Targeting cholesterol synthesis increases chemoimmuno-sensitivity in chronic lymphocytic leukemia cells
© Benakanakere et al.; licensee BioMed Central Ltd. 2014
Received: 28 July 2014
Accepted: 14 September 2014
Published: 26 September 2014
Cholesterol plays an important role in cancer development, drug resistance and chemoimmuno-sensitivity. Statins, cholesterol lowering drugs, can induce apoptosis, but also negatively interfere with CD-20 and rituximab-mediated activity. Our goal is to identify the alternative targets that could reduce cholesterol levels but do not interfere with CD-20 in chemo immunotherapy of chronic lymphocytic leukemia (CLL).
MEC-2 cells, a CLL cell line, and the peripheral blood mononuclear cells (PBMCs) from CLL patients were treated with cholesterol lowering agents, and analyzed the effect of these agents on cholesterol levels, CD-20 expression and distribution, and cell viability in the presence or absence of fludarabine, rituximab or their combinations.
We found that MEC-2 cells treated with cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475) reduced 20% of total cellular cholesterol levels, but also significantly promoted CD-20 surface expression. Furthermore, treatment of cells with fludarabine, rituximab or their combinations in the presence of BIBB-515, YM-53601 or TAK-475 enhanced MEC-2 cell chemoimmuno-sensitivity measured by cell viability. More importantly, these cholesterol lowering agents also significantly enhanced chemoimmuno-sensitivity of the PBMCs from CLL patients.
Our data demonstrate that BIBB-515, YM53601 and TAK-475 render chemoimmuno-therapy resistant MEC-2 cells sensitive to chemoimmuno-therapy and enhance CLL cell chemoimmuno-sensitivity without CD-20 epitope presentation or its downstream signaling. These results provide a novel strategy which could be applied to CLL treatment.
KeywordsFludarabine Rituximab Cholesterol lowering agent Chronic lymphocytic leukemia
Chronic lymphocytic leukemia (CLL) is the most prevalent hematologic malignancy affecting Caucasian adults in Western countries. In the United States, about 15,000 new cases are diagnosed every year . CLL is characterized by the accumulation of mature CD5, CD19, and CD23 B-lymphocytes in peripheral blood, bone marrow, lymph nodes and spleen . The disease typically occurs in elderly patients (the average age at the time of diagnosis is around 72 years). Overall survival at 5 and 10 years ranges between 87% and 73% for low-risk patients and 29% and 16% for high risk patients . CLL is widely heterogeneous in terms of progression, therapeutic response and outcome . Approximately one-third of patients with CLL survive many years without requiring treatment, whereas others need multiple therapies early in the course of the disease . Although the entire pathogenesis of CLL has not been elucidated, the widely held concept is that CLL is associated with a defective regulation of apoptosis, rather than uncontrolled cell proliferation .
Over the last three decades, chlorambucil has been the mainstay for the treatment of CLL; however, the complete response rate with chlorambucil is only about 10% [7–9]. Purine analogues, particularly fludarabine, are effective agents to treat CLL, resulting in a higher complete response rate than chlorambucil or alkylating-based chemotherapies (20-30% versus 10%) and a longer disease-free interval; survival, however, is not prolonged . Rituximab and alemtuzumab also result in a higher complete response rate than chlorambucil in chemonaïve patients [11, 12]. Recent advances have now moved away from mono-therapy to combination including the chemoimmuno-therapy [13–17]. The combination of fludarabine, cyclophosphamide and rituximab treatment achieves complete responses in only 24% to 39% of patients [15, 16]. Bendamustine, an alkylating agent, plus rituximab is the other commonly used regimen for CLL, achieving a response rate of 88% and a complete response rate of 26% . In spite of some progress in therapy, CLL is still considered an incurable disease because most patients generally relapse and eventually develop drug-resistance . It is clear that new approaches to overcoming drug-resistance and potentiating the action of conventional therapy are urgently needed.
