Original Article


Fenofibrate induces effective apoptosis in mantle cell lymphoma by inhibiting the TNFα/NF-κB signaling axis

Published online:


Mantle cell lymphoma (MCL) is a type of aggressive B-cell non-Hodgkin's lymphoma characterized by frequent resistance to conventional chemotherapy. In this study we provided evidence that fenofibrate, which is widely known as an agonist for peroxisome proliferator-activated receptor-α (PPARα), can induce effective apoptosis in treating MCL cells. Addition of fenofibrate to MCL cell lines significantly decreased the number of viable cells by 50% at approximately 20 μM at 72 h. This decrease in cell growth was due to apoptosis, as evidenced by the cleavage of caspase 3 and poly(ADP-ribose) polymerase. The fenofibrate-mediated effects were not significantly affected by GW6471, a specific PPARα antagonist. Using an apoptosis pathway-specific oligonucleotide array, we found that fenofibrate significantly downregulated several pro-survival genes, including tumor necrosis factor-α (TNFα). Importantly, addition of recombinant TNF-α conferred partial protection against fenofibrate-induced apoptosis. Fenofibrate also decreased the nuclear translocation of nuclear factor (NF)-κB-p65 and significantly inhibited the DNA binding of NF-κB in a dose-dependent manner. To conclude, fenofibrate shows efficacy against MCL, and the mechanism can be attributed to its inhibitory effects on the TNF-α/NF-κB signaling axis. In view of the documented safety of fenofibrate in humans, it may provide a valuable therapeutic option for MCL patients.


Fenofibrate belongs to the fibrates class of lipid-lowering drugs and it has been used for >20 years to treat endogenous hyperlipidemias, hypercholesterolemias and hypertriglyceridemias.1 In these settings, the pharmacological actions of fibrates are believed to be mediated through direct activation of peroxisome proliferator-activated receptor-α (PPARα).2 It has been shown that PPARα exerts its biological effects by acting as a transcriptional factor, forming a heterodimer with the retinoid X receptor, and binding to a peroxisome proliferator-response element located in the promoter region of the target genes.3, 4 PPARα is known to modulate the expression of multiple target genes involved in lipid metabolism, fatty acid oxidation and glucose homeostasis.1 More recent studies suggest that PPARα can modulate various signaling pathways involved in inflammatory responses. Specifically, activated PPARα inhibits the production of inflammatory response markers, such as endothelin-1, inter-cellular adhesion molecule-1 and vascular cell adhesion molecule-1 in endothelial cells and tissue factor, and matrix metalloproteinase-9 and tumor necrosis factor-α (TNFα) in macrophages.5 In human aortic smooth muscle cells, PPARα activation inhibits interleukin-1 stimulated interleukin-6 secretion and decreases interleukin-6 and cyclooxygenase-2 gene transcription.6 Moreover, PPARα has been shown to inhibit the nuclear factor (NF)-κB pathway.7, 8 Specifically, it physically interacts with the p65 subunit of NF-κB, which inhibits NF-κB-dependent transactivation.6, 9 In addition, fenofibrate treatment significantly reduced the plasma interferon-γ and TNFα levels in dyslipidemic patients.8, 10

Compared with their lipid-lowering effects, the anticancer therapeutic potential of fibrates is less commonly reported, and the reason may be related to some of the earlier reports describing that PPARα agonists may have carcinogenic effects in rodents.11, 12 However, no carcinogenic effect of fibrates have been found in patients who are treated with fibrates for hyperlipidemia,13 possibly because the expression of PPARα on human hepatocytes is much lower than that in the rodent hepatocytes. Furthermore, it is well documented that activation of mouse and human PPARα often results in opposite physiological effects in the liver.12, 14 In humans, PPARα-specific agonists have been recently reported to have antitumor effects in a number of various cancer types such as acute myeloid leukemia,15, 16 chronic lymphocytic leukemia17 and malignant tumors of the ovary,18 liver,19 skin, breast and lung.20 Recently, fenofibrate was found to be the most potent drug among all of the fibrates in inhibiting the growth of various cell lines derived from melanoma, lung carcinoma, glioblastoma and fibrosarcoma in a xenograft mouse model.20

