WO2017181799A1 - 藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途 - Google Patents

藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途 Download PDF

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WO2017181799A1
WO2017181799A1 PCT/CN2017/077350 CN2017077350W WO2017181799A1 WO 2017181799 A1 WO2017181799 A1 WO 2017181799A1 CN 2017077350 W CN2017077350 W CN 2017077350W WO 2017181799 A1 WO2017181799 A1 WO 2017181799A1
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glioma
cells
gambogic acid
acid amide
medicament
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French (fr)
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彭小忠
胡艳
韩为
强伯勤
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中国医学科学院基础医学研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 

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  • the invention relates to the field of tumor treatment, in particular to the use of gambogic acid amide for the preparation of a medicament for treating human glioma.
  • Glioma is the most common primary intracranial malignant tumor derived from the neuroepithelial. It has high malignancy, rapid growth, strong invasiveness and high morbidity. Although the current comprehensive treatment technology for cancer has made great progress, it is located in an important position in the brain, accompanied by microvascular proliferation, showing obvious invasive growth characteristics. Traditional surgery and radiotherapy cannot be completely cured, resulting in high recurrence rate. High mortality and low cure rate.
  • the drugs for clinical chemotherapy are mainly alkylated drugs, but the traditional alkylating agents have large side effects and are easy to produce drug resistance; the new alkylating agent temozolomide (TMZ) has small side effects and long-term drug tolerance.
  • TMZ new alkylating agent temozolomide
  • MDS myelodysplastic syndrome
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • Inhibitors such as EGFR gefitinib and erlotinib have been shown to be highly susceptible to drug resistance after administration, and patients with efficacy are very rare, and progression-free survival has not been extended after taking the patient.
  • EGFR/Her2 inhibitor lapatinib, EGFR monoclonal antibody cetuximab has been confirmed to have a relatively small effect on glioma; in addition to mTOR related inhibitors: rapamycin, for Sirolimus, everolimus, dexamethasone, deoxyacetylase (Histone deactylase, HDAC) inhibitors: valerinostat alone or in combination with temozolomide have been shown to have no or minimal effect .
  • the study also developed a number of anti-angiogenic molecules such as bevacizumab and cediranib that have been shown to have partial effects in response to the rich microvascular formation of gliomas, which can rapidly reduce para-carcinoma and effectively It prolongs the patient's progression-free survival, but it does not reverse the progression of the disease.
  • Patients who received anti-vascular therapy eventually failed to cure successfully, nor could they change the status of death in most patients months after diagnosis.
  • no new and effective drug molecules have been found.
  • glioma stem cells are the root cause of glioma development, and glioma stem cells can induce tumor angiogenesis by activating Wnt signaling pathway, enhancing tumor hypoxia stress and invasion ability.
  • Wnt signaling pathway enhancing tumor hypoxia stress and invasion ability.
  • Bao et al. confirmed that glioma stem cells can also produce chemoradiation resistance.
  • the chemotherapeutic drugs of glioma only target the whole tumor tissue, and the patient has a certain effect after shrinking the tumor tissue after receiving the treatment for a period of time, but the glioma stem cells resistant to chemotherapy can be further formed by proliferation and differentiation. New tumor tissue that promotes tumor recurrence.
  • Keqiang Ye Lab discovered a chemically stable neurotrophic factor (NGF), an analog of Nerve growth factor (NGF), which was found to be intracellularly proximal to the NGF-specific receptor TrkA.
  • NGF nerve growth factor
  • the membrane regions bind to each other to dimerize TrkA and autophosphorylate, thereby activating TrkA and its downstream Akt and MAPKs signaling pathways.
  • gambogic acid can significantly inhibit neuronal cell cell death at the level of in vitro and in vivo through the above pathway, and has an effect of promoting neurogenesis of PC12 cells. It suggests that it may play a role in the treatment of neurodegenerative diseases and stroke.
  • gambogic acid also specifically promotes transcription and translation of TrkA.
  • NGF can rapidly activate TrkA and its downstream signaling pathways, but after 60 minutes, due to the significant degradation of TrkA, the phosphorylation level of TrkA and its downstream related proteins decreases, while gambogic acid can promote the transcription and translation of TrkA. Activation of TrkA and its downstream signals can be maintained for a longer period of time (>72 hours).
  • Keqiang Ye found that gambogic acid and NGF could inhibit the proliferation of K562 cells by inhibiting the binding of AcinS and CtBP2 and inhibiting the transcription of cyclin A1, and significantly inhibiting the tumor growth of K562 cells in nude mice.
  • the amide has a certain antitumor effect.
  • gambogic acid amide has a specific inhibitory effect on the four glioma stem cell lines isolated from the laboratory at the cellular level relative to the glioma cell line and the human normal astrocyte cell line.
  • the present invention verifies at the cellular level that gambogic acid amide can specifically inhibit the self-renewal ability and tumor ball formation ability of glioma stem cells; and gambogic acid amide is verified by zebrafish and nude mouse glioma transplantation model. Therapeutic effect on glioma.
  • gambogic acid amide is likely to provide a new potent therapeutic drug for the treatment of glioma.
  • the present invention provides a use of gambogic acid amide for the preparation of a medicament for treating human glioma.
  • the medicament treats the human glioma by inhibiting the cellular activity of glioma stem cells.
  • the medicament treats the human glioma by inhibiting self-renewal of glioma stem cells, tumor ball formation, and tumor formation in vivo.
  • the drug treats the human glioma by inducing apoptosis of glioma stem cells.
  • the inhibitory effect of the drug on glioma stem cells can be partially restored by the TrkA inhibitor.
  • the glioma stem cells include, but are not limited to, U87MG SLC, U251 SLC, GSC2, GSC5.
