Identification Of Anti-NPC Activity From Herbal Extracts Of31Plants Grown In Guangdong And Investigation Of The Mechanisms Of Action Of Nerium Oleander Extracts As An Anti-NPC Agent

Background and ObjectiveNasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in Southeast Asia and southern China, and the5years survival rate for stageⅣ NPC is only45%-50%after radiotherapy and chemotherapy, despite of significant improvements on the diagnosis and treatment modalities in the past decades. Radioresistance chemoresistance and severe seriously complications lower the effect of combining treatment for NPC. The search for more effective and less toxic anti-NPC drugs is an urgent need to address the problem. Medicinal plants from China are widely used in tranditional Chinese medicine to treat a range of conditions including cancers,and may thus be useful sources of novel anti-tumor compounds. In this study, we screened the effects of31species of herbal plants grown in Guangdong province for their anti-tumor activity and have identified extracts of3plants with potent antitumor activity. We further examined the anti-NPC and anti-cancer stem-like cell activity of Nerium oleander that exhibited the most potent effect among the31plants as well as the underlying molecular mechanisms of action. Material and 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. Cultures were maintained in a humidified incubator at37°C in5%CO2air atmosphere.MTT assay.Cells were seeded in96-well microplates at a density of2,000cells per well, and were treated with increasing concentrations of drugs 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.Apoptosis assay.CNE2cells were treated with different concentrations of drugs for18-48hours, and then were digested into single cells.Then Annexin V-FITC Kit was used according to the manufacture procedure.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.Hoechst staining and SP cells assay.Cells were suspended in DMEM/2%fetal bovine serum at1x106/ml cells and stained with Hoechst33342dye for90min at37°C. Following this incubation, cells were washed with ice-cold PBS, stained with propidium iodide label and exclude dead cells. Cells were maintained at4°C for flow cytometry analyses and for sorting of SP fraction using a FACSAria Flow cytometer.Tumorsphere culture.CNE2cells were plated in Ultra Low Attachment plates in serum-free DMEM-F12supplemented with different concentration of drugs. In these conditions cells grew as suspension spherical clusters.2weeks after treatment, the number of tumor sphere was counted and diameter of tumor sphere was measured in microscope. In the second passage, cells from primary tumorsphere were digested and cultured in a density of800-1000cells/ml without drugs.Result1、Daphinpyllum calycinum, Vitex negundo and Nerium oleander extracts have potent anti-NPC activity.2、Nerium oleander extract inhibited the growth and proliferation of CNE2cell,as well as induced apoptosis of CNE2cells in a dose dependent manner. Western blot assay revealed that, in CNE2cell, the Nerium oleander extract treatment decreased the amount of phosphorylated PI3K and AKT significantly, however, had no significant influence on the level of expression of total PI3K and AKT. At the same time,the expression of Bcl-2was decreased significantly by drug treatment, while the expression of Bad and cleaved caspase-3were increase, with no significant influence on the level of expression of Bcl-XL.These datas showed that Nerium oleander extract suppress CNE2cell growth and induce its apotosis probably through PI3K/Akt signal transduciong pathway,which triggers apoptosis of NPC cell largely through the intrinsic pathway.3、Nerium oleander extract inhibited the primary tumor sphere and the second tumor sphere dose-dependently (p<0.05), as well as the SP cells(p<0.05).Western blot assay revealed that, in CNE2cell, the Nerium oleander extract treatment decreased the amount of phosphorylated β-catenin significantly, however, had no significant influence on the level of expression of total β-catenin. At the same time,the expression of c-myc was decreased significantly by drug treatment, with no significant influence on the level of expression of Nanog and Oct4. These datas showed that Nerium oleander extract suppress the self-renewal of cancer stem cells in CNE2cell lines probably through inhibiting the expression of p-β-catenin and c-myc.Conclusion1、Daphinpyllum calycinum, Vitex negundo and Nerium oleander extracts have potent anti-NPC activity.2、Nerium oleander extract inhibited the growth and proliferation of NPC. This effect is probably exerted by inhibiting PI3K/Akt pathway.3、Nerium oleander extract suppressed the self-renewal of cancer stem cells in NPC by inhibiting the expression of p-β-catenin and c-myc.