Mantle cell lymphoma (MCL) is a distinct type of B-cell non-Hodgkin's lymphoma defined by a constellation of pathologic, cytogenetic and clinical features.21 One of the characteristic features of MCL is the recurrent chromosomal translocation, t(11;14)(q13;q32), which brings the cyclin D1 gene under the control of the enhancer of the immunoglobulin heavy chain gene, leading to overexpression of the cyclin D1 protein. Although it is widely accepted that cyclin D1 has an important role in the pathogenesis of MCL, accumulating evidence suggests that MCL often has defects in many other cellular processes, such as those involved in cell-cycle regulation, apoptosis and DNA repair.22, 23 With regard to apoptosis, MCL is well known to be resistant to apoptosis induced by a variety of conventional chemotherapeutic agents.23 Recent studies have revealed a number of biochemical defects that may contribute to its relatively high resistance to apoptosis,24 including constitutive activation of the NF-κB pathway,25, 26, 27 overexpression of several anti-apoptotic proteins and the absence of the Fas receptor.28 Aberrant cellular signaling, such as the phosphoinositide 3-kinase/Akt pathway, may also contribute to the chemoresistance of MCL.29, 30 Despite the advent of several new therapeutic agents,31 a significant proportion of MCL patients continues to have a relatively poor clinical outcome.23 Thus, there is a need to continue to develop new therapeutic strategies for this disease.

In this study, we tested whether fenofibrate has any therapeutic potential in MCL. Our data showed that fenofibrate induced effective apoptosis in MCL cell lines. Furthermore, our data showed that these observed effects cannot be directly attributed to the stimulation of PPARα.

Materials and methods

MCL cells, tissue culture and compounds

Three previously described MCL cell lines, Jeko-1, Mino and SP53, were used in this study.32 In brief, the three cell lines are positive for cyclin D1, and they carry the mature B-cell immunophenotype and the t(11;14)(q13; q32) cytogenetic abnormality. These three cell lines are negative for the Epstein–Barr virus nuclear antigen and they were grown in RPMI-1640 supplemented with 10% fetal bovine serum and glutamine. MCL cells were plated at a density of 1 × 105 cells/ml and treated with control diluent (dimethylsulfoxide (DMSO)) or different concentrations of fenofibrate (Sigma-Aldrich, St Louis, MO, USA) ranging from 0 to 100 μM. The number of viable and dead cells was determined every 24 h for up to 3 days by vital dye exclusion assay using Trypan blue (Sigma-Aldrich), and CellTiter-Blue fluorescence was used to monitor cell viability according to the manufacturer's protocols (Promega, Madison, WI, USA) for the indicated time point. The PPARα ligand Wy-14643 and the PPARα antagonist GW6471 were purchased from Alexis Biochemicals (Berlin, Germany). Recombinant human TNFα was obtained from eBioscience (San Diego, CA, USA). MCL cells were serum starved overnight before the addition of TNFα and fenofibrate.

Measurements of apoptosis

Detection of apoptosis has been determined by phosphatidylserine externalization and propidium iodide, respectively, an early and late marker of cell apoptosis. Phosphatidylserine externalization has been detected by a standard flow cytometry technique using an Annexin V staining kit (BD Biosciences Pharmingen, San Diego, CA, USA). Propidium iodide staining was determined by a usual previously described protocol.33 Mitochondrial membrane potentials were determined using 3,3′-dihexyloxacarbocyanine iodide (DiOC6; Molecular Probes, Carlsbad, CA, USA) staining. In brief, cells treated for 16 h with different doses of fenofibrate were incubated with 5 nM DiOC6 for 15 min. The cells were harvested, washed in phosphate-buffered saline and immediately analyzed on a Becton Dickinson (Franklin Lakes, NJ, USA) FACSCalibur flow cytometer. Cells with intact mitochondrial membrane potential incorporate DiOC6 into the mitochondria.

Fenofibrate-treated primary MCL human cells were subjected to caspases 3/7 activities measurement with Caspase-3/7 Apo-One kit (Promega). In brief, 100 μl of Caspase-3/7 Apo-One reagent was added to 100 μl of cells culture treated with or without fenofibrate. The plate was then incubated at room temperature for 2 h. The fluorescence of each sample was measured in a fluorescent plate-reading FLUOstar Optima (BMG Labtechnologies, Offenburg Germany). The experiments were performed in triplicate and repeated on two separately initiated cultures.