  • the medicament further treats the human glioma by inhibiting glioma cells.
  • the glioma cells include, but are not limited to, U251, U87MG, N3.
  • the human glioma is in grades I to IV.
  • Glioma stem cells exist in a small number of cells with self-renewal ability, multiple differentiation ability, and tumor formation ability in glioma tissues. It develops, relapses and colloids with glioma. Tumors are closely related to the formation of resistance to chemotherapy and chemotherapy.
  • MTS Method A detection reagent for detecting the number of viable cells in cell proliferation and cytotoxicity experiments by colorimetry.
  • MTS is a novel tetrazole compound and PMS is an electronic coupling agent. PMS has enhanced chemical stability which allows it to be mixed with MTS to form a stable solution.
  • MTS is biologically reduced to a colored formazan product that is directly soluble in the medium. This transformation is most likely accomplished by the action of NADPH or NADH produced by dehydrogenase in metabolically active cells. The amount of formazan product detected at 490 nm is directly proportional to the number of viable cells in the culture.
  • Self-renewal refers to the process by which stem cells (here specifically referred to as glioma stem cells) produce at least one stem cell-deficient daughter cell through symmetric or asymmetric division. Self-renewal can maintain the potential of stem cells to differentiate, for tissue-specific stem cells. In other words, self-renewal is the basis for maintaining the potential for differentiation throughout life.
  • Tumor sphere forming ability refers to the ability of tumor stem cells (here specifically referred to as glioma stem cells) to be cultured in a medium containing EGF and bFGF in vitro to form a tumor sphere.
  • the present invention finds that gambogic acid amide can be used for the preparation of a medicament for treating human glioma, thereby providing a new powerful drug for the treatment of human glioma, with significant social and economic benefits.
  • FIG. 1 Gambogic acid amide specifically inhibits the cellular activity of glioma stem cells.
  • MTS method was used to detect four strains of glioma stem cell lines (U87MG SLC, U251SLC, GSC2, GSC5) at different concentrations (0.03 ⁇ M, 0.1 ⁇ M, 0.3 ⁇ M, 1 ⁇ M, 3 ⁇ M, 10 ⁇ M, 30 ⁇ M).
  • FIG. 2A to 2D Gambogic acid amide specifically inhibits glioma stem cells.
  • the green fluorescently labeled GSC2-GFP was mixed with the non-fluorescently labeled HA 1:1 in the system, and the hypoxanthine was applied at a concentration of 0.1 ⁇ M, 0.3 ⁇ M, 1 ⁇ M, and 3 ⁇ M for 4 hours (Fig. 2A). 12 hours (Fig. 2B), 24 hours (Fig. 2C), 48 hours (Fig. 2D), and the flow cytometry analysis of the ratio of GSC2-GFP, U87MG SLC, HA in different treatment groups, combined with co-culture models in different Cell viability at different concentrations and time, statistical analysis of relative cell viability of each cell component in the co-culture model.
  • 3A to 3C Gambogic acid amide specifically inhibits glioma stem cells.
  • the green fluorescently labeled U87MG SLC-GFP was mixed with the non-fluorescently labeled HA 1:1 in the system, and the hyalinamide was applied at 0.1 ⁇ M, 0.3 ⁇ M, 1 ⁇ M, and 3 ⁇ M for 12 hours (Fig. 3A). ), 24 hours (Fig. 3B), 48 hours (Fig. 3C), and the flow cytometry analysis of the ratio of GSC2-GFP, U87MG SLC, HA in different treatment groups, combined with co-culture model at different concentrations, different time Cell viability, statistical analysis of the relative cell viability of each cell component in the co-culture model.
  • FIG. 6 Gambogic acid amide inhibits glioma stem cell self-renewal ability.
  • GSC5 cells were diluted to 25, 50, 100, 200/100 uL, gambogic acid amide was treated at a concentration of 0.1 ⁇ M for 2 weeks, and then the percentage of tumor-free spherical pores was counted, and the ordinate indicates the logarithm of the percentage of tumor-free spherical pores.
  • FIG. 7 Gambogic acid amide affects glioma stem cells at low concentrations (0.1 ⁇ M, 0.3 ⁇ M) for 4 hours, and has little effect on cell viability, only at high concentrations (1 ⁇ M, 3 ⁇ M) for glioma stem cells. Has a certain impact.
  • the GSC2 cells were plated in a pre-coated 96-well cell culture plate at 10,000 cells/well, and the drug was added overnight, so that the concentration of gambogic acid amide was 0.1 ⁇ M, 0.3 ⁇ M, 1 ⁇ M, 3 ⁇ M, and the MTS after 4 hours of drug action. Relative cell viability under each treatment condition was tested.
  • Fig. 8A to Fig. 8C-5 Gambogic acid amide against glioma stem cells at low concentration (0.1 ⁇ M, 0.3 ⁇ M) for 4 hours had little effect on cell viability, only at high concentration (1 ⁇ M, 3 ⁇ M) for colloidal The cell viability of tumor stem cells has an effect, but it can significantly inhibit the ability of glioma stem cells to form secondary tumor spheres.
  • Gambogic acid was used to treat GSC2 cells at concentrations of 0.1 ⁇ M, 0.3 ⁇ M, 1 ⁇ M, and 3 ⁇ M for 4 hours. The drug was removed, washed twice with PBS, and counted by trypan blue staining. Cells were re-plated in 96-well cells at each concentration.
  • Fig. 8A and Fig. 8B The formation of tumor spheres in each treatment group was counted under light microscope (Fig. 8A and Fig. 8B), and the results of observation under light microscope were shown in Fig. 8C-1 to Fig. 8C-5, respectively, which were the control group (Fig. 8C-1) and Garcinia Cambogia.
