The Study Of Extras WADM-3 From Asparagus Densiflourus Targeting Glioma Stem Cells

Objective: Cancer Stem Cells(CSCs) are the main reason of tumorigenesis, tumor drug resistance and tumor metastasis. CSCs are defined as the target cells for cancer therapy. Glioblastoma Multiforme(GBM) which comprises high death rate and low survival rate is recognized as high malignant grade brain tumor. Drugs extract from plants play important roles in the study of tumor therapy. Asparagus densiflorus, a family member of vines, grows in tropical and temperate regions. This plant is widely distributed in our country. Via screening activities from plants, asparagus densiflorus extras WADM-3 specific target GSCs has been found by our research team. This present study was performed to analysis the function and the mechanism of WADM-3 targeting Glioma Stem Cells(GSCs). We evaluate WADM-3 specific target GSCs and research the possible machanism on cell level. Animal model displays a great degree of phenotype and function, we also successfully establish orthotopic transplantation of GBM mouse model. Therefore, the study aims to offer the theoretical basis for finding active pharmaceutical components from plants which specific target GSCs.Methods: 1. Drug Screening The activities drug to NSCs、GBM、T98G cell、GBM U-87 MG cell. Observe cell status and record cell toxic reaction. Implant 200 ul cell suspension into 96-well plates that coated with laminin. Add 10ug/ul WADM-3 into plates after cultured for 24 hours. Culture GSCs with WADM-3 and GSCs and observe cell status in 24 h, 48 h and 72 h. 2 Extra from Asparagus densiflorus targets GSCs 2.1 Act Asparagus densiflorus extras on GSCs and NSCs to observe the cellular culture and morphology. The cytotoxicity experiment performed on GSCs and the normal Neural stem cells(NSCs) at the same time. Thus, the specific antineoplastic activity can be confirmed. Adherent culture GSCs and normal NSCs in 24-well plates(20000cells per well). Drug the 10ug/ml of extras to GSCs and NSCs after 24 hours. After 24 h, 48 h, 72 h, two cell lines growth inhibiting were detected by observing cell morphology. Evaluating the ability of asparagus densiflorus extras lyse GSCs and NSCs to clarifed the specific targeting. 2.2 The cytotoxicity experiment of asparagus densiflorus extras to GSCs and IC50 analytic method. Digest cells and implant cells in 96-well plates(20000 cells per well), Drug Asparagus densiflorus extras to cells after 24 hours. The final concentration is 20ug/ml, make the 2 times gradient dilution, mix with the culture medium and then add the mixture to the culture plates. After 72 hours, dispute the culture medium, add 100 ul MTS which is diluted 10 times with culture medium, put the plates in the incubator for 1 hours. Measure the absorbance values in 490 nm by Thermo Scientific Microplate Reader and calculate IC50. 2.3 Asparagus densiflorus extras target GSCs and Colony efficiency(CE) assay GSCs were mixed with soft agar in 6-well plates and culture in the incubator, change the culture medium at 5 days interval, after colonies formed, add 10ug/ml, asparagus densiflorus into cell culture plates and the same concentration of Dimethyl Sulfoxide(DMSO) was drugged into the control plates. After 10 days, fix cells into paraformaldehyde with 0.05% crystal violet, Calculate the number of the colonies. Observe the colonies and evaluate CE which indicates the inhibition and the ability of specific targeting GSCs. 3. Research the mechanisms of asparagus densiflorus extras directed toward selectively targeting GSCs 3.1 Assay the affection of asparagus densiflorus extras on GSCs proliferation Implant GSCs into 24-well plates with cell density 6×104 after cell digestion. After 48 hours, add 5ug/ml extras into culture plates. GSCs behave cytotoxic phenotype after co-culture 24 hours. The Brd U antibody staining labels the GSCs cellular proliferation, Trace the proliferous cells by immunofluorescence technique. Stain normal cells by DAPI, Observe cell proliferation after double staining. Via evaluating cell proliferation, we can further understand if the extras target GSCs by inhibiting cell growth, and find out the machanism 3.2 Assay the affection of asparagus densiflorus extras on GSCs cell apoptosis Implant GSCs into 24-well plates with cell density 6×104 after cell digestion. After 48 hours, add 5ug/ml extras into culture plates. GSCs behave cytotoxic phenotype after co-culture 24 hours. Use Caspase-3 antibody to stain GSCs cells, Trace the apoptotic cells by immunofluorescence technique. Stain normal cells by DAPI. Observe cell apoptosis after double staining. Via evaluating cell apoptosis, we can find out whether the extras specific target GSCs by inducing cell apoptosish and research the machanism. 