Cell-cycle analysis by flow cytometry

Cells were treated with 0–100 μM of fenofibrate or with DMSO for the indicated times. Cells were then fixed with ice-cold 70% ethanol and analyzed after RNase treatment and propidium iodide staining. DNA content was determined using a FACSCalibur flow cytometer (BD Biosciences). Data acquisition was gated to exclude cell doublets, and cell-cycle phase distribution was analyzed using the CellQuest program (Becton Dickinson) (25 000 events were counted). Apoptotic cells appeared as the sub-G0-G1 peak on the DNA histogram.

Western blot analysis

Cells were harvested after the indicated time, washed with ice-cold phosphate-buffered saline and lysed in buffer containing 1% Triton X-100 and a complete protease and phosphatase inhibitor cocktail. Protein samples were electrophoresed through 10% SDS-polyacrylamide gels and transferred to nitrocellulose, and stained with 0.05% Ponceau S (Sigma-Aldrich) to ensure equivalent protein loading per lane. Probing with antibodies were performed overnight, at 4 °C with anti-cyclin D1, poly(ADP-ribose) polymerase-cleaved, caspase 3-cleaved, caspase 7-cleaved (Cell Signaling Technology, Beverly, MA, USA), PPARα (Rockland Immunochemicals, Gilbertsville, PA, USA), anti-NF-κB-p65, anti-β-actin and γ-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactivity was detected using peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G and visualized using enhanced chemiluminescence (Pierce, Rockford, IL, USA).

Reverse transcriptase-PCR

Reverse transcriptase-PCR was used for detecting PPARα mRNA in MCL cell lines. Total RNA was prepared with the Trizol (Invitrogen) in accordance with the manufacturer’s suggested protocol. In brief, complementary DNA synthesis was carried out for 50 min at 42 °C using the superscript reverse transcriptase II (Invitrogen, Carlsbad, CA, USA). The PCR was performed for 30 cycles in a thermal cycler (Applied Biosystems, Streetville, Ontario, Canada), with each consisting of denaturation (94 °C for 1 min), primer annealing (58 °C for 1 min) and DNA extension (72 °C for 1 min 30 s for 30 cycles). Amplified products were electrophoresed in 2% agarose gel containing ethidium bromide and visualized using Alpha Imager 3400 (Alpha Innotech, San Leandro, CA, USA). The following PPARα set primers was used: PPARα, forward 5′-AGATTTCGCAATCCATCGGC-3′; PPARα, reverse 5′-GCGTGGACTCCGTAATGATA-3′ (expected size, 276 bp); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control, GAPDH housekeeping primers forward 5′-AAGGTCATCCCTGAGCTGAA-3′, reverse 5′-CCCTGTTGCTGTAGCCAAAT-3′ (expected size, 316 bp).

Expression of apoptotic genes studied using oligonucleotide arrays

We used the RT2 Profiler PCR Array Human apoptosis Signaling Pathway array purchased from SuperArray Bioscience Corporation (Frederick, MD, USA). The complete gene list is available at www.superarray.com (no. PAHS-012). Total RNA from Mino cells treated without or with 40 μM fenofibrate for 24 h were isolated using Trizol (Invitrogen). First-strand complementary DNA synthesis reaction was performed as follows: 2 μg of extracted RNA was mixed with 10 μl of the SuperArray RT cocktail mix. The products were then incubated at 37 °C for 1 h and heated at 95 °C for 5 min. Quantitative SYBR green PCR was performed using an ABI 7900HT instrument (Applied Biosystems) and the following thermal cycling condition was used: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. The cycle threshold values, which are defined as the fractional cycle number at which the fluorescence passes an arbitrarily set threshold, were analyzed using the SDS (version 2.2.2) program (Applied Biosystems). The cycle threshold value of each gene was normalized to five housekeeping genes, α-2-microglobulin, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L13a, glyceraldehyde-3-phosphate dehydrogenase and β-actin, which are included in this commercially available kit. Triplicate experiments were performed and the statistical significance between the fenofibrate-treated cells and the negative controls were assessed by the Superarray Analysis software using Student’s t-test.

Assessment of the DNA binding of NF-κB and the nuclear translocation of the p65 subunit

Extraction of nuclear proteins was performed using a kit from Panomics (Fremont, CA, USA) as per the manufacturer’s suggested protocol. Protein quantification was determined by the BCA protein assay kit (Pierce). The NF-κB Transcription Factor ELISA Kit from Panomics was used, by following the instruction of the company, to assess the DNA binding of NF-κB. The colorimetric signals were measured using a Bio-Rad spectrophotometer (Bio-Rad Life Science Research, Mississauga, ON, Canada) at an absorbance of 450 nm.

