Abstract
Background/Aim: Recent innovations in the treatment of multiple myeloma have enriched our therapeutic repertoire regarding the treatment of multiple myeloma during the last decades. However, despite today's therapies many multiple myeloma (MM) patients experience relapse of disease and eventually remain incurable. Wnt/β-catenin signaling has been demonstrated in lymphoma and MM, rendering related signaling molecules promising therapeutic targets. Fenofibrate, an extensively scrutinized and widely used drug for primary hypercholesterolemia or mixed dyslipidemia, has proven anticarcinogenic properties mediated by peroxisome proliferator-activated receptor-alpha (PPARα) agonism, thereby also influencing WNT-associated signaling molecules. Materials and Methods: The antitumor apoptotic effect of fenofibrate at doses ranging from 0.1-200 μM was investigated on a total of seven human, two murine myeloma/lymphoma cell lines and two healthy control cell lines, as determined by 3’3-Dihexyloxacarbocyanine iodide (DiOC6) and propidium iodide (PI) staining in flow cytometry. Results: Fenofibrate significantly reduced viability due to apoptosis induction in all investigated myeloma and lymphoma cell lines in a dose-dependent manner, whereas healthy control cells were less sensitive. Conclusion: Our results provide a rationale for future in vitro and in vivo studies with fenofibrate as a safe and well-tolerated agent in MM and lymphoma treatment.
Multiple myeloma (MM) is a hematological malignancy induced by monoclonal malignant secretory plasma cells in the bone marrow and is usually associated with monoclonal protein in peripheral blood and/or urine (1, 2). Innovative therapy strategies including immunomodulatory drugs (IMiDs) like bortezomib, lenalidomide and thalidomide as well as advanced cell-based treatment approaches improved treatment outcome and patient survival (3, 4). Supportive therapies like localised radiotherapy can additionally improve patients' quality of life in advanced disease stages (5).
Wnt signaling, in general restricted solely to the embryonic development, represents a tumor-specific signaling cascade causing and perpetuating carcinogenic effects in both the oncogenesis as well as the propagation of lymphoma and MM (6-15). A targeted inhibition of the Wnt pathway, therefore, impedes tumor progression, and thus renders Wnt signaling molecules a promising therapeutic target for MM (15, 16).
In recent studies, we confirmed the efficacy of multiple agents, inter alia clofibrate as an analogue of fenofibrate, by targeting Wnt/beta-catenin signaling molecules, particularly in hematopoietic malignancies (17-32). Fenofibrate is chemically related to other known Wnt inhibitors and has already proven anticarcinogenic properties in numerous neoplasms (33). Herein we demonstrated, for the first time, that fenofibrate is efficacious in the in vitro treatment of MM through inducing significant apoptosis.
Materials and Methods
Cell lines and culture conditions. Cell lines were obtained from DSMZ (Braunschweig, Germany) or ATCC (LGC Standards, Wesel, Germany) and incubated at 37°C with 5% CO2 at 90% humidity. The human myeloma cell lines KMS 18, OPM-2, RPMI-8226 and U-266 (all obtained from DMSZ) were cultured in RPMI-1640-medium (PAA, Pasching, Austria), supplemented with 5% heat-inactivated fetal calf serum FCS (Invitrogen, Darmstadt, Germany) and 1% penicillin-streptomycin (Seromed, Jülich, Germany). Human lymphoma cell lines Raji, SU-DHL-4 and Oci Ly 8 Lam 53 cells were cultured under identical conditions as human myeloma cell lines. MPC-11 is a murine plasmocytoma cell line and RAW 264,7 is a leukaemia monocyte macrophage cell line. Cells were cultured in RPMI-1640 medium supplemented with 5% heat-inactivated FCS and 1% penicillin/streptomycin. RAW 264,7 cells were harvested using 0.05% trypsin-EDTA solution (Invitrogen).
The human colon fibroblast cell line CCD-18Co was obtained from ATCC and cultured in ATCC-formulated Eagle's Minimum Essential Medium (LGC Standards) supplemented with 15% of heat-inactivated FCS and 1% penicillin-streptomycin. Cells were harvested by 0.05% trypsin-EDTA solution (Invitrogen), centrifuged at 1,200 × g for 7 min and re-suspended in 1 mL media to define the cell count. Media were renewed at least every 3 days.
Human samples. Peripheral blood lymphocytes (PBLs) were isolated from blood samples of healthy volunteers using Ficoll density gradient centrifugation (Lymphoprep; Nycomed, Oslo, Norway). Blood from buffy coats was diluted 1:2 with phosphate-buffered saline (PBS)/1% bovine serum albumin (BSA) (both from PAA) and used for a Ficoll gradient (Lymphoprep). The leukocyte layer was transferred to new tubes after centrifugation at 800 × g for 30 minutes. Cells were washed three times with PBS/1%BSA and resuspended in RPMI-1640 medium supplemented with 10% FCS, 1% penicillin/streptomycin and 2.5% HEPES buffer solution (PAA).