  • the amide concentration was 0.1 ⁇ M (Fig. 8C-2), 0.3 ⁇ M (Fig. 8C-3), 1 ⁇ M (Fig. 8C-4), and 3 ⁇ M (Fig. 8C-5).
  • Figure 9 The effect of gambogic acid amide on glioma stem cells is somewhat irreversible. After 4 hours of gambogic acid treatment of GSC2 cells, the drug was withdrawn, the cells were plated again, 5000 cells/well, and naturally grown for 4 days, MTS was used to measure the cell survival rate of each treatment group.
  • FIG. 10 Gambogic acid amide can significantly inhibit the ability of glioma stem cells to self-renew.
  • Gambogic acid amide was treated with 0.1 ⁇ M, 0.3 ⁇ M, 1 ⁇ M, and 3 ⁇ M for 4 hours, and then the drug was removed. After trypan blue staining, the cells were diluted to 25, 50, 100, 200/100 uL, and naturally grown for 8 days. Then, the percentage of tumor-free spherical pores was counted, and the ordinate indicates the logarithm of the percentage of tumor-free spherical pores.
  • Fig. 11A and Fig. 11B After cell digestion, trypan blue After staining, the cell count (the cell viability was found to be very low at 1 ⁇ M and 3 ⁇ M, so the subsequent experiments were not continued), and the cells were re-plated in 96-well cell culture plates at 5000 cells/well at each concentration. After 4 days of natural growth.
  • the formation of tumor spheres in each treatment group was counted under light microscope (Fig. 11A and Fig. 11B), and the results of observation under light microscope were shown in Fig. 11C-1 to Fig. 11C-3, wherein Fig. 11C-1 is the control group, and Fig. 11C-2 is the control group.
  • Fig. 11C-3 is gambogic acid amide 0.3 ⁇ M.
  • Figure 12A and Figure 12B The effect of gambogic acid on glioma stem cells is somewhat irreversible.
  • Drug treatment GSC2 24 hours, 48 hours after the withdrawal of the drug, replaced with fresh medium to continue to culture 48 After the hour, the cell viability of each treatment group was measured by the MTS method, and the cell activity changes of the cells treated with the drug treatment group were compared.
  • “1 day” and “2 days” means MTS detection after 24 hours or 48 hours of drug treatment; "drug continued action” means withdrawal of the drug after 24 hours and 48 hours of drug treatment, and continued to add fresh medium culture with the corresponding drug concentration 48 After the hour, the MTS test; “withdrawal of the drug” means that the drug was withdrawn after 24 hours and 48 hours of drug treatment, and the fresh medium containing no drug was added to continue the culture for 48 hours after the MTS test.
  • FIG. 13A and Figure 13B Pretreatment of GSC2 at a concentration of gambogic acid at 1 ⁇ M significantly inhibited the ability of in situ tumor sphere formation in nude mice.
  • Gambogic acid amide was pretreated with GSC2 at a concentration of 1 ⁇ M for 24 hours, and then the viable cells were collected. The cells were counted after trypan blue staining, and the cells were intracranially tumorigenic, 10 5 viable cells/only (5 nude mice).
  • the DMSO control group (5 nude mice), the DMSO treatment group (Fig. 13A), and the gambogic acid pretreatment group (Fig. 13B) were subjected to HE staining after in situ tumor formation.
  • FIG 14A and Figure 14B Gambogic acid amide significantly inhibited tumor growth in a zebrafish glioma xenograft model.
  • the yolk sac of the 48-hour zebrafish embryo was injected with 3000 green-fluorescent-labeled U87MG SLC-GFP, and after 48 hours of natural growth, it was treated with gambogic acid 1 ⁇ M concentration (Fig. 14B) for 48 hours, and the zebrafish was detected by fluorescence microscopy. The intensity of green fluorescence in the yolk sac was used to determine the antitumor effect of gambogic acid on the zebrafish xenograft model.
  • Figure 14A is the control group.
  • FIG. 15A to 15C Gambogic acid amide significantly inhibited tumor growth in a nude mouse xenograft model with less side effects.
  • GSC2 cells were inoculated into 15 subcutaneous subcutaneouss of 7-week-old Balb/C nude mice, and the number of inoculations was 5*10 4 per mouse.
  • the gambogic acid drug-administered group was administered at a dose of 2 mg/kg, administered intraperitoneally for 11 days, during which the volume of tumors of the nude mice (Fig. 15A and Fig. 15B) and body weight (Fig. 15C) were recorded. Variety.
  • apoptosis-related proteins PARP, Caspase3, Cleaved caspase3
  • cell cycle-associated antibodies p27, p21
  • FIG 17A-1 to Figure 17A-5 Glycinamide was induced to induce apoptosis of glioma stem cells by Annexin V/PI double staining.
  • Gambogic acid amide at 0 ⁇ M (control group, Figure 17A-1), 0.1 ⁇ M (Figure 17A-2), 0.3 ⁇ M (Figure 17A-3), 1 ⁇ M ( Figure 17A-4), 3 ⁇ M ( Figure 17A-5)
  • Treatment of GSC2 for 12 hours, Annexin V/PI double staining, flow analysis of cell apoptosis in each treatment group, Figure 17A-1 to Figure 17A-5 (Gate: P2) found that gambogic acid can significantly induce glioma stem cells Apoptosis.
  • FIG. 17B-1 to Figure 17B-5 Glucosamine was induced to induce apoptosis of glioma stem cells by Annexin V/PI double staining.
  • Gambogic acid amide at 0 ⁇ M (control group, Figure 17B-1), 0.1 ⁇ M (Fig. 17B-2), 0.3 ⁇ M (Fig. 17B-3), 1 ⁇ M (Fig. 17B-4), 3 ⁇ M (Fig. 17B-5) Handling GSC2 24 small
  • FIG. 18 The TrkA inhibitor GW44176 partially restored the inhibition of glioma stem cells by gambogic acid.