Using a commercially available nuclear extraction kit (Panomics), nuclear and cytoplasmic proteins from MCL cell lines treated with fenofibrate for 24 h were extracted. The p65 subunit was detected using a monoclonal antibody from Santa Cruz Biotechnology. Anti-α-tubulin and γ-tubulin (Santa Cruz Biotechnology) were used to assess the efficiency of the nuclear/cytoplasmic fractionation and the total proteins loaded in each lane, respectively.

Enzyme-linked immunosorbent assay (ELISA) for human TNFα secretion

TNFα secretion was monitored using a commercially available ELISA kit (Cayman Chemical Co., Ann Arbor, MI, USA). Aliquots of culture medium from MCL cell lines treated without or with fenofibrate for 24, 48 and 72 h were centrifuged at 15 000 g, and the supernatant was assayed for TNFα levels, as per the manufacturer’s protocol.

MCL patient blood samples

Peripheral blood mononuclear cells (PBMCs) from four MCL patients with >5 × 109/l absolute lymphocyte count were obtained from the Department of Laboratory Medicine and Pathology, Cross Cancer Institute, with the approval by the institutional ethics committee. PBMCs were separated by Ficoll-Hypaque density centrifugation and washed three times and plated at a density of 1 × 105 cells/ml in RPMI-1640 supplemented with 10% fetal bovine serum. PBMCs were treated with control diluent DMSO or different concentrations of fenofibrate for 24 h and cell morphological analyses were visualized using Zeiss LMP microscope (Oberkochen, Germany).


Fenofibrate significantly decreases the growth of MCL cells in vitro

Addition of fenofibrate induced a significant reduction in the number of viable cells in all three MCL cell lines, as assessed by Trypan blue exclusion assay as well as the CellTiter-blue assay. Results from three representative experiments are illustrated in Figure 1a. At 24 h, the inhibitory concentration at 50% (IC50) for Mino, SP53 and Jeko-1 were approximately 40, 60 and 70 μM, respectively. At 72 h, the IC50 for all these three cell lines was 20 μM. Thus, fenofibrate inhibited cell growth (Figure 1a) and increased the number of dead cells (inserts in Figure 1a) of MCL cell lines in a dose- and time-dependent manner. The viability of PBMCs from three healthy volunteers treated with fenofibrate up to 100 μM showed no significant change (Figure 1b).

Figure 1
Figure 1

(a) Effects of fenofibrate-induced cell growth arrest and cell death on MCL cells. Three MCL cell lines, Mino, SP53 and Jeko-1 (1 × 105 cells/ml), were cultured for 24, 48 and 72 h in the presence of 0–100 μM of fenofibrate. Cell growth and dead cells (inserts histograms) were assessed by Trypan blue exclusion. Results are expressed as a percentage of control (without treatment). Values are mean±s.d. Triplicate experiments were performed. (b) PBMCs from three different healthy volunteers were treated with fenofibrate (0–100 μM) for the indicated period of time.

Fenofibrate effectively induces apoptosis in MCL cells in vitro

To examine whether cell-cycle arrest and/or apoptosis was responsible for fenofibrate-induced growth inhibition in MCL cell lines, two MCL cell lines (Mino and Jeko-1) were treated with various concentrations of fenofibrate for 24 h and examined by flow cytometry. Triplicate experiments were performed and results from a representative experiment are shown in Figure 2. Both Jeko-1 and Mino cells showed a dose-dependent increase in the size of sub-G0/1 cell population. It is noteworthy that Mino cells seemed to be more sensitive to fenofibrate than Jeko-1 cells, and this finding is in accordance with the results of the Trypan blue study described above. Owing to the extensive apoptosis (as reflected by the sub-G0/1 cell population), the cell-cycle status could not be fully assessed.

Figure 2
Figure 2

Fenofibrate alters cell-cycle progression. (a) Jeko-1 and (b) Mino cells were treated with fenofibrate at various concentrations for 24 h. Cell-cycle distribution was determined by flow cytometry. The percentages of cells in various phases of cell-cycle are shown. The results illustrated are representative of three independent experiments.

