Salinomycin Inhibits Cancer Stem Cells Of Nasopharyngeal Carcinoma Through Upregulating MiRNA-200c

BackgroundNasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in Southeast Asia and southern China, with an incidence of25-50per100,000populations. The pathological mechanisms responsible for the initiation and progression of NPC have been attributed to EBV infection, genetic predisposition and exposure to certain chemical carcinogens. Despite of significant improvements on the diagnosis and treatment modalities in the past decades, the5-year survival for stage IV NPC is only30%and poor survival is often associated with local, regional and systemic recurrences as well as remote metastasis.In recent years, accumulating evidence has suggested that tumors are driven by a subpopulation of undifferentiated cells termed cancer stem cells (CSCs) that possess an unique capacity to self-renewal and differentiate into the multiple lineages of the heterogeneous tumor mass. CSCs are resistant to chemotherapy and may contribute to tumor metastasis and tumor recurrence after treatment. Therefore, eradication of CSCs is critical for successful anti-cancer therapy.Salinomycin is a polyether anticoccidial drug produced by a Streptomyces albus strain. Recently, salinomycin was reported to possess potent anti-CSC activity. Compared to paclitaxel, a chemotherapeutic drug, salinomycin reduced the proportion of CSCs by>100-fold in breast cancer.In this study, we examined the effect of salinomycin on CSCs of NPC, compared to the commonly used chemotherapeutic drug cisplatin. We found that salinomycin significantly inhibited CSCs of NPC, as evidenced through side-population analysis, Aldefluor staining, and tumor sphere formation assay. Furthermore, we revealed that the salinomycin inhibits CSCs through regulating expression of miRNA-200c. Thus our results provide important insight into the mechanisms that salinomycin acts as an anti-CSC agent.Objective1. Characterize CSC properties of NPC tumor spheres;2. Evaluate anti-CSC effecacy of salinomycin in NPC;3. Explore the molecular mechanisms by which salinomycin inhibits CSCs.MethodsCells and culture conditions.All cell lines were preserved in our laboratory. The human NPC cell line was cultured in DMEM medium supplemented with10%fetal bovine serum and100U/ml penicillin/streptomycin. Cultures were maintained in a humidified incubator at37℃in5%CO2air atmosphere.Tumorsphere culture and differentiation.Single cells were plated in Ultra Low Attachment plates in serum-free DMEM-F12supplemented with10ng/mL bFGF,10ng/mL EGF and B27. In these conditions cells grew as suspension spherical clusters. To propagate spheres in vitro, primary cell spheres were to obtain single-cell suspension and replated to evaluate self-renewal by formation of secondary tumor spheres. To guide differentiation of spheres in vitro, spheroids were cultured in DMEM supplemented with10%fetal bovine serum without growth factors.Immunofluorescent staining.For immunofluorescent staining, adherent or semi-differentiated spheroid cells were grown on the surface of cover slides. Spheroids staining was performed in96-well microplates. The cells were fixed with4%paraformaldehyde. After rehydration in KB, cells were incubated with respective primary antibodies at37℃for45min. Slides or spheroids were then washed with KB for15min and secondary antibodies were incubated at37℃for45min. The nuclei were stained with DAPI. Sections were examined with a confocal microscopy.10×KB:0.1M Tris, pH7.5;1.5MNaCl;1.0%BSA.MTT assay.Cells obtained from adherent or spheroid cells were seeded in96-well microplates at a density of2,000cells per well. Cells were treated with increasing concentrations of drug as indicated. MTT assay was performed to determine the viability of the cells. The number of living cells is directly proportional to the absorbance at490nm.Hoechst staining and SP cells assay. Cells obtained from adherent or spheroid cells were suspended in DMEM/2%fetal bovine serum at1×106/ml cells and stained with Hoechst33342dyefor90min at37℃. Following this incubation, cells were washed with ice-cold PBS, stained with propidium iodide label and exclude dead cells. Cells were maintained at4℃for flow cytometry analyses and for sorting of SP fraction using a FACSAria Flow cytometer.Aldefluor assay by FACS. The ALDEFLUOR kit was used to analyze the population with a high ALDH enzymatic activity. Cells obtained from adherent or spheroid cells were suspended in ALDEFLUOR assay buffer containing ALDH substrate and incubated during40min at37℃. As negative control, for each sample of cells an aliquot was treated with50mmol/L diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor. FACS was performed using a FACSAria Flow cytometer.Western blot analysis. Cells were lysed and equal amount of protein were subjected to electrophoresis on a SDS-PAGE gel. The separated proteins were transferred to PVDF membranes and probed with appropriate primary antibodies. After extensive washing, the membranes were incubated with peroxidase-conjugated secondary antibodies and protein bands were detected by enhanced chemiluminescence reagents according to manufacturer’s instructions. Immunohistochemistry.For immunohistochemistry, paraffin-embedded sections were deparaffinized in xylene and rehydrated in graded alcohol. Antigen retrieval was done by boiling the slides in10mM sodium citrate buffer, pH6.0. Staining was done using EliVision Plus Kit according to the