3.3 Asparagus densiflorus extras effect on GSCs cell cycle Add asparagus densiflorus extras into the GSCs culture plates and co-culture for 48 hours, After PI staining. Analyze cell cycle by Flow Cytometry(FCM) technology, describe the extras affection of GSCs cell cycle and its mechanisms. 4. Establish orthotopic transplantation of GBM mouse model 4.1 Adherent culture GSCs in vitro The GSCs cell line that we use is established from the clinical tumor by the researchers in our lab. GSCs which adherent cultured in vitro offer the fundationmental basis on the overall and cell level research. 4.2 Develope orthotopic transplantation tumor of GBM mouse model Combined immunodeficiency(NOD/SCID) mice were used to make the GBM model. Anesthetize mice expose the cranium. According to the cross sharp on the cranium, line up to 1mm, left to 2mm and depth to 3mm is striatum. With the speed of 0.2ul/2min inject total 2ul, 30000 GSCs cells into brain striatum on the left by using the stereotaxic instrument for brain surgery. After injection, suture the mice and keep observing. 4.3 Pathological examination on orthotopic transplantation tumor of GBM mouse model The brain tissue of orthotopic transplantation tumor of GBM mouse model was analyzed by hematoxylin-eosin staining(HE) and histochemical immune staining. Paraffin blocks stained with HE were observed with the pathology of hemorrhages, necrosis and infiltrative growth of tumor. Ki67 is the antigen that marks cell proliferation, Ki67 antibody was used to stain the brain tissue by histochemical immune staining, DAPI was used to stain the normal cells at the same time, observe the tumor cells proliferation via the result from double staining.Result: 1. Drug Screening There are 10 components have false positive to GSCs from 52 extraxs. The results 42 components are toxic to GSCs. There are 3 components has no toxic effect to NSCs out 42 activity components. There are 3 components has toxic effect on GBM T98 G cell and GBM U-87 MG cell. 2 Evaluate the ability of asparagus densiflorus extras specific targeting on GSCs 2.1 Compare with normal NSCs control, the extras has significant cytotoxicity to GSCs, normal NSCs were barely affected with the same concentration. 2.2 IC50 values the concentration of extras is 2.2-2.8ug/ml, far less than the normal NSCs IC50(>40ug/ml). The date indicates the extras selective target on GSCs through cytotoxic effection. 2.3 Asparagus densiflorus extras damage GSCs colony efficiency. Colony formation in vitro reveal asparagus densiflorus extras damage GSCs colony efficiency, the extras not only blockade GSCs colony growing but also damage formed colonies, and eventually lyse GSCs colony. Comparing with the control which was added DMSO, the colony has not been damaged. 3. Study on the mechanisms of asparagus densiflorus extras selective targeting GSCs 3.1 Assay the efficacy of asparagus densiflorus extras on GSCs proliferation by Brd U antibody staining, the result shows GSCs proliferation was barely affected by asparagus densiflorus extras(P=0.32). GSCs growth fraction(GF) is 28.5%, the control GF is 27.1%. 3.2 GSCs apoptosis was assessed by Caspase-3 pathway, the result supports the active extras significant induce GSCs apoptosis(p=0.0002). The apoptosis ratio is 20%, for control, the apoptosis ratio is 5.1%. The result reveals that asparagus densiflorus extras specific target GSCs was relate to inducing GSCs apoptosis effect but not inhibiting cell growth. 3.3 Analysis the GSCs cell cycle after asparagus densiflorus extras affect cells for 72 hours,we found that asparagus densiflorus extras is invalid to cell cycle(P=0.48). 4. Develop and evaluate the GBM mouse model Four weeks after inject GSCs into brain, The GBM mouse model signs obviously tumor. The pathological section demonstrated the tumor qualified the characteristic of GBM and the model can be used to study on the activity of asparagus densiflorus extras.Conclusion: 1. There are 3 components target GSCs after drug screening, WADM-3 is monomer. 2. The asparagus densiflorus extras target GSCs. 3. Asparagus densiflorus extras significant induce GSCs apopsis but has no inhibition GSCs proliferation effection. 4. The pathological feature of orthotopic transplantation tumor of GBM mouse model is similar with clinic. This GBM mouse model can be used on GBM research.