To further examine whether the effect of fenofibrate-induced inhibition of MCL cells growth is attributed to apoptosis, we used three different approaches. First, we performed a double staining for Annexin V and propidium iodide detected by flow cytometry. As shown in Figures 3a–c, we detected a dose-dependent increase in the number of Annexin V-positive cells in all MCL cell lines. However, Mino cells were found more sensitive than Jeko-1 and SP53 cells. Second, we performed flow cytometry to assess the mitochondrial transmembrane potential using the DiOC6 fluorescent probe. As shown in Figure 3d, the percentage of depolarized SP53 cells reached 13.96 and 25.26% after 16 h of incubation with 50 and 100 μM of fenofibrate, respectively (P<0.05 for both versus DMSO-treated cells). Finally, we were able to show a dose-dependent activation of caspases by fenofibrate. As shown in Figure 3e, there was cleavage of caspase-3 and poly(ADP-ribose) polymerase at 24 h after treatment in a dose-dependent manner. DMSO-treated cells (that is, at 0 μM) did not show detectable evidence of cleavages of these two proteins.

Figure 3
Figure 3

Fenofibrate induces apoptosis in MCL cells. Dose-dependent induction of apoptosis in three MCL cell lines: Jeko-1 (a), SP53 (b) and Mino (c). Cells were treated with different doses of fenofibrate for 24 h and apoptosis was assayed by Annexin V-binding and propidium iodide (PI). (d) A loss of mitochondrial membrane potential, an indicator of apoptosis,33 was determined by a decrease in DiOC6 fluorescence. Results of the mitochondrial membrane potential for SP53 cells exposed to different doses of fenofibrate for 16 h are shown. The percentage of SP53 cells with a decrease in DiOC6 fluorescence is shown in the lower panel. * Statistically significant P-value (P<0.05) when compared with DMSO-treated cells. (e) Fenofibrate-induced activation and cleavage of caspase 3 and poly(ADP-ribose) polymerase (PARP). Mino and Jeko-1 cells were cultured with fenofibrate at various concentrations for 24 h. Whole-cell lysates were subjected to western blot analysis.

Fenofibrate treatment resulted in a decrease in cyclin D1 expression

As cyclin D1 overexpression is believed to have an important pathogenetic role in MCL, we assessed whether fenofibrate mediates any effects on the expression of cyclin D1 in MCL cell lines. MCL cells were treated with various concentrations of fenofibrate for 24 h and then examined for cyclin D1 protein expression by western blot analysis. As shown in Figures 4a and b, fenofibrate decreased the expression of cyclin D1 at 24 h in a dose-dependent manner in Mino and SP53. Although we did not observe any appreciable difference in the expression of cyclin D1 in Jeko-1 cells after 24 h of fenofibrate treatment (at 40 μM), cyclin D1 expression in these cells was substantially decreased in a time-dependent manner (Figure 4c).

Figure 4
Figure 4

Fenofibrate induces downregulation of cyclin D1 expression. Mino (a) and SP53 cells (b) were incubated with or without fenofibrate (0–100 μM for 24 h) and Jeko-1 cells (c) were incubated with 40 μM of fenofibrate for up to 72 h. Cell lysates were examined for cyclin D1 expression by western blot, and there was a dose-dependent decrease in the cyclin D1 protein level.

Effects of another PPARα agonist Wy-14643 in MCL cells

To further elucidate the mechanism of fenofibrate-induced cell growth inhibition on MCL, we sought to determine whether the effects of fenofibrate on MCL cells are mediated through the PPARα receptor. In this regard, we found that MCL cells expressed the gene transcript of PPARα receptor (Figure 5a). However, the levels of PPARα protein in MCL cell lines were substantially lower than those of a number of epithelial cell lines examined (Figure 5b). In addition, western blot studies revealed that all five MCL frozen tumors examined had no detectable expression of PPARα protein (Figure 5c). We then examined whether the biological effects of fenofibrate in MCL cells are dependent on PPARα activation. Thus, we examined the biological effects of another agonist of PPARα, Wy-14643, in MCL cells. As shown in Figure 5d, addition of Wy-14643 also resulted in a significant decrease in cell growth, although the effect was less than that of fenofibrate. Specifically, the IC50's at 72 h were 60, 80 and 80 μM for Mino, SP53 and Jeko-1, respectively, when compared with 20 μM of fenofibrate for these three cell lines. Importantly, Wy-14643 did not induce significant cell death in MCL cells, as shown by the inserts in Figure 5d (that is, Trypan blue exclusion test). Thus, activation of the PPARα receptor does not fully account for the biological effects of fenofibrate in MCL cells, especially regarding its ability to induce apoptosis.

