Salinomycin Suppresses PDGFR�, MYC, and Notch Signaling in Human Medulloblastoma

Research Article

Austin J Pharmacol Ther. 2014; 2 (3). 1020

Salinomycin Suppresses PDGFRβ, MYC, and Notch Signaling in Human Medulloblastoma

Shuang Zhou1, Fengfei Wang1, Ying Zhang1, Max R Johnson2, Steven Qian1, Min Wu3, Erxi Wu1*

1Department of Pharmaceutical Sciences, North Dakota State University, USA

2Retina Consultants Ltd and University of North Dakota, USA

3Department of Basic Sciences, University of North Dakota, USA

*Corresponding author: : Erxi Wu, Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND, USA

Received: February 15, 2014; Accepted: April 20, 2014; Published: April 22, 2014


Medulloblastoma (MB) is the most common childhood brain tumor. Despite improved therapy and management, approximately 30% of patients die of the disease. To search for a more effective therapeutic strategy, the effects of salinomycin were tested on cell proliferation, cell death, and cell cycle progression in human MB cell lines. The results demonstrated that salinomycin inhibits cell proliferation, induces cell death , and disrupts cell cycle progression in MB cells. Salinomycin was also tested on the expression levels of key genes involved in proliferation and survival signaling and revealed that salinomycin down–regulates the expression of PDGFRβ, MYC, p21 and Bcl–2 and up–regulates the expression of cyclin A. In addition, the results reveal that salinomycin suppresses the expression of Hes1 and Hes5 in MB cells. Our datashed light on the potential of using salinomycin as a novel therapeutic agent for patients with MB.

Key words: Salinomycin; Medulloblastoma; PDGFRβ; MYC; Notch Signaling


PDGFRβ: Beta–type Platelet–Derived Growth Factor Receptor; Bcl–2: B–cell Lymphoma 2; DLL1: Delta–Like 1 (Drosophila); Dll3: Delta–Like 3 (Drosophila); Hes1: Hairy And Enhancer Of Split 1 (Drosophila); Hes5: Hairy And Enhancer of Split 5 (Drosophila); Hey1: Hairy⁄Enhancer–of–Split Related with Yrpw Motif 1; Hey2: Hairy⁄Enhancer–of–Split Related with Yrpw Motif 2; Dtx1: Deltex Homolog 1 (Drosophila); Dtx2: Deltex Homolog 2 (Drosophila); MAML1: Mastermind–Like 1 (Drosophila); MAML2: Mastermind– Like 2 (Drosophila); MAML3: Mastermind–Like 3 (Drosophila); RBPJ: Recombination Signal Binding Protein for Immunoglobulin Kappa J Region; MTS: 3–(4, 5–Dimethylthiazol–2–Yl)–5–(3– Carboxymethoxyphenyl)–2–(4–Sulfophenyl)–2H– Tetrazolium, Inner Salt; DMSO: Dimethyl Sulfoxide.


Medulloblastoma (MB), an embryonal neuroepithelial tumor of the cerebellum, is the most common malignant brain tumor in children [1]. This highly invasive tumor has a tendency to disseminate throughout the central nervous system early in its course. Although, the medical treatment outcome for children with MB has improved over the past several decades, approximately one–third of patients with MB tumors remain incurable. Moreover, current medical treatments have associated toxicities that can cause significant disabilities in long–term survivors [2].Thus, more effective drugs are needed for treating patients with MB.

Salinomycin is a 751 Da mono carboxylic polyether antibiotic which is widely used as an anti–coccidial drug. Recently, salinomycin has been found to reduce the proportion of breast cancer stem cells (CSC) by more than 100–fold compared to paclitaxel, a common drug used for breast cancer [3]. Cumulative findings strongly suggest that salinomycin is a selective killer of human CSC and an effective killer of multi–drug resistant human CSC–like cells [4–11]. The action mechanism of salinomycin in cancer and CSCs has been shown to modulate multiple signaling pathways including the Wnt, NF–κB, and p38 MAPK pathways [12–14].