Drugs and chemical reagents. Fenofibrate was purchased from Sigma-Aldrich (Steinheim, Germany) and tested at concentrations ranging from 0.1-200 μM for 72 h.
3’3-Dihexyloxacarbocyanine iodide (DiOC6) and propidium iodide (PI) staining. Reduced mitochondrial transmembrane potential is known to occur late in the apoptotic process. We used DiOC6 staining and flow cytometry to assess the mitochondrial transmembrane potential. Therefore, 1×105 cells were plated in 3 ml medium in 6-well plates. Fenofibrate was dissolved in dimethyl sulfoxide (DMSO) (Invitrogen) and added to the medium at different concentrations for three days. Staining with DiOC6 for detecting viable cells and with PI, which binds to DNA in necrotic cells, was used for the apoptosis assay, measured by a fluorescence-activated cell sorter (FACS; BD FACSCanto II, Becton Dickinson Biosciences, Franklin Lakes, NJ, USA). The medium containing drug-treated cells was transferred from each well into a glass tube. Then cells were centrifuged at 800 × g for 7 min, washed with phosphate buffered saline (PBS, pH 7.4) (Roti-Stock 10x; CarlRoth, Karlsruhe, Germany) and stained after repeated centrifugation by adding 500 μl staining solution (RPMI-1640, 0.5% bovine serum albumin [BSA], 80 nM DiOC6) for 15 min at 37°C. After another washing step with PBS/1% BSA, cells were re-suspended in 500 μl PBS/1% BSA. FACS analysis was performed immediately after the addition of 5 μl PI solution (100 μg/ml) with a FACS (BD FACSCanto II, Becton Dickinson Biosciences, Franklin Lakes, NJ, USA). Approximately 10,000 counts were made for each sample. In this assay, viable cells show high fluorescence intensity for DiOC6 and a low fluorescence for PI. Necrotic cells fluoresce in an opposite manner, with high intensity for PI and a low intensity for DiOC6. Early apoptotic cells show low fluorescence for both DiOC6 and PI. Cells with high fluorescence intensity for both DiOC6 and PI correspond either to late apoptotic cells as apoptotic bodies or debris.
Cell viability assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT). The efficacy of fenofibrate in CCD-18Co cells was determined by cell viability in the MTT assay. Viable cells convert the yellow MTT (Sigma Aldrich, Steinheim, Germany) into purple formazan when taken-up into mitochondria. Previously, cells were plated at 1×104 well/100 μl in 96-well plates, left to adhere overnight in the incubator. 24 h later media were removed and renewed containing various concentrations of fenofibrate. After 69 h 1 μL MTT (5 mg/ml) was added to each well and incubated for another 3 h. Then 80 μL of the media were removed and 50 μl of acidified isopropanol was added for cell lysis. After shaking for 10 min the amount of formazan was measured at 565 nM. The measured amount of formazan in treated cells was compared to untreated cells.
Inhibitory concentration (IC50) of fenofibrate for human lymphoma, human and murine multiple myeloma, murine leukemia and control cell lines. CCD-18Co cells and peripheral blood lymphocytes (PBL) served as controls. A total of 1×105 cells were cultured with different concentrations of fenofibrate for 72 h. Cell viability and apoptosis was measured by 3’3-Dihexyloxacarbocyanine iodide (DiOC6) and propidium iodide (PI) staining in flow cytometry. CCD-18Co cells were investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay. Results represent the mean of data from at least three independent experiments each.
Statistical analysis. Values are given as mean±standard deviation (SD). At least three separate and independent experiments were performed with each cell line. Paired, two-tailed Student's t-test was used for statistical analysis. A p-value less than 0.05 was considered significant.
Results
Titration of fenofibrate. The mean 50% inhibitory concentration (IC50) after 72 h was calculated following titration. Fenofibrate concentrations inducing a significant decrease in viability were, therefore, determined. PBLs and CCD-18Co colonic fibroblasts served as healthy controls. All except CCD-18Co cells were investigated by DiOC6 and PI staining in flow cytometry. CCD-18Co cells were investigated by MTT. IC50 values of fenofibrate employed after 72 h of incubation are given in Table I.
Effect of fenofibrate on viability of Raji, SU DHL 4 and Oci Ly 8 Lam 53 human lymphoma cells. Cells were cultured with fenofibrate for 72 h. Cell viability and apoptosis was measured by 3’3-Dihexyloxacarbocyanine iodide (DiOC6) and propidium iodide (PI) staining in flow cytometry. Results represent data from three separate experiments. Data are shown as mean±SD. *p<0.05 compared to untreated cells.