Figure 5
Figure 5

Fenofibrate effect on cell proliferation is independent of PPARα. (a) PPARα expression on MCL cells, reverse transcriptase-PCR (RT-PCR) to detect PPARα gene expression in three MCL cell lines (Jeko-1, Mino and SP53). Western blot analysis revealed that the expression level of PPARα was relatively low in MCL cell lines (b) and primary MCL tumor samples (c) when compared with a number of epithelial cell lines. (d) Three MCL cell lines, Mino, SP53 and Jeko-1 (1 × 105 cells/ml), were cultured for 24, 48 and 72 h in the presence of 0–80 μM of Wy-14643. Cell growth and dead cells (inserts) were assessed by Trypan blue exclusion. (e) MCL cells were incubated with antagonist for PPARα (GW6471, 20 μM) in the absence or presence of fenofibrate (40 and 60 μM). Results were expressed as a percentage of control (without treatment). Values are mean±s.d. of three independent experiments.

Effects of fenofibrate are not dependent on the transcriptional activity of PPARα

Next, we examined whether the DNA binding of PPARα is important for the biological activity of fenofibrate in MCL cells. Thus, we used GW6471, a PPARα-specific inhibitor of the DNA binding of PPARα.34 As shown in Figure 5e, exposure of MCL cells to 20 μM of GW6471 alone did not significantly affect the cell growth in SP53 and Jeko-1. Importantly, in the presence of fenofibrate, addition of GW6471 failed to prevent the cell growth-inhibitory effects of fenofibrate in these cells. Thus, the biological effects of fenofibrate do not seem to be dependent on the transcriptional activity of PPARα.

Fenofibrate significantly changes the expression of specific apoptotic-related genes

To further investigate the mechanism by which fenofibrate induced apoptosis in MCL, we used an apoptotic pathway-specific oligonucleotide arrays to compare MCL cells treated with DMSO versus those treated with fenofibrate. Mino cells were treated with fenofibrate for 24 h at 40 μM (that is, its IC50 at 24 h), and the results are illustrated in Figure 6a and summarized in Figure 6b. Only 5 of the 86 apoptotic-associated genes in the array showed statistically significant differences. They included BCL2A1, BCL2L1, HIAP1/BIRC3, CD40 and TNFα (−3.99-fold, −2.12-fold, −2.91-fold, −2.17-fold and −3.91-fold, when compared with DMSO-treated cells, respectively). Using an ELISA-based assay, we confirmed a significant decrease in TNFα secretion by Jeko-1 cells treated with fenofibrate (Figure 6c; P<0.01, compared with DMSO-treated cells).

Figure 6
Figure 6

Oligonucleotide array studies using MCL cells treated with fenofibrate. (a) Mino cells treated without or with 40 μM of fenofibrate were subjected to oligonucleotide array studies. Untreated cells were used as negative controls. Of the 86 genes, 5 were found to be significantly downregulated by fenofibrate, including BCL2A1, BCL2L1, HIAP1/BIRC3, CD40 and TNFα. (b) The statistical significance of these five genes are summarized. (c) Dose- and time-dependent inhibition of TNFα secretion in Jeko-1 cells treated with fenofibrate. TNFα was measured using a commercially available ELISA kit. Results are expressed as a percentage of the negative controls. Values represent mean±s.d. Triplicate experiments were performed. *Statistical significance (that is, P <0.01) compared with DMSO-treated cells. (d) Serum-starved Mino cells treated with 10 or 100 ng/ml of human TNFα in the presence of 40 μM of fenofibrate. Data represent mean±s.d. (e) NF-κB DNA-binding activity in Jeko-1 cells was significantly decreased with fenofibrate treatment and the relative levels of DNA binding of NF-κB are expressed as absorbance at 450 nm. Data represent mean±s.d. Triplicate experiments were performed. *Statistical significance (P&lt;0.01) when compared with DMSO-treated cells. (f) Nuclear expression of NF-κB-p65 in MCL cell lines was substantially decreased in MCL cells 24 h after fenofibrate treatment. Ponceau S, α-tubulin and γ-tubulin were used as loading controls and results shown are from one of two independent experiments.

