Knockdown of Nrf2 (Fig. 6A) dramatically attenuated clofibrate-induced HO-1 promoter activity, whereas knockdown of PPARα (Fig. 6B) had no effect (Fig. 6C). This further indicates that the Nrf2 pathway mediates clofibrate-induced HO-1 expression.

The exclusion of PPARα involvement in this event was also evident by the use of PPARα specific antagonist GW6471 or a general PPAR antagonist GW9662, both inhibited clofibrate-stimulated PPARα signaling [5] but had no effect on clofibrate-induced HO-1 promoter activity in A2780 cells (Fig. 7A). In addition, treatment with fenofibrate, another PPARα ligand, or troglitazone (TGZ), PPARγ ligand, had no effect on HO-1 promoter activity (Fig. 7B).

Nrf2 signaling is often activated by oxidative stress leading to induction of HO-1 gene transcription [9]. To test the involvement of oxidative stress in clofibrate-induced HO-1 expression, we analyzed ROS generation in A2780 cells after clofibrate treatment (Fig. 7C). Indeed, clofibrate significantly enhanced ROS levels as detected by the Carboxy-H₂DCFDA probe. Furthermore, we pre-treated A2780 cells with an antioxidant, N-Acetyl-L-cysteine (NAC) for 15 min, followed by treatment with clofibrate for 48 hours. A concentration dependent suppression by NAC of the clofibrate-induced HO-1 promoter activity was observed (Fig. 7D), supporting the involvement of oxidative stress in this process.

The key finding of the present study is that clofibrate, a well-established PPARα activator, induces HO-1 gene transcription through a PPARα-independent and Nrf2-dependent cellular mechanism, indicating that clofibrate does not act solely as a PPARα ligand, and it may have more broad activity in human cancer cells. Given that clofibrate has been used in humans for many years and recently considered as an anticancer agent, this finding provides novel insight into clofibrate's activity in cancer cells and may assist the development of clofibrate into clinical oncology practice.

There are two PPRE elements identified in the HO-1 gene promoter, which have been shown to mediate HO-1 gene transcription [14]. We initially believed that clofibrate might induce HO-1 gene expression through activation of the PPARα pathway that targets the PPRE elements in the HO-1 gene promoter. However, our experimental results clearly demonstrate that clofibrate induces HO-1 expression through a PPARα-independent mechanism. The key evidences to support this conclusion include the deletion of PPRE elements in the HO-1 gene promoter that does not alter clofibrate-induced HO-1 promoter reporter gene activity, and the knockdown of PPARα expression that does not compromise clofibrate-induced HO-1 gene transcription. Furthermore, fenofibrate, another well-known PPARα activator, could not alter HO-1 gene expression, and a well-established PPARα inhibitor, GW6741, could not attenuate clofibrate-induced HO-1 gene transcription in our model system. These results strongly indicate that other signaling pathways are involved in clofibrate-induced HO-1 gene expression. It is interesting to note that PPARα-independent activity of clofibrate has been recent described in rodent liver [20].

HO-1 gene expression has been known to be regulated by different signaling pathways [21]. One of the best characterized pathways that regulate HO-1 gene expression is the Nrf2 signaling pathway [9]. We found in our model system that the Nrf2 signaling pathway plays a critical role in clofibrate-induced HO-1 gene transcription. First, deletion of the two ARE elements in the HO-1 gene promoter completely reversed clofibrate-induced HO-1 gene promoter reporter activity in A2780 cells, indicating that the Nrf2 signaling pathway is indispensable in clofibrate-induced HO-1 gene expression in A2780 cells. Note that the ARE elements are known DNA elements bound by Nrf2 to activate gene expression in many target genes of the Nrf2 signaling pathway [22]. Second, in contrast to that of PPARα, knockdown of Nrf2 expression attenuated clofibrate-induced HO-1 gene transcription, further supporting the involvement of this pathway in clofibrate-induced HO-1 expression. Last, clofibrate was found to enhance ROS levels in A2780 cells, and application of NAC, a well-established antioxidant, compromised clofibrate-induced HO-1 gene transcription, indicating the involvement of oxidative stress in clofibrate-induced HO-1 gene expression, consistent with the fact that the Nrf2 signaling pathway primarily mediates cellular response against oxidative stress [22].

We have recently reported that clofibrate and docoxahexaenoic acid, both being PPARα ligands, suppress SOD1 gene transcription via a PPARα-mediated cellular mechanism in A2780 cells [7]. Since SOD1 is a primary antioxidant enzyme, suppression of its expression will consequently enhance cellular oxidative stress [8], which could be associated with the activation of the Nrf2 signaling pathway and the induction of HO-1 gene expression by clofibrate. This assumption is supported by the fact that the suppression of SOD1 gene transcription by clofibrate took place rather rapidly, peaking within 4 hours of stimulation [8], whereas the induction of HO-1 expression by clofibrate was much delayed with a peak at 48 hours after treatment (Fig. 2). This temporal difference in SOD1 suppression and HO-1 induction by clofibrate reflects a fine-tuned antioxidant system in eukaryotic cells that cooperatively responds to extracellular stimuli. However, because both SOD1 and HO-1 are considered as antioxidant enzyme and molecular targets for cancer therapy [11,12,13,23], the suppression of SOD1 expression and the induction of HO-1 expression by clofibrate depicts a rather complicated scenario as to how much each event contributes to the net outcome of clofibrate's anticancer activity, which merits further characterization.

In conclusion, we have demonstrated that clofibrate induces HO-1 gene expression in A2780 cells through a PPARα-independent mechanism and Nrf2 signaling is indispensable for this event. These findings provide novel insight into clofibrate's anticancer activity, thus may facilitate the development of clofibrate as an anticancer agent in clinical oncology practice.

AREs (Antioxidant response elements); HO-1 (Heme oxygenase 1); NAC (N-Acetyl-L-cysteine); Nrf2 (nuclear factor (erythroid-derived 2)-like 2); PPREs (PPAR response elements); TGZ (Troglitazone).

This work was supported in part by grants from the American Cancer Society (WQD, CNE-117557); the Susan G. Komen for the Cure Foundation (WQD, KG081083); the NIH OK-INBRE program (WQD, 3P20RR016478-09S2); the National Natural Science Foundation of China (ZJ, 81372433); and the Natural Science Foundation of Jiangsu Province (ZJ, BK20131149).