MB cells have the potential to differentiate to neuronal and⁄or glial cells [15,16], indicating that MB cells are of stem cell origin. Growing evidence points toward the existence of a CSC–like population that may contribute to MB therapy resistance [17–19]. Notch signaling is critical for cell differentiation and proliferation and plays a fundamental role in MB initiation and progression by regulating downstream effectors, e.g., MYC [20–22]. The Notch pathway inhibitors, e.g., γ–secretase inhibitor MK–0752, suppress the cleavageof Notch, eliminate the stem cell like population [23–25], reduce cell proliferation and increase apoptosis [23–25], which implicates Notch signaling as a target that may constitute an additional promising treatment strategy for MB patients. In the present study, for the first time, we determined the anticancer effects of salinomycin in 3 MB cell lines. We also measured the effects of salinomycin on the expression of a few genes critical in cell proliferation, survival, and differentiation in MB cells.

Materials and methods

Cell Culture and Chemicals

MB cell lines (Daoy and D283) were obtained from ATCC and D425 cells were a gift from Dr. Darell D. Bigner [26]. Daoy and D283 MB cells were maintained in minimum Essential Medium (MEM) (Cellgro) supplemented with 4 mM L–glutamine, 100 units⁄ ml penicillin, 100 µg⁄ml streptomycin, 1% sodium pyruvate, 1% nonessential amino acids, and 10% fetal bovine serum (FBS) at 37°C with 5% CO2. D425 cells were maintained in improved MEM (Zinc Optioin 1 x) (GIBCO) supplemented with 10% FBS at 37°C with 5% CO2. Salinomycin and propidium iodide (PI) were obtained from Sigma.

Cell Proliferation Analysis

MB cells (Daoy 1x104⁄well, D283 5 x104⁄well, D425 1 x105⁄well) were placed in 96–well plates overnight. Salinomycin solution or identical volume of control (DMSO) was added to the appropriate wells. After 48 hours of treatment, a 20–µl of MTS solution (Promega) was added to each well. The cell number in each condition was determined by measuring the optical densities at 490 nm after 4–hour of incubation. The results were expressed as the percentages of control cultures.

Cell Death and Cell Cycle Analysis

MB cells (5 x105 ⁄well) were placed in 6–well plates overnight. Salinomycin or identical volume of control was added to the appropriate wells. After 24 hours of treatment, the cells were stained with PI and analyzed for cell death and cell cycle distribution using the Cell Lab Quanta TM SC system (Beckman Coulter) followed by Flow Cytometry Analysis (FACS).

Western Blotting Analysis

MB cells were harvested at the 24–hour time point for protein by adding Tris–triton cell lysis buffer (1% Triton, 50 mM Tris–HCl pH 7.4, 10% glycerol, 150 mM NaCl) supplemented with protease inhibitor cocktail (Roche Applied Science) and phosphates inhibitor cocktail 100x (cell signaling). The protein samples were separated using a 10%–12% SDS–PAGE gel, and then transferred onto a nitrocellulose membrane. Immunoblots were probed with antibodies specific for cyclin A, Bcl–2, p–21 (Santa Cruz), PDGFRβ (Epitomic), and MYC (Cell signaling). β–actin (Sigma) served as a loading control. Signals of the specific proteins were detected by using the Immun– Star HRP peroxide Luminol⁄Enhancer kit (BIO–RAD) and recorded on KODAK Biomax light film.

RT–PCR Analysis

RNA was isolated from MB cells using the RNeasy Plus (Qiagen) by following the manufacturer’s protocol. The quantity and purity of RNA were determined using a Nano Drop 1000 spectrophotometer (Thermo Scientific). 1 µg of total RNA was used to prepare cDNA using Super Script first–strand synthesis system (Invitrogen) by following the instructions provided by the manufacturer. Primers used in this study are listed in Table 1 and synthesized by Integrated DNA Technology. Semi–quantitative PCR was achieved by amplifying genes using an equal amount of cDNA and limited number of cycles. For detection of basal levels, 35 cycles were used for all genes except GAPDH. For detection the effects of salinomycin on gene expression, PCR conditions are listed in Table 1. PCR amplification was performed using GoTaq Hot Start Colorless Master Mix (Promega) and MJ Mini Personal Thermal Cycler (Bio–Rad).The results were visualized by analyzing the samples using DNA gel.