Cholesterol contributes to chemotherapy resistance in hepatocellular carcinoma , breast cancer cells  and prostate cancer cells . Statins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase which catalyzes the conversion of HMG-CoA into mevalonate, a rate-limiting step in the cholesterol biosynthetic pathway . Statins are commonly prescribed medications that lower serum cholesterol and prevent cardiovascular diseases [22, 23]. Beyond their cholesterol-lowering properties, statins have also been shown to induce apoptosis through extrinsic and intrinsic pathways [24–26], and exhibit important antitumor activity in colorectal cancer, breast cancer, lung cancer, prostate cancer, pancreatic cancer and many other malignancies [27–29]. Although statins can induce apoptosis of acute myelogenous leukemia cell lines  and CLL cells , they are unsuitable for CLL therapy because statins interfere with the detection of CD-20 and impair rituximab-mediated complement-dependent cytotoxicity .
To avoid the negative interference of statins with CD-20 molecules and rituximab-mediated activity and to explore whether cholesterol levels contribute to chemoimmuno-sensitivity and drug-resistance of cancer cells, we chose several agents that inhibit squalene synthase or oxidosqualene cyclase, downstream steps in the cholesterol biosynthetic pathway, and used a CLL cell line (MEC-2) and the peripheral blood mononuclear cells (PBMCs) from CLL patients to investigate the effect of cholesterol lowering agents on the cholesterol levels, CD-20 expression and distribution, and cell viability with fludarabine, rituximab or their combinations. Our results indicate that the inhibitors of squalene synthase or oxidosqualene cyclase can significantly enhance CLL cell chemoimmuno-sensitivity.
Effect of chemoimmuno-therapeutic drugs in MEC-2 cell viability and apoptosis
Lovastatin enhances chemo-sensitivity in fludarabine-treated MEC-2 cells
Effect of BIBB-515 and YM-53601 on MEC-2 cell chemoimmuno-sensitivity
TAK-475 enhances chemoimmuno-sensitivity in MEC-2 cells
BIBB-515 and TAK-475 enhance chemoimmuno-sensitivity in the PBMCs
Cholesterol plays an essential role in the stability and architecture of the plasma membrane and in the involvement of vesicle traffic and receptor signaling [40, 41]. Emerging data from profiling of cancer tissues and animal models [42–44] as well as in vitro cancer cell lines  demonstrate that cholesterol is capable of regulating cell proliferation, migration, and signaling pathways in carcinogenesis, tumor development and chemotherapy resistance. Recognizing cholesterol as an important factor contributing to cancer development, many researchers focus on manipulating cholesterol metabolism as novel targets for cancer therapy [18–25]. Statins, cholesterol lowering agents, inhibit mevalonate metabolism and exhibit antitumor effects against various cancer cell lines [27–29]. Using lovastatin, we reported here that lowering cholesterol exhibited increased chemosensitivity in fludarabine-treated MEC-2 cells, but had no effect on immunotherapy in rituximab-treated cells (Figure 3). Earlier data from in vitro  and in vivo  also showed that statins do not influence clinical response and B cell depletion after rituximab treatment because statins induce conformational changes in CD-20 molecules and impair rituximab-mediated complement-dependent cytotoxicity.
CLL cell killing by rituximab requires cross linking of CD-20, which induces redistribution of CD-20 to lipid rafts. Lipid rafts are specialized microdomains of the plasma membrane that are enriched in sphingolipid and cholesterol, and play an important role in the initiation of many anticancer drug-induced signaling pathways and toxicological effects. Anticancer drugs are able to suppress cell growth and induce apoptosis of tumor cells through disrupting lipid raft integrity [32, 47]. The redistribution of CD-20 may result in lipid raft disruption, activate or deactivate raft-associated proteins, such as death receptors and protein kinases in apoptotic pathway which are correlated with efficiency of complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. Rituximab and other anti-CD-20 antibodies are currently key drugs in CLL chemoimmuno-therapy. Although statins have antitumor activity, they are clearly unsuitable for enhancement of chemoimmuno-sensitivity of lymphomas and leukemias.
To search for new agents that can lower cellular cholesterol, and do not negatively interfere with CD-20 and rituximab-mediated activity, we chose three compounds that can inhibit squalene synthase (YM-53601 and TAK-475) or oxidosqualene cyclase (BIBB-515). These enzymes are further downstream steps in cholesterol biosynthetic pathway (Figure 2). By confocal microscopy, our results indicate that treatment of MEC-2 cells with YM-53601, BIBB-515 or TAK-475 did significantly up-regulate CD-20 surface expression and membrane-association (Figures 4 and 6). One recent study has shown that farnesyltransferase inhibitors may up-regulate CD-20 at mRNA and protein levels and improve anti-CD-20 monoclonal antibody-mediated activation of complement-dependent cytotoxicity . Earlier reports demonstrated that cytokines (interleukin-4, granulocyte-macrophage colony-stimulating factor, or interferon-α) also increase CD-20 surface expression [46, 49]. Taken together, our data and those results indicate that the agents inhibiting downstream steps of cholesterol biosynthesis and other cholesterol-associated pathways could positively regulate CD-20 expression and its antibody-mediated signaling.