  • Glioma stem cells were pre-treated with GW44176 (800 nM), K252a (30 nM) for 2 hours, and then 11# drugs at different concentrations were added to the glioma stem cell line (GSC2) for 4 hours, and then detected by MTS method. Cell viability.
  • a, DPBS sequentially add 0.2g KCl, 8.0g NaCl, 0.2g KH 2 PO 4 , 1.15g Na 2 HPO 4 to 1L, adjust the pH to 7.35, then add 0.1gMgCl 2 ⁇ 6H 2 O, mix well until the solution is clear, then add After 0.133 g of CaCl 2 ⁇ 2H 2 O, it was thoroughly mixed here until the solution was clear. After sterilizing twice with a 0.2 ⁇ M filter, the mixture was stored at 4 ° C.
  • PMS solution Prepare a 0.92 mg/mL PMS solution using DPBS, filter and sterilize twice with a 0.2 ⁇ M filter, and dispense into an EP tube wrapped with tin-plated paper, and store at -20 °C in the dark.
  • MTS/PMS solution MTS and PMS were separately thawed, incubated at 37 ° C for 15 min, then dissolved in 2.1 mL of DPBS according to 4.2 mg of MTS powder, and 0.1 mL of PMS was added at the time of use.
  • Example 2 Eukaryotic cell lentivirus infection
  • the U87MG SLC cells stably expressing green fluorescence were screened with the corresponding concentration of blasticidin, and the GSC2 cells stably expressing green fluorescence were screened with the corresponding concentration of puromycin, and the method of selecting the monoclonal was selected.
  • HA cells stably expressing red fluorescence were screened.
  • 6-well and 96-well cell culture plates were pre-coated with 100 ug/mL polylysine, washed overnight at 37 ° C, washed twice with physiological saline, and air-dried.
  • GSC2-GFP cells stably expressing green fluorescence and HA cells stably expressing red fluorescence were digested into single cells, resuspended in fresh medium, and counted in cells, diluted to 5 ⁇ 10 4 cells/mL, and the two cells were 1:1.
  • Mix well divide into five tubes, add gambogic acid amide, and mix them to a concentration of 0 ⁇ M, 0.1 ⁇ M, 0.3 ⁇ M, 1 ⁇ M, and 3 ⁇ M.
  • 6-well cell culture plates (2 mL) and 96-well cell culture plates (100 uL, 3 replicate wells per group) were added, respectively, for 4 hours, 12 hours, 24 hours, and 48 hours, respectively.
  • MTS method was used to detect 96-well cell culture.
  • the cells in each treatment group were plated, and the cells in 6-well cell culture plates were collected, washed twice with PBS, and resuspended with 100 uL each.
  • Flow cytometry analysis of the proportion of GSC2-GFP and HA-RFP in living cells of each treatment group The relative cell viability of each cell component in the co-culture model was statistically analyzed by combining the co-culture model with the changes in cell viability after drug treatment.
  • Co-culture model - U87MG SLC-GFP + HA-RFP operates as above;
  • Gambogic acid amide can specifically inhibit the cell viability of glioma stem cells in a co-culture model relative to human normal astrocytes.
  • the suspension cultured GSCs tumor cells were collected into a 15 mL centrifuge tube, and 0.5 mL of ACCUTASE enzyme (purchased from sigma) was added for digestion at 37 ° C for 5 mins, and the cells were blown into single cells and centrifuged. Clearing
  • the cells were seeded in a 96-well plate, 100 uL/well, 37 ° C, 5% CO 2 , cultured in an incubator, and the proportion of tumor-free spherical wells was counted after 2 weeks;
  • Gambogic acid was used to treat GSC2 cells at a concentration of 0.1 ⁇ M, 0.3 ⁇ M, 1 ⁇ M, and 3 ⁇ M for 4 hours.
  • the cells were collected by centrifugation to remove the drug medium, washed twice with PBS, and resuspended in fresh medium, and partially stained with trypan blue. After the cells were counted, the cells in each treatment group were plated in 96-well plates, with 3 replicate wells in each group and 5000 live cells per well. After 4 days, the formation of tumor spheres in each treatment group was observed under light microscope, and the tumor spheres of each treatment group were counted. number.
  • Example 7 Three-time (Thirdly, 3 rd ) tumor ball formation experiment
  • Gambogic acid was used to treat GSC2 cells at a concentration of 0.1 ⁇ M, 0.3 ⁇ M, 1 ⁇ M, and 3 ⁇ M for 4 hours.
  • the cells were collected by centrifugation to remove the drug medium, washed twice with PBS, and resuspended in fresh medium to allow the cells to grow naturally to passable. The cells were counted by trypan blue staining.
  • the cells of each treatment group were plated in 96-well plates, with 3 replicate wells in each group and 5000 live cells per well. After 4 days, the formation of tumor spheres in each treatment group was observed under light microscope. The number of tumor spheres in each treatment group was counted.
  • MTS detects cell viability, the drug acts for 48 hours, and then the test recovers for 48 hours, the operation is as above;
  • the cells were pretreated for 24 hours at a concentration of 1 ⁇ M of gambogic acid amide, washed twice with PBS, partially stained with trypan blue, and counted.
  • the cells were injected in situ into the intracranial striatum of Balb/C nude mice at 5-7 weeks old, 10 5 cells/only, 5 rats in each group, and a DMSO control group was set up.
  • Example 10 Administration of zebrafish glioma transplantation model
  • the GSC2 single cells resuspended in PBS after centrifugation were inoculated into 15 subcutaneous subcutaneouss of 7-week-old Balb/C nude mice, and the number of inoculations was 5*10 4 per mouse.