Effect of fenofibrate on viability of human lymphoma cells. Fenofibrate treatment with concentrations from 50 μM and above significantly decreased lymphoma cell viability in all tested cell lines. The IC50 of Raji, SU-DHL-4 and Oci Ly 8 Lam 53 was attained after treatment with 33 μM, 42 μM and 30 μM, respectively. Figure 1 presents the respective results. Figure 2 (Panel A) presents exemplary flow cytometry results.
Effect of fenofibrate on viability of human myeloma cells. Comparably, the viability of the investigated myeloma cells decreased in a concentration-dependent manner after the addition of fenofibrate. In RPMI-8226 cell concentrations starting from 10 μM were required for significant apoptosis induction. All remaining human myeloma cell lines except KMS-18 cells significantly underwent apoptosis following the treatment with 50 μM of fenofibrate for 72 h. KMS-18 myeloma cells showed the lowest sensitivity towards fenofibrate treatment with an IC50 of 125 μM. Results were as shown in Figure 3. Figure 2 (Panel B and C) shows the corresponding flow cytometry results.
Effect of fenofibrate on viability of murine cells. The described induction of apoptosis for human myeloma and lymphoma cells was also reproducible in murine myeloma and leukaemia monocyte macrophage cells. Fenofibrate concentrations starting from 50 μM were required for a significant decrease of viability in MPC-11 cells. However, RAW 264,7 cells were less sensitive towards fenofibrate with an IC50 of 93 μM. Results are given in Figure 4.
Effect of fenofibrate on viability of healthy controls. CCD-18Co colon fibroblasts and PBLs were chosen to investigate the toxic potential of fenofibrate towards healthy stroma cells and lymphocytes, respectively. CCD18-Co cells and PBLs tolerated far higher concentrations of fenofibrate compared to human and murine myeloma and lymphoma cell lines tested with an IC50 >400 μM and 97 μM, respectively. Results are shown in Figure 5.
Discussion
MM represents a systemic hematologic malignancy due to degenerated plasma cells, caused by frequent gene mutations and/or chromosomal translocations (7). Today's therapy regimens consist of a primary initiated high-dose chemotherapy along with a facultative hematopoietic stem cell transplantation (34-37). However, most MM patients experience relapse of disease following chemotherapy (3, 35-41).
The development of MM cells relies on the bone marrow microenvironment. Herein, bone marrow stromal cells were shown to supply specific Wnt ligands facilitating and maintaining an inordinate propagation of MM cells (9, 39-41). Targeting the Wnt signaling cascade promoting tumor differentiation and proliferation might, thus, represent a promising therapeutic approach in MM research and treatment, since its inhibition can prevent MM progression (11-15, 39).
In recent studies, we revealed various drugs as potent inducers of apoptosis in lymphoma and myeloma cells in vitro and partially proved in vivo efficacy in subsequent animal studies. Several of these agents were demonstrated as efficacious in MM and lymphoma treatment due to an inhibition of the Wnt pathway as the underlying mechanism of action (17-32, 42, 43).
Exemplary results generated by flow cytometry. The relative number of cells is given within the quarters. (A) Oci Ly 8 Lam 53 cells before and after treatment with 200 μM fenofibrate. (B) RPMI-8226 cells before and after treatment with 200 μM fenofibrate. (C) U-266 cells before and after treatment with 100 μM fenofibrate. Cells were treated with fenofibrate at different concentrations. Seventy-two hours after incubation, flow cytometry was performed.
Fenofibrate is one of the most commonly prescribed fibrates since 1975. Like other fibrates it is used for the treatment of hypercholesterolemia and hypertriglyceridemia, predominantly in people at risk of cardiovascular disease. Lipid levels are lowered by interaction with the peroxisome proliferator-activated receptor alpha (PPARα). PPARα, in return, induces lipoprotein lipase and reduces apoprotein CIII, leading to increased lipolysis and elimination of triglyceride-rich particles within the plasma. PPARα represents a nuclear receptor of the steroid hormone receptor superfamily that has been shown to possess anti-inflammatory properties and to exert various anticancer properties (33, 44, 45). Multiple studies investigated a potential utilization in cancer therapy, particularly by ligands of PPARα, beyond their prescription as well-tolerated and extensively scrutinized lipid-lowering drugs (45).
