Research On The Anti-NPC Effect Of SangA,an Extract From Mulberry Leaves

BackgroundNasopharyngeal carcinoma (nasopharyngeal, carcinoma, NPC) as a nasopharyngeal malignant epithelial tumor, is one of the most common malignant tumors in Southeast Asia and southern China, patients usually have no obvious symptoms because of concealed position, so the proportion of early clinical diagnosis and treatment is relatively low, About 70% patients were in terminal as they are diagnosed, who have been lost the best treatment period, the standard treatment is radiotherapy combined with chemotherapy in clinical, chemotherapy is generally taken in combination with platinum-based drug, five year survival rate is between 50%-60%. poor survival is often associated with local, regional and systemic recurrences as well as remote metastasis. Therefore, there is an urgent need to discover novel and effective approaches for the prevention and/or treatment of pancreatic cancer. Therefore, there is an urgent need to discover novel and effective approaches for the treatment of nasopharyngeal cancer.The concept of cancer stem cell (CSC) was introduced manyyears ago to explain tumor cell heterogeneity, and recent studies suggest that CSC may be responsible for tumorigenesis andcontribute to some individuals’ resistance to cancer therapy. As present, Nasopharyngeal cancer stem cell research is in the leading edge of nasopharyngeal cancer research field, more and more experiment shown cancer stem cells, is the basis of the development、occurrence and metastasis of nasopharyngeal carcinoma in nasopharyngeal carcinoma cells and nasopharyngeal carcinoma tissue.Because the proportion of tumor stem cells in tumor cells is very few, and in vitro, tumor stem cells do not amplify, but rapidly proliferate.So the way to screen anti-tumor stem cell drug by large-scale culture of tumor stem cell is not realize. The critical point of screening anti-tumor stem cell drug is able to get stable cell subpopulation which rich in tumor stem cell in vitro.Muberry leaf is Moraceae Morus mulberry(Morus alba L.) leaves,cold,sweet,with the evacuation of wind-heat, clearing lung-heat and moistening dryness removing liver-fire for improving eyesight, is usually used in traditional Chinese medicine, and widely planted in all parts of our country. Modern pharmacological studies have shown, mulberry leaf not only has hypoglycemic. Scavenging free radicals, antibacterial, antiviral and other pharmacological effects, but also has antitumor activity.Mainly reported that flavonoids, alkaloids and other several compound monomer, has an inhibitory effect on the proliferation of tumor cells, the evaluation of mulberru in animal experiment in very few. The screening of the active ingredients of Chinese medicine targeted tumor stem cell has not been reported.In this study, we examined the effect of mulberry leaf extract A on CSCs of NPC, compared to the commonly used chemotherapeutic drug cisplatin. We found that mulberry leaf extract A significantly inhibited CSCs of NPC, as evidenced through side-population analysis, and tumor sphere formation assay. Thus our results provide important conclusion that mulberry leaf extract A acts as an anti-CSC agent.Objective1. Evaluate the effect of SangA on inhibition of proliferation invasion and metastasis in NPC2. Evaluate anti-CSC effecacy of SangA in NPC in vivo and in vitro Methods Cells and culture conditions.All cell lines were preserved in our laboratory.The human NPC cell line was cultured in RPMI-1640 medium supplemented with 10% bovine serum and 100 U/ml penicillin/streptomycin. Cultures were maintained in a humidified incubator at 37℃ in 5%　CO2 air atmosphere. Tumorsphere culture.Single cells were plated in Ultra Lo w Attachment plates in serum-free DMEM-F12 supplemented with 20 ng/mL bFGF,20 ng/mL EGF Penicillin-streptomycin (1:100) and B27(1:50). In these conditions cells grew as suspension spherical clusters, the appearance of primary spheres was evaluated under inverted microscope after 5-7days. MTT assay.Cells obtained from adherent cells were seeded in 96-well microplates at a density of 10,000 cells per well. Cells were treated with decreasing 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 at 490 nm. Hoechst 33342 staining and SP cells assay.Cells obtained from adherent cells were suspended in RPMI-1640 at 5×105/ml cells and stained with Hoechst 33342 dye for 90min at 37℃. Following this incubation, cells were washed with ice-cold PBS, stained with propidium iodide label and exclude dead cells. Cells were maintained at 4℃ for flow cytometry analyses and for sorting of SP fraction using a FACS 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 probe d 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.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 of 100 ul of culture mediummixed 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 two day with a slide calliper rule. Tumor volume was calculatedusing 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-1640 medium mixed with collagenase/hyaluronidase at 37℃ for 60 minutes. The tissues were further dissociated by pipette trituration and then passed through a 40-n m nylon mesh to produce a single-cell suspension, which was used for further experiment.Result1. Anti-proliferation, invasion and metastasis of SangA on NPC(1).The inhibition of a serial concentration of SangA on CNE2 and 5-8F cells were lower than DMSO group(all P<0.05) by MTT detection;(2).In vitro, the proliferation of CNE2 and 5-8F cells by SangA lower than by DMSO(P=0.000);(3).Compared to DMSO group, migration in vitro experiment results show that the cell migration ability of CNE2 and 5-8F cells by SangA was lower (P=0.000)(4). Compared to DMSO group, invasion in vitro experiment results show that the cell invasion ability of CNE2 and 5-8F cells by SangA was lower (P=0.000)2. Anti-CSC effect of SangA on NPC(1). SangA inhibited tumor-sphere forming capability. Treatment with SangA not only reduced the number of tumor-spheres but also decreased the size of the spheres as compared to cells treated with DMSO (P=0.000);(2). SangA and cisplatin reduced the proportion of SP;(3). SangA and DDP inhibited the expression of β-catenin、Bmi-1、c-myc、 ABCG2;(4). In vivo, SangA in certain concentration inhibited the growth of tumor in vivo compared with blank group(P=0.036,0.041), and no significant effect on the body weight of nude mice (P=0.998,0.956,1.000), but DDP reduced the body weight of nude mice(P=0.017).(5).Median suvival in the nude mice with NPC was 57days, with the SangA or DDP,median suvival period was increased significantly.Conclusion1.SangA inhibits the proliferation, invasion and metastasis of NPC2. SangA selectively inhibits CSCs of NPC.