Our observations indicate for the first time that BIBB-515, YM-53601 and TAK-475 significantly inhibit their respective enzymes in the pathway of cholesterol biosynthesis to lower cellular cholesterol levels, and that targeting these enzymes could positively affect CD-20 distribution and its antibody-mediated activation in contrast to statins. Lowering cellular cholesterol levels with statins has been shown to induce apoptosis, [24–26] and have important antitumor activity in some malignancies [27–29]. Our experiments demonstrate that the cholesterol lowering agents BIBB-515, YM-53601 and TAK-475 render chemoimmuno-therapy resistant cells sensitive to chemoimmuno-therapy, and enhance cell chemoimmuno-sensitivity and apoptosis (Figures 5 and 6). These results validate the concept that cholesterol reduction using enzymatic inhibitors is different to the action of statins, probably due to different mechanism of activation of CD-20 dependent pathways.
We have also observed the effect of BIBB-515 and TAK-475 on chemoimmuno-sensitivity in the PBMCs from CLL patients. These cells from different patients show differences in response to three-day chemoimmuno-therapeutic drug treatment. The PBMCs from two patients (total 14 patients) weakly responded to fludarabine, and three patients are very weak in response to rituximab; however, all of these patient cells showed a 5-30% increase of chemoimmuno-sensitivity with cholesterol lowering agents. In these patients, two patients had undergone fludarabine treatment and one had undergone a round of chlorambucil and rituximab treatment. These PBMCs also show increased chemoimmuno-sensitivity with fludarabine, rituximab or their combinations in the presence of cholesterol lowering agents. Although cells treated with multiple drugs could cause pleiotropic effects, it is important that the combinations of BIBB-515, YM-53601 or TAK-475 and chemoimmuno-therapeutic drugs are synergistic effects. Our findings regarding the role of cholesterol on chemoimmuno-therapy along with recent reports in different types of cancer cells [18–29] highlight the relevance of cholesterol modulation in cancer therapy.
Decreased cell growth by lowering cellular cholesterol levels with statins in different cancer cells have been previously shown, we demonstrate that the mechanism of cholesterol modulation by other inhibitors is important because statins negatively affect CD-20 expression and antibody-mediated cell killing through CD-20. Our data demonstrate that BIBB-515, YM-52601 and TAK-475 acting at squalene synthase or oxidosqualene cyclase reduce cellular cholesterol levels, and inhibit cell growth leaving a CD-20 mediated pathway intact. Compensatory changes in membrane lipid composition as a result of treatment with these cholesterol synthesis inhibitors are an area we are investigating. Taken together, our results demonstrate that membrane lipid alteration in cancer cells is a novel and viable approach to both cancer therapeutics and reversal of chemoimmuno-therapy resistance.
Material and methods
All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA) unless specified otherwise. Ficoll-Paque Plus was obtained from Amersham Biosciences (Piscataway, NJ, USA). The media for cell culture were purchased from GIBCO (Grand Island, NY, USA). The anti-CD-20 APC antibody was obtained from eBiosciences (San Diego, CA, USA). CellTiter 96® Non-Radioactive Cell Proliferation Assay kit (MTT) was purchased from Promega (Madison, WI, USA). Fludarabine was obtained from Teva Parenteral Medicines, Inc (Irvine, CA, USA). Rituximab was obtained from Genentech (South San Francisco, CA, USA). BIBB-515 and YM-53601 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). TAK-475 was obtained from Chemzon Scientific (Montreal, Quebec, Canada).
Cell culture, PBMC isolation and cell treatment
MEC-2 cells were cultured in DMEM/F-12 media with 10% FBS. Cells were grown at 37°C in 5% CO2. Cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475) were added to the cultures at 10 μM final concentration in the presence or absence of fludarabine (10 μM), rituximab (10 μg/ml) or their combinations for 3 days. Treated cells were used for analysis according to experimental design.