Effect of TNFα on MCL cell growth treated with fenofibrate

To assess whether TNFα is indeed relevant in the context of fenofibrate-induced apoptosis, we added human recombinant TNFα (at 10 or 100 ng/ml) to Mino cells treated with fenofibrate. As shown in Figure 6d, TNFα treatment significantly improved the cell viability, in a dose-dependent manner, after 24 and 48 h of treatment with 40 μM of fenofibrate.

The DNA binding of NF-κB is decreased by fenofibrate in MCL cells

As NF-κB has been shown to be important for the pathogenesis of MCL, and because PPARα ligands has been shown to inhibit the NF-κB activity and expression in other cell types,9 we examined whether fenofibrate has any significant effects on this signaling pathway. Using an ELISA-based assay, we assessed the DNA-binding assay of NF-κB in the presence or absence of fenofibrate in Jeko-1. As shown in Figure 6e, we found a significant decrease in the DNA binding of NF-κB in Jeko-1 cells treated with various concentrations of fenofibrate, compared with DMSO-treated cells (P<0.01). In addition, the decrease in the DNA binding of NF-κB seemed to be dose dependent. Using subcellular fractionation and western blots, we found a dose-dependent decrease of nuclear NF-κB-p65 protein level in MCL cells line treated with fenofibrate (Figure 6f).

Effects of fenofibrate in primary MCL cells

To determine whether fenofibrate induces cell death in primary MCL cells, we treated PBMCs, harvested from four MCL patients, with increasing concentrations of fenofibrate. After 24 h of treatment, cells were visualized using phase contrast microscopy (Figure 7a). As shown in Figures 7a and b, fenofibrate treatment induced a decrease in the number of viable primary MCL cells and an increase in the apoptotic-appearing cells in a dose-dependent manner. In Figure 7c, in vitro addition of fenofibrate for 24 h induced a significant reduction in the number of viable cells, as assessed by Trypan blue exclusion. In the same patient (patient 4), we detected a significant increase in caspases 3/7 activities after fenofibrate treatment.

Figure 7
Figure 7

Fenofibrate induces cell death of PBMCs from MCL patients. (a) Representative pictures of peripheral blood mononuclear cells from three different MCL patients showing a dose-dependent induction of cell death. (b) Flow cytometric analysis of Annexin-V-positive cells from patient MCL 3, treated by fenofibrate for 24 h. Data are expressed as ratios compared with the negative controls. (c) Viable and dead cells were assessed by Trypan blue assay using PBMCs from patient MCL 4, at 24 h after fenofibrate treatment. (d) Fenofibrate activated caspases 3/7 activities, as assessed with the commercially available Caspase-3/7 Apo-One kit. Data represent mean±s.d. Triplicate experiments were performed. *Statistical significance (P<0.05) when compared with DMSO-treated cells.


MCL remains a challenging disease to treat and the clinical outcome for most patients is poor. There is a need to develop and explore new approaches for the treatment of this lymphoma. In this study, we have shown that fenofibrate, a widely known PPARα agonist, induces effective apoptosis in MCL cells. However, we found that its mechanism of action in MCL cells is not fully dependent on the transcriptional activity of PPARα, as these effects were not significantly blocked by the PPARα antagonist GW6471. Consistent with this concept, we found a relatively low level of PPARα protein expression in MCL cell lines and tumors. In addition, we found that Wy-14643, a PPARα agonist, failed to produce a potent apoptotic effect as observed with fenofibrate in MCL cell lines. Our observations are in accordance with the previous reports that have also suggested that the antitumor activity of PPARα agonists may involve unknown pathways that are independent of blocking PPARα transcriptional activity and have been described as ‘off-target’ effects.15, 17, 20

Cyclin D1 overexpression is a hallmark of MCL cells. It has been recently reported that inhibition of cyclin D1 in MCL cells blocks cell proliferation but is not sufficient to induce cell death in these cells.35 Downregulation of cyclin D1 by PPARα agonist has been described in studies of human smooth muscle cells,36 kidney cells37 and cancer cells.16, 38 We decided to investigate whether fenofibrate has an effect on the regulation of cyclin D1. Our results show that fenofibrate treatment decreased cyclin D1 protein expression in MCL cell lines. On the basis of our literature search, we are not aware of any known consensus sequence for PPARα binding in the promoter region of cyclin D1. Although the mechanism of how fenofibrate decreases cyclin D1 in MCL cells is uncertain, we cannot completely rule out the possibility that the occurrence of fenofibrate-induced apoptosis may have contributed to the apparent downregulation of cyclin D1 in these experiments.