  • the tumor size of nude mice was recorded (nude mice).
  • Tumor volume length * width 2 /2), when the subcutaneous tumor of nude mice grows to about 100 mm 3 , it is divided into DMSO group and gambogic acid drug-administered group at a dose of 2 mg/kg, intraperitoneally injected. The drug was administered for 11 days, during which time the volume and body weight of the tumors of the nude mice were recorded.
  • Example 13 Annexin V/PI double staining for detection of apoptosis
  • Annexin V/PI double staining kit used was purchased from BD pharmingen with product lot number 556547.
  • GW44176 (Selleck, 800nM), K252a (abcam, 30nM) cell suspension was added to a 96-well cell culture plate, 100uL per well;
  • TrkA inhibitor GW44176 partially restored the inhibition of glioma stem cells by gambogic acid.
  • the present invention demonstrates gambogic acid amide relative to glioma cells (U251, U87MG, N3 (primary glioma cells isolated from patient tumor tissue)), human normal astrocytes (HA) at the cellular level.
  • Human neural stem cells H1P56NSC
  • H1P56NSC Human neural stem cells have significant specific inhibitory effects on four glioma stem cells (U87MG SLC, U251SLC, GSC2, GSC5) isolated from the laboratory (IC 50 values for 48 hours of action: 0.01 to 0.64) uM).
  • the present invention utilizes a zebrafish glioma xenograft model and a nude mouse glioma subcutaneous xenograft model to demonstrate the therapeutic effect of gambogic acid on glioma at the body level.
  • the present invention finds that as a human neurotrophic factor NGF analog, the inhibitory effect of gambogic acid on glioma stem cells can be partially restored by the TrkA inhibitor, which explains to some extent that gambogic acid amide exerts an inhibitory effect on glioma stem cells. It exerts a mechanism of anti-tumor effect on glioma.
  • the present inventors have found that the small molecule compound gambogic acid can inhibit human glioma stem cells by activating TrkA and its downstream signaling pathway, thereby achieving the purpose of treating human glioma. It is well known that small molecule compounds rely on related key groups to function in the body, and on the basis of this, other group changes can produce different compounds, but they can also perform their intended functions. Therefore, any modification to the modification of gambogic acid amide on the basis of this, and the research which achieves the same effects as the present invention are all used in the present invention.