Blood was obtained from CLL patients as defined by NCI96 criteria 28  following receipt of written informed consent under an IRB protocol approved by the University of Missouri-Columbia. PBMCs were isolated from whole blood immediately following donation using Ficoll density gradient centrifugation. Isolated cells were treated with test compounds as experimental designed and incubated at 37°C and 5% CO2 in RPMI-1640 media for 3 days. Cells were counted manually on a hemocytometer with Trypan blue solution before treatment, and at 1, 2, and 3 days after treatment to determine cell viability.
Total cholesterol analysis
Three million MEC-2 cells in the experiments were harvested, and 6 μl of standard cholesterol (1 mg/ml of chloroform) was added to the samples. Total cholesterol was extracted by the method of Bligh and Dyer . The resulting organic lower phase was withdrawn and evaporated under a stream of N2. One milliliter of methanolic potassium hydroxide was added to the tubes, and the samples were heated at 80°C for 1 hour. The tubes were sonicated several times during saponification. After cooling down, 2 ml of HPLC-grade water was added and the sterols were recovered by extracting the mixture 3 times with 3 ml hexane. The hexane extracts were pooled and dried down under N2. After complete dry, the samples were re-dissolved in 70 μl pyridine. The samples were derivatized by adding 30 μl N-trimethylsilyl-N-methyltrifluoroacetamide with trimethylchlorosilane and incubating at 50°C for 1 hour. The solvents were completely dried and re-dissolved in 75 μl of chloroform. Gas chromatography mass spectrometry (GC/MS) analysis was performed at the Kansas Lipidomics Research Center on an Agilent 6890 N GC coupled to an Agilent 5975 N quadrupole mass selective detector. The data were collected and processed with Agilent Chemstation.
MEC-2 cells were treated with various compounds for 3 days, washed in cold medium, and fixed with 4% paraformaldehyde in 100 mM phosphate buffer on ice. The fixed cells were washed twice with 1% BSA in PBS (BSA buffer). The cells were blocked for 15 minutes in 5% mouse serum in PBS and washed with BSA buffer. The cells were stained for 1 hour with anti-CD-20 APC antibody and cholera toxin-B FITC. Wet-mount slides were prepared, and the samples were viewed by a Zeiss 510 META laser scanning confocal microscope and photographed by LSM 4.2 software.
Cell viability assays
MEC-2 cells were plated in 96-well assay plates at a concentration of 10,000 cells per well in 100 μl of culture medium with test compounds according to experimental designed. After adding the agents, the cells were cultured at 37°C for 3 days, and cell viability was determined using Promega’s CellTiter 96® Non-Radioactive Cell Proliferation Assay kit (MTT) according to the manufacturer’s instructions. Absorbance at 570 nm was recorded using a Biotek Synergy H4 Hybrid Reader (Winooski, VT). MEC-2 cells and PBMCs were stained with Trypan blue solution, and dead and alive cells were counted after treatment to determine cell viability.
Measurement of DNA fragmentation
According to experimental designed, treatment of MEC-2 cells were harvested and centrifuged at 1,500 g for 2 min, and the pellets were resuspended in 0.5 ml of lysis buffer containing 5 mM Tris-HCl, pH 8.0, 20 mM EDTA, and 0.5% Triton X-100 and placed on ice for 15 min. The samples were then centrifuged at 12,000 g for 20 min, and the supernatant containing DNA cleavage products in the same amount of cellular proteins was precipitated overnight using isopropyl alcohol. The samples were centrifuged at 24,446 g for 20 min. Pellets were resuspended in Tris-EDTA buffer and digested with 0.2 mg/ml proteinase K and 1 mg/ml RNase A for 60 min at 48°C. DNA fragments were separated on a 1.5% agarose gel, visualized with ethidium bromide, and photographed using the Bio-Rad image system.
Statistical analysis was done using Sigma plot 12. The difference in the mean values among treatment groups to the controls were analyzed by one way analysis of variance.
Chronic lymphocytic leukemia
Peripheral blood mononuclear cell
We thank the Kansas Lipidomics Research Center for Lipid analysis, Dr. Alexander Jurkevic at the Molecular Cytology Research Core Facility at the University of Missouri-Columbia for the help of confocal microscopy, and clinical trials office Lynn walker for patient bloods.
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