manufacturer’s protocol. DAB was used as a substrate for peroxidase.In vivo tumorigenicity assay. Animal studies were conducted in strict accordance with the principles and procedures approved by the Committee on the Ethics of Animal Experiments of Southern Medical University. Nude mice were fed autoclaved water and laboratory rodent chow. A volume of100μl of culture medium mixed with Matrigel containing cells was transplanted into the flanks of mice by subcutaneous injection. The mice were monitored daily and tumor volumes were measured every three day with a slide calliper rule. Tumor volume was calculated using V=1/2(width2×length). After several weeks, animals were humanely euthanized and tumors were harvested. To obtain a single-cell suspension, tumors were minced by a scalpel and incubated in RPMI-1640medium mixed with collagenase/hyaluronidase at37℃for60minutes. The tissues were further dissociated by pipette trituration and then passed through a40-μm nylon mesh to produce a single-cell suspension, which was used for further experiment.Result1. Cultivation and characterization of NPC tumorspheres1. By performing Immunofluorescent staining and western blot analysis, we found that the cancer stem cells markers Oct3/4, Bmi-1, Nanog, Sox2, EpCAM and mesenchamal marker vimentin were highly expressed, while the epithelial marker cytokeratin5/6was down-regulated in tumorsphere compared with differentiated, attached cells.2. A single PKH26-positive cell can form tumorsphere, suggesting that PKH26-positive cells represent cancer stem cells; Immunofluorescent staining showed that a few of cells in tumorspheres co-express PKH26and stem cell marker Sox2, CD133, and ABCG2. 3. The proportion of ALDH-postive cells in tumorsphere (18.1%,31.9%) was higher than that in differentiated cells (1.0%,1.6%). Similarily, the proportion of side populatin cells in tumorsphere (6.1%) was more than that in differentiated cells (2.3%).4. Tumorsphere cells had faster proliferation rate than differentiated cells. Tumorsphere cells exhibited increased IC50value (>5-fold) to cisplatin relative to differentiated cells.5. Tumorsphere cells had higher tumor initiating capability than differentiated cells, When1×104cells derived from tumor-spheres were injected into nude mice,2of3mice developed tumors. In contrast, innoculation of the same number of differentiated cells did not give rise to any tumors.2. Anti-CSC effect of salinomycin on NPC.1. Salinomycin inhibited tumor-sphere forming capability. Treatment with salinomycin not only reduced the number of tumor-spheres but also decreased the size of the spheres as compared to untreated cells or cells treated with cisplatin (P=0.000);2. Tumorsphere cells were more sensitive to salinomycin than to cisplatin, while differentiated cells exhibited similar sensitivity to salinomycin and cisplatin;3. The proportion of SP cells were decreased by salinomycin and increased by cisplatin. In a similar way, the proportion of ALDH-positive cells was decreased by salinomycin (2μM, P=0.005;5μM, P=0.003), but not by cisplatin treatment (P=0.421);4. The expression of c-myc, Oct3/4, Nanog was decreased by salinomycin treatment;5. Salinomycin treatment was more potent in inhibiting tumor growth than cisplatin was.(P=0.000). Compared with cisplatin, salinomycin is more effective in delaying tumor formation (P=0.011);6. Salinomycin reduced the proportion of SP (0.9%vs.2.1%) and ALDH-positive cells (0.9%vs.8.6%) in primary cells derived from xenografts when compared with cisplatin. The ability of tumorsphere formation of tumor cells derived from mice treated with salinomycin was decreased;7. Tumor tissue from mice treated with salinomycin underwent necrosis, expressed high level of E-cadherin and low levels of vimentin and Oct3/4.3. Molecular mechanisms of salinomycin selectively inhibiting CSCs1. Salinomycin treatment increased expression of E-cadherin and decreased expression of vimentin, SUZ12and Slug;2. C-666and6-10B cells have higher expression levels of ABCG2than CNE2cells does. Intriguely, C666and6-10B cells were more sensitive to salinomycin than CNE-2cells were. Salinomycin inhibited the expression of ABCG2and p-glycoprotein;3. Salinomycin can inhibit the expression of β-catenin, cyclinD1and P-AKT;4. Salinomycin induced apoptosis in a dose-dependent manner (P<0.05).4. Salinomycin inhibited CSCs by regulating miR-200c1. Salinomycin treatment resulted in dramatic increase of miR-200c compared with the control (>10fold);2. miR-200c-inhibitor increased SP proportion and tumorsphere forming capability. The effects of salinomycin treatment in suppressing SP and tumor-sphere forming capability were attenuated by simultaneous treatment with miR-200c-inhibitor;3. Cells treated with miR-200c-inhibitor exhibited decreased sensitivity to cisplatin, and increased sensitivity to salinomycin;4. Treatment with miR-200c-inhibitor increased the expression of the CSC marker SUZ12and Bmi-1. This effect of miR-200c-inhibitor could be counteracted by simultaneous treatment with salinomycin.Conclusion1. Nasopharyngeal carcinoma tumorsphere contains cells with cancer stem-like properties, with upregulated expression of stem cell markers and increased tumorigenicity.2. Salinomycin selectively inhibits CSCs of NPC. This effect is exerted by inhibiting EMT and Wnt/(3-catenin pathway, down-regulating the expression of drug resistance-associated protein.3. Salinomycin inhibits CSCs through up-regulating expression of miR-200c.