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Abstract

本发明公开了藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途，所述藤黄酰胺是一种TrkA激动剂和神经营养因子NGF类似物，相对于人神经胶质瘤细胞及人正常星形胶质细胞，可显著特异性抑制胶质瘤干细胞活性，且经藤黄酰胺处理后胶质瘤干细胞的自我更新、肿瘤球形成、裸鼠体内成瘤能力也明显降低。经体内实验，利用斑马鱼胶质瘤移植瘤模型、裸鼠胶质瘤移植瘤模型，在体验证该药物对胶质瘤的治疗效果。具体而言，本发明发现了小分子化合物藤黄酰胺可通过靶向性抑制胶质瘤干细胞达到用于制备治疗人神经胶质瘤的药物目的。本发明为人神经胶质瘤的治疗提供了新的治疗药物。

Description

藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途 技术领域
本发明涉及肿瘤治疗领域，具体涉及藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途。
背景技术
神经胶质瘤是最常见的来源于神经上皮的原发性颅内恶性肿瘤，其恶性程度高、生长迅速、浸润性强，具有高发病率的特点。虽然目前肿瘤综合治疗技术已取得长足进步，但因其位于脑部重要位置，且伴随微血管增生，呈现明显的浸润性生长等特点，传统的手术、放疗都无法彻底根治，而导致其复发率高、病死率高和治愈率低。目前临床上化疗的药物以烷基化药物为主，但传统烷化剂副作用大，易产生耐药性；新型烷化剂替莫唑胺(temozolomide，TMZ)虽毒副作用小，且长时间用药耐受性好，但TMZ治疗后引起的骨髓增生异常综合征(myelodysplastic syndrome，MDS)、急性髓性白血病(acute myeloid leukemia，AML)以及急性成淋巴细胞性白血病(acute lymphoblastic leukemia，ALL)的报道与日俱增，具体原因也在进一步研究中。因此，寻找新一代治疗神经胶质瘤的有效药物，改善神经胶质瘤的治疗迫在眉睫。
近年来，科学家们结合胶质瘤的发生发展的机理，在其新药探索与开发方面做出了诸多努力。研究显示，神经胶质瘤的发生发展主要与RTK/RAS/PI3K通路，p53通路，RB通路中的相应关键分子的异常表达或突变相关。因而近年来的研究都在开发相应的药物分子的抑制剂用以抵消某种关键分子的作用效果，但都只是在临床前研究结果中表现出强的治疗前景，在Ⅰ、Ⅱ期临床结果，或是批准上市后表现得不尽人意或疗效颇微。如EGFR的抑制剂：吉非替尼和埃罗替尼被证实患者服用后极易产生耐药，而且有疗效的患者非常罕见，并且病人服用之后无进展生存期皆未有所延长。同时EGFR/Her2抑制剂拉帕替尼，EGFR的单克隆抗体西妥昔单抗都被证实对胶质瘤的治疗效果颇微；除此以外还有mTOR相关抑制剂：雷帕霉素、替西罗莫司、依维莫司、地磷莫司，脱乙酰酶(Histone deactylase，HDAC)抑制剂：伏立诺他等单独治疗或与替莫唑胺联合治疗都被证实没有或只有极小的作用效果。与此同时，研究还针对胶质瘤微血管形成丰富的特点，开发了被证实有部分效果的抗血管生成的分子如贝伐单抗、西地尼布，它们可以快速的减少癌旁水肿，有效的延长了患者的无进展生存期，但是也无法逆转病程的进展。接受抗血管疗法的患者最终也无法成功治愈，还是无法改变大多数患者在诊断后数月死亡的现状。总体而言，尽管国内外科学家，药物公司在胶质瘤的新药探索中付出诸多人力物力，目前都没有找到一种新的，有效的药物分子。由于胶质瘤复杂 的发病机制，目前我们尚不知道在不同发病机理导致的胶质瘤患者中哪个关键分子需要被抑制。此外，另一些科学家提出，胶质瘤组织内细胞存在异质性，以上探索的药物分子之所以结果颇微，可能与我们没有针对对胶质瘤的发生、发展起到重要作用的细胞类型有关。
1994年，Lapidot等从白血病患者身上首次分离获得肿瘤干细胞，肿瘤干细胞理论逐渐被接受。随后Singh等在胶质瘤中分离得到极少数具有自我更新及分化能力的CD133+胶质瘤干细胞，且其相对于CD133-的细胞具有更强的体内成瘤能力，仅100个细胞就可在裸鼠内诱导形成肿瘤。Jian Chen等通过细胞体内示踪技术，从在体的水平，证明了胶质瘤干细胞的存在。随着研究的深入，胶质瘤干细胞的特点性质，以及与胶质瘤的关系逐渐被揭示。研究表明胶质瘤干细胞是胶质瘤发生发展的根本原因，且胶质瘤干细胞可通过激活Wnt信号通路诱导肿瘤血管形成，增强肿瘤的低氧应激以及侵袭能力等。不仅如此，Bao等证实胶质瘤干细胞也能产生放化疗抗性。然而，目前神经胶质瘤的化疗药物只针对整个肿瘤组织，病人在接受一段时间的治疗后，肿瘤组织缩小取得一定的效果，但是对化疗产生抵抗的胶质瘤干细胞可通过增殖和分化进一步形成新的肿瘤组织，从而促使肿瘤复发。由此可见，胶质瘤干细胞与胶质瘤的发生发展，及放化疗抗性的形成，肿瘤的复发息息相关，是胶质瘤难以攻克的一个重要因素。因此，我们设想，是否可以通过获得靶向性胶质瘤干细胞的药物，并结合传统的放化疗，达到最终彻底消灭整个肿瘤组织的目的？
近几年，国内外科学家分别在不同肿瘤中报道了几种能靶向肿瘤干细胞的药物，其中包括一些新的药物分子和已在临床上使用的药物分子。如天然的多酚白藜芦醇，具有抗菌活性的离子载体盐霉素，衣霉素，灭绦虫活性的药物氯硝柳胺，以及用以治疗糖尿病的药物二甲双胍及用以治疗肝癌的药物索拉非尼，都利用裸鼠成瘤模型，验证了药物相对于目前的一线药物TMZ更具抑瘤效果。但是以上药物都无法在保证安全性的前提下，发挥靶向肿瘤干细胞的功能，因而效果有限。