To further understand how fenofibrate induces apoptosis in MCL, we investigated its effect on the TNFα/NF-κB pathway axis. It has been previously reported that fenofibrate can modulate the NF-κB signaling, a constitutively active signaling pathway in MCL. Thus, we speculated that fenofibrate can promote apoptosis, in part, by blocking this pathway. Using multiple assays, we found that fenofibrate downregulates the level of TNFα and the NF-κB in MCL cells. In keeping with the observation in other cell types,39, 40, 41 our results suggest that fenofibrate may exert its effect by inhibiting the TNFα/NF-κB signaling axis.

To further elucidate the mechanism of fenofibrate-mediated apoptosis in MCL, we used an ‘apoptosis array’ to examine and decipher the most important apoptotic regulators that are affected by fenofibrate treatment. Our results show that fenofibrate downregulates a very specific and small group of anti-apoptotic proteins. BCL2A1 and BCL2L1 belong to the BCL-2 family proteins and BCL2L1 is located at the mitochondrial membrane and regulates cell death by maintaining mitochondrial membrane potential and blocking cytochrome c release through voltage dependent anion channel (VDAC).42 Our results show that fenofibrate induces a downregulation of BCL2L1 and reduction of mitochondrial membrane potential. It has recently been reported that BCL-2 inhibition can induce apoptosis in MCL cells;28 our results indicate that fenofibrate may induce apoptosis, in part, by regulating mitochondrial membrane potential through BCL2L1. BCL2A1, another member of the BCL-2 family, has been reported to be a direct transcription target of NF-κB. We also found downregulation of this protein in fenofibrate-treated cells. As we have earlier shown that fenofibrate also downregulates NF-κB in MCL, it is likely that NF-κB downregulation reduced BCL2A1 expression, which consequently leads to a loss of mitochondrial membrane potential and activation of caspases. However, we cannot completely exclude the possibility that fenofibrate also induces other forms of cell death such as necrosis or autophagy.

To ascertain the efficiency of fenofibrate as a potential therapy of MCL, we treated primary cells from MCL patients. Our results show that fenofibrate exert the same effect in vitro and ex vivo. Fenofibrate treatment induced cell death of primary MCL cells and we further showed that fenofibrate-mediated cell death involved induction of apoptosis, as reflected by the activation of caspases. Fenofibrate did not induce cell death of PBMCs from normal individuals. These results are in keeping with the previous studies that have shown the safety of fenofibrate therapy in humans. As all our primary MCL samples were derived from patients in leukemic phase, further studies using primary MCL cells from patients without leukemia may be of interest.

In summary, our study shows that fenofibrate effectively induces apoptosis in MCL cells by inhibiting the TNFα/NF-κB pathway axis, which is known to be important in the pathogenesis of MCL. We show that fenofibrate is able to modulate the expression of key anti-apoptotic members that are expressed in MCL. This study has underlined that fenofibrate treatment may hold potential in overcoming the apoptosis resistance observed in many MCL tumors. The half-life of fenofibrate has been reported to be 20 h in individuals with normal renal functions, and the level of fenofibrate required to achieve IC50 for MCL is within the therapeutic range that is used to treat hyperlipidemia.43, 44, 45, 46 In light of the excellent safety, tolerability and affordability of fenofibrate, there is merit in investigating the possibility of extending the clinical use of fenofibrate, either as a sole agent or in combination with conventional chemotherapy, in the treatment of MCL.


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ZZ and PG are recipients of the Research Fellowship Award of the Alberta Cancer Research Institute. This study is supported by a research operating grant awarded by the National Cancer Institute of Canada to RL.

Author information


  1. Department of Laboratory Medicine and Pathology, Cross Cancer Institute and University of Alberta, Edmonton, Alberta, Canada

    • Z Zak
    • , P Gelebart
    •  & R Lai


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Competing interests

The authors declare no conflict of interest.

Corresponding author

Correspondence to R Lai.