Fenofibrate showed to exert direct antitumor and antiendothelial effects in vitro and significantly reduced melanoma, glioblastoma and fibrosarcoma cell growth in murine tumor models (33). In hepatocellular carcinoma cells, fenofibrate suppressed cell growth through a PPARα independent mechanism by blocking Akt activation due to an increase of C-terminal modulator protein (CTMP) as the supposed mechanism of action (46). In glioblastoma cells treatment with 25 μM fenofibrate effectively repressed cell growth by G (1) arrest, whereas cells treated with 50 μM fenofibrate underwent a delayed and massive apoptosis after 72 h that was preceded by FoxO3A nuclear accumulation and expression of FoxO-dependent apoptotic protein (47). Interestingly, fenofibrate was also shown to inhibit the transcriptional activity of nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-ĸB) and to disrupt its association with hypoxia inducible factor 1 alpha (HIF1α) required for the binding of NF-ĸB to the pyruvate kinase muscle isozyme M (PKM) promoter and PKM2, causing mitochondrial damage in glioblastoma cells (48). Similarly, in breast cancer fenofibrate induced apoptosis by activation of the NF-ĸB pathway since the effect was reversible by a NF-ĸB-specific inhibitor, and additionally, cell-cycle arrest at G0/G1 phase accompanied by down-regulation of cyclin D1 was observed. Subsequent in vivo experiments showed that tumor growth was attenuated in a xengograft breast cancer mouse model (49). Of major importance, especially in the context of hematological malignancies, fenofibrate was shown to significantly downregulate several pro-survival genes in mantle cell lymphoma including tumor necrosis factor-alpha (TNF-a) and NF-ĸB in a dose-dependent manner (50).
Effect of fenofibrate on viability of KMS-18, OPM-2, RPMI-8226 and U-266 human myeloma cells. Cells were cultured with fenofibrate for 72 h. Cell viability and apoptosis was measured by 3’3-Dihexyloxacarbocyanine iodide (DiOC6) and propidium iodide (PI) staining in flow cytometry. Results represent data from three independent experiments. Data are shown as the mean±SD. *p<0.05 compared to untreated cells.
Effect of fenofibrate on viability of MPC-11 and RAW 264,7 murine myeloma and leukemia cells, respectively. Cells were cultured with fenofibrate for 72 h. Cell viability and apoptosis was measured by 3’3-Dihexyloxacarbocyanine iodide (DiOC6) and propidium iodide (PI) staining in flow cytometry. Results represent data from at least three separate experiments. Data are shown as mean±SD. *p<0.05 compared to untreated cells.
Our results demonstrated for the first time, that fenofibrate also reduces the proliferative capacity of MM and several lymphoma subtypes since it significantly reduced the viability of all tested myeloma and lymphoma cell lines by apoptosis induction due to reduced mitochondrial membrane potentials. Both human and murine cells were equally affected in a dose-dependent manner. Doses of approximately 50 μM led to a significant decrease in cell viability in most myeloma and lymphoma cell lines. Interestingly, CCD-18Co colonic fibroblasts and PBLs, both serving as healthy controls, tolerated far higher fenofibrate concentrations, which, in turn, confirms the well-known safety profile of fenofibrate as a widely-prescribed drug.
Effect of fenofibrate on viability of CCD-18Co cells and peripheral blood lymphocytes (PBL) that served as healthy controls. Cells were cultured with fenofibrate for 72 h. For CCD-18Co cells, viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay. For PBL viability and apoptosis was measured by 3’3-Dihexyloxacarbocyanine iodide (DiOC6) and propidium iodide (PI) staining in flow cytometry. Results represent data from three separate experiments. Data are shown as mean±SD. *p<0.05 compared to untreated cells.
Despite recent treatment innovations in myeloma treatment research, there still exists considerable scope in the identification of novel, and more importantly, more effective and sustainable therapeutic agents. Particularly, addressing specific genetic mutations as within the Wnt pathway in MM represent a promising treatment strategy and might improve treatment outcome and reduce therapy-associated adverse reactions due to a targeted inhibition of proliferation-enhancing effects. Due to its influence on Wnt-associated signaling molecules in mantle cell lymphoma and several solid neoplasms, fenofibrate might also interfere with signaling molecules embedded in the Wnt and associated signaling pathways in lymphoma and MM. Fenofibrate demonstrated a significant cytotoxic potential towards both MM and lymphoma cells by apoptosis induction and slightly attenuated the propagation of healthy controls.
Since fenofibrate is efficacious at low effective doses in in vitro treatment of MM and as a widely-prescribed lipid-lowering drug, it might represent a valuable agent for the utilization in MM treatment. Further in vitro studies using fenofibrate should be taken into consideration to delineate the exact mechanisms of action associated with the various patterns defining cellular survival in MM. Subsequent animal myeloma models are warranted to clarify its in vivo efficacy and toxicity in order to path a way for an initial clinical application in MM patients.
Hence, our results provide a rationale for advanced in vitro and in vivo studies using fenofibrate as a well-tolerated agent for the treatment of MM and lymphoma.
- Received May 22, 2017.
- Revision received May 30, 2017.
- Accepted May 31, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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