| Research Background:Nasopharyngeal carcinoma(NPC)is the most frequent head and neck tumor in South China,but is rare in Western Europe and North America.NPC is a highly malignant cancer which often invades adjacent regions and metastasizes to regional lymph nodes and distant organs.Many people agree with the tumor stem cell hypothesis that the tumor cells contain a small number of cells with the ability of unlimited self-renewal and producing heterogeneous tumor cells.These special cells resistant to radiotherapy and chemotherapy are the root cause of the tumor development.The chemical medicines mainly platinum for the current treatment of nasopharyngeal carcinoma,but the platinum chemicals exhibit high toxicity,so we urgently need to seek better chemical drugs for the treatment of nasopharyngeal carcinoma.In recent years,it has been reported that the anti-tumor active components are extracted from mulberry leaves.However,there are few reports about anti-tumor stem cells.In our previous study,we found SangA which was extracted from mulberry leaves by systematic separation has significant anti-tumor stem cell activity,but the main active components are remains unknown,and its mechanism of anti-tumor stem cells needs further studyObjective1.To isolate the active fractions from SangAand examined whether they can effectively eliminate NPC stem cell-like population by tumorsphere formation assay.2.To evaluate Whether the active fractions can selectively inhibit NPC stem cells.Methods1.To isolate the active fractions from SangA by Column Chromatography and Thin Layer Chromatography(TLC),and analysis the main components in each fraction by high performance liquid chromatography spectrometry.2.To evaluate the anti-tumorsphere formation activity of each fraction.3.To evaluate whether the active fractions can selectively inhibit NPC stem cellsby comparing the activity on tumorspheres and bulk tumor cells.Results1.20 fractions were separated from the mulberry leaf extract SangA and divided into four groups(S1?S2?S3?S4).2.Compared to the control,the active fraction S3 and S4 can significantly inhibit the formation of tumorspheres in NPC CNE2 cells in high concentration group.The disruptionof sphere number and diameter were observed.3.Chemical composition analysis indicated that S3 and S4 mainly contain flavonoids such as moracin and its isomers.4.Compared with the control group,the plate clone formation rate of adherent CNE2 and 5-8F cells treated with S3 and S4 decreased significantly.5.The scratch test of the adherent cells shows that compared with the control group,cell migration rate of adherent CNE2 and 5-8F cells treated with S3 and S4 was decreased.6.S3 and S4 can significantly inhibit the proliferation of nasopharyngeal carcinoma cells,but have little effect on normal nasopharyngeal epithelial cells(NP69).7.The inhibition activity of S3 or S4 on the adherent NPC cells and tumorspheres was assessed,the results indicated that the tumorspheres were more sensitive to S3 and S4 treatment.In a low dosage,the tumorspheres formation was significantly reduced while the viability of the adherent cells was not affected.8.After treated with S3 or S4,the number and diameter of the secondary generation tumorspheres decreased in NPC CNE2 and 5-8F cell lines.9.After treated with S3 or S4,the clone formation of the secondary generation tumorspheres decreased in NPC CNE2 and 5-8F cell lines.Conclusion1.S3 and S4 were the active fractions which were able to selectively inhibit NPC stem cells.The main chemical compounds were flavonoids such as moracin and its isomers.2.Both S3 and S4 inhibited the formation of tumorspheres and reduced clonogenicity significantly while had little effect on the viability of the bulk NPC cells,which indicated the selective effect of S3 and S4 towardsCSCs. |
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