2007年Keqiang Ye实验室通过药物筛选发现的一种化学特征稳定的神经营养因子(Nerve growth factor，NGF)的类似物——藤黄酰胺，发现其可NGF特异性的受体TrkA的胞内近膜区域相互结合而使TrkA发生二聚化进而自磷酸化，从而激活TrkA及其下游的Akt、MAPKs信号通路。并证明藤黄酰胺可通过以上途径在体外体内水平明显的抑制神经元细胞细胞死亡，并具有促进PC12细胞的神经发生作用。提示其可能在神经退行性疾病、脑卒中的治疗发挥一定作用。与NGF不同，藤黄酰胺还可特异性地促进TrkA的转录和翻译。实验显示，NGF可迅速激活TrkA及其下游信号通路，但是60分钟后会由于TrkA的显著降解，TrkA及其下游相关蛋白的磷酸化水平下降，而藤黄酰胺可促进TrkA的转录和翻译，其对TrkA及其下游信号的活化可维持较长时间(>72小时)。该实验通过体外体内实验表明，藤黄酰胺可明显的抑制神经元细胞细胞死亡及促进PC12细胞的神经发生的作用效 果。随后Keqiang Ye实验室发现藤黄酰胺、NGF可通过促进AcinS与CtBP2结合而后抑制周期蛋白(cyclin)A1的转录明显抑制K562细胞的增殖，并明显抑制K562细胞裸鼠移植瘤的肿瘤生长，提示藤黄酰胺具有一定的抑瘤效果。
本发明发现相对于胶质瘤细胞株、人正常星形胶质细胞株，藤黄酰胺在细胞水平对实验室分离得到的四株胶质瘤干细胞株具有特异性抑制效果。在此基础上，本发明从细胞水平验证了藤黄酰胺可特异性抑制胶质瘤干细胞的自我更新能力，肿瘤球形成能力；并通过斑马鱼、裸鼠胶质瘤移植模型验证了藤黄酰胺对胶质瘤的治疗效果。由此，藤黄酰胺将很有可能成为神经胶质瘤的治疗提供新的有力治疗药物。
发明内容
基于上述目的，本发明提供了一种藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途。
优选的，所述药物通过抑制胶质瘤干细胞的细胞活性以治疗所述人神经胶质瘤。
优选的，所述药物通过抑制胶质瘤干细胞的自我更新、肿瘤球形成，及体内肿瘤形成的能力以治疗所述人神经胶质瘤。
优选的，所述药物通过诱导胶质瘤干细胞凋亡以治疗所述人神经胶质瘤。
可选的，所述药物对胶质瘤干细胞的抑制效果可被TrkA抑制剂部分回复。
可选的，所述胶质瘤干细胞包括但不限于：U87MG SLC、U251 SLC、GSC2、GSC5。
可选的，所述药物还通过抑制胶质瘤细胞以治疗所述人神经胶质瘤。
可选的，所述胶质瘤细胞包括但不限于：U251、U87MG、N3。
可选的，所述人神经胶质瘤处于I到IV级。
术语的定义
“胶质瘤干细胞”存在于胶质瘤组织内存在的一类极少数的具有自我更新能力，多项分化能力，体内成瘤能力的细胞，它与胶质瘤的发生发展，复发以及胶质瘤对放疗化疗抗性形成密切相关。
“MTS方法”：一种用比色法来检测细胞增殖和细胞毒实验中的活细胞数量的检测试剂。MTS是一种新型四唑化合物，PMS是一种电子偶联剂。PMS具有增强的化学稳定性，这使它可与MTS混合形成稳定的溶液。MTS被细胞生物还原成为一种有色的甲臜产物，可直接溶解于培养基中。这种转化很可能是在代谢活跃的细胞中的脱氢酶产生的NADPH或NADH的作用下完成的。在490nm处检测到的甲臜产物的量与培养中的活细胞数成正比。
“相对细胞存活率”：运用据上MTS方法检测吸光度值后，根据吸光值A计算相对细胞存活率，相对细胞存活率＝(实验组A/对照组A)×100％。
“自我更新”：指干细胞(此处特指胶质瘤干细胞)通过对称或者不对称分裂产生至少一个保留干细胞特性子细胞的过程，自我更新能够维持干细胞具有多分化的潜能，对于组织特异性干细胞而言，自我更新是维持其终生具有分化潜能的基础。
“肿瘤球形成能力”：指肿瘤干细胞(此处特指胶质瘤干细胞)在体外含EGF，bFGF的培养基中培养，会形成肿瘤球的能力。
综上，本发明发现藤黄酰胺可用于制备治疗人神经胶质瘤的药物，从而为人神经胶质瘤的治疗提供新的有力药物，具有显著的社会效益和经济效益。
附图说明
图1：藤黄酰胺特异性抑制胶质瘤干细胞的细胞活性。MTS方法检测藤黄酰胺在不同浓度(0.03μM、0.1μM、0.3μM、1μM、3μM、10μM、30μM)下对四株胶质瘤干细胞株(U87MG SLC、U251SLC、GSC2、GSC5)，三株胶质瘤细胞株(U251、U87MG、N3(从病人肿瘤组织分离得到的原代胶质瘤细胞))、人神经干细胞(H1P56NSC)作用48小时后细胞存活率，“——”表示IC50值>106μM。
图2A至图2D：藤黄酰胺特异性抑制胶质瘤干细胞。将带由绿色荧光标记的GSC2-GFP与不带荧光标记的HA 1:1混匀于体系中，而后藤黄酰胺分别在0.1μM、0.3μM、1μM、3μM浓度下作用4小时(图2A)、12小时(图2B)、24小时(图2C)、48小时(图2D)，而后流式细胞仪分析不同处理组下GSC2-GFP、U87MG SLC、HA的比例，并结合共培养模型在不同浓度、不同作用时间下的细胞存活率，统计分析共培养模型中各细胞成分的相对细胞存活率。
图3A至图3C：藤黄酰胺特异性抑制胶质瘤干细胞。将带由绿色荧光标记的U87MG SLC-GFP与不带荧光标记的HA 1:1混匀于体系中，而后藤黄酰胺分别在0.1μM、0.3μM、1μM、3μM浓度下作用12小时(图3A)、24小时(图3B)、48小时(图3C)，而后流式细胞仪分析不同处理组下GSC2-GFP、U87MG SLC、HA的比例，并结合共培养模型在不同浓度、不同作用时间下的细胞存活率，统计分析共培养模型中各细胞成分的相对细胞存活率。
图4A至图4F：藤黄酰胺抑制胶质瘤干细胞肿瘤球的生长与维持。96孔细胞培养皿包被后每孔加入100uL含5000个GSC2单细胞悬浮液，自然生长4天后，吸去原培养基，每孔加入含各浓度药物的100uL新鲜培养基，并使其药物浓度分别为：0μM(对照，图4A)、0.03μM(图4B)、0.1μM(图4C)、0.3μM(图4D)、1μM(图4E)、3μM(图4F)，药物作用时间为48小时，利用体外实验模拟临床上胶质瘤干细胞形成肿瘤后检测藤黄酰胺的抑制效果。藤黄酰胺可显著减小肿瘤球的体积、数量，并使肿瘤球边缘细胞死亡直至整个肿瘤球，图4A至图4F为光镜下检测形态改变。
图5：图4A至图4F中实验的数据统计结果。
图6：藤黄酰胺抑制胶质瘤干细胞自我更新能力。将GSC5细胞稀释为25、50、100、200个/100uL，藤黄酰胺在0.1μM浓度下处理2周，而后统计无肿瘤球孔百分比，纵坐标表示无肿瘤球孔百分比的对数值。
图7：藤黄酰胺在低浓度(0.1μM、0.3μM)下对胶质瘤干细胞作用4小时对其细胞活性影响较小，仅高浓度(1μM、3μM)下对胶质瘤干细胞的细胞活性具有一定影响。将GSC2细胞铺于预先包被的96孔细胞培养板，10000个细胞/孔，过夜后加药，使藤黄酰胺的浓度分别为0.1μM、0.3μM、1μM、3μM，药物作用4小时后MTS检测各处理条件下的相对细胞存活率。
图8A至图8C-5：藤黄酰胺在低浓度(0.1μM、0.3μM)下对胶质瘤干细胞作用4小时对其细胞活性影响较小，仅高浓度(1μM、3μM)下对胶质瘤干细胞的细胞活性具有一定影响，但皆可显著抑制胶质瘤干细胞的二次肿瘤球形成能力。藤黄酰胺分别在0.1μM、0.3μM、1μM、3μM浓度下处理GSC2细胞4小时，撤去药物，PBS洗两遍，台盼蓝染色后细胞计数，各浓度下各细胞重新铺于96孔细胞培养板，5000个细胞/孔。自然生长4天后。光镜下统计各处理组肿瘤球形成情况(图8A和图8B)，图8C-1至图8C-5为光镜下观察的结果，其中分别为对照组(图8C-1)、藤黄酰胺浓度0.1μM(图8C-2)、0.3μM(图8C-3)、1μM(图8C-4)、3μM(图8C-5)组。
图9：藤黄酰胺对胶质瘤干细胞的影响在一定程度上是不可逆的。藤黄酰胺处理GSC2细胞4小时后，撤药物，重新将细胞铺板，5000个细胞/孔，自然生长4天后，MTS检测各处理组的细胞存活率。
图10：藤黄酰胺可明显抑制胶质瘤干细胞自我更新能力。藤黄酰胺分别在0.1μM、0.3μM、1μM、3μM浓度下处理GSC2细胞4小时，而后撤去药物，台盼蓝染色后将细胞稀释为25、50、100、200个/100uL，自然生长8天，而后统计无肿瘤球孔百分比，纵坐标表示无肿瘤球孔百分比的对数值。
图11A至图11C-3：藤黄酰胺在低浓度(0.1μM、0.3μM)下对胶质瘤干细胞作用4小时对其细胞活性影响较小，仅高浓度(1μM、3μM)下对胶质瘤干细胞的细胞活性具有一定影响，但皆可显著抑制胶质瘤干细胞的三次肿瘤球形成能力。藤黄酰胺分别在0.1μM、0.3μM、1μM、3μM浓度下处理GSC2细胞4小时，撤去药物，PBS洗两遍，继续培养细胞至形成约100μm肿瘤球，收集细胞，细胞消化后，台盼蓝染色后细胞计数(发现1μM、3μM浓度下细胞存活率很低，故不再继续后续实验)，各浓度下各细胞重新铺于96孔细胞培养板，5000个细胞/孔。自然生长4天后。光镜下统计各处理组肿瘤球形成情况(图11A和图11B)，图11C-1至图11C-3为光镜下观察的结果，其中图11C-1为对照组，图11C-2为藤黄酰胺0.1μM，图11C-3为藤黄酰胺0.3μM。
图12A和图12B：藤黄酰胺对胶质瘤干细胞的作用效果一定程度上是不可逆的。药物处理GSC2 24小时，48小时后分别撤药物，换上新鲜培养基继续培养48 小时后利用MTS的方法检测各处理组细胞存活率，并比较其与一直用药物处理组的细胞活性改变。“1天”、“2天”表示药物处理24小时或48小时后MTS检测；“药物继续作用”表示药物处理24小时、48小时后撤药物，并继续加入相应药物浓度的新鲜培养基培养48小时后MTS检测；“撤去药物”表示药物处理24小时、48小时后撤药物，并加入不含药物的新鲜培养基继续培养48小时后MTS检测。
图13A和图13B：藤黄酰胺1μM浓度下预处理GSC2，可明显抑制其裸鼠原位肿瘤球形成能力。藤黄酰胺在1μM浓度下对GSC2预处理时间24小时，而后收集活细胞，取部分细胞台盼蓝染色后细胞计数，细胞颅内成瘤，105个活细胞/只(5只裸鼠)，并设置DMSO对照组(5只裸鼠)，DMSO处理组(图13A)，及藤黄酰胺预处理组(图13B)裸鼠原位成瘤后HE染色结果。
图14A和图14B：藤黄酰胺可显著抑制斑马鱼胶质瘤移植瘤模型的肿瘤生长。48小时斑马鱼胚胎的卵黄囊内注射3000个带有绿色荧光标记的U87MG SLC-GFP，自然生长48小时后，藤黄酰胺1μM浓度(图14B)下对其治疗48小时，荧光显微镜检测斑马鱼卵黄囊部位绿色荧光的强弱，用以判断藤黄酰胺对斑马鱼移植瘤模型的抑瘤效果，图14A为对照组。
图15A至图15C：藤黄酰胺可显著抑制斑裸鼠移植瘤模型的肿瘤生长，且副作用较小。将GSC2细胞接种于15只7周大的Balb/C裸鼠腋下皮下，接种数目为5*104个/只，当裸鼠皮下肿瘤长至约100mm3，将其分为DMSO组、及藤黄酰胺药物给药组，给药剂量为2mg/kg，腹腔注射给药，给药时间为11天，期间记录两组裸鼠肿瘤的体积(图15A和图15B)及体重(图15C)变化。
图16A至16B：藤黄酰胺可诱导胶质瘤干细胞凋亡，对胶质瘤干细胞的细胞周期影响不大。(图16A)藤黄酰胺分别在0.1μM、0.3μM、1μM、3μM浓度下处理四株胶质瘤干细胞株4小时，而后western blot检测凋亡相关抗体(PARP、胱天冬酶3(Caspase3)、活化的半胱天冬酶3(Cleaved caspase3))、细胞周期相关抗体(p27、p21)蛋白的表达，发现经藤黄酰胺处理后凋亡相关抗体的表达明显上调(图16B)藤黄酰胺在0.3μM浓度下处理胶质瘤干细胞株GSC2不同时间后，凋亡相关蛋白(PARP、Caspase3、Cleaved caspase3)、细胞周期相关抗体(p27、p21)蛋白的表达，发现经藤黄酰胺处理后凋亡相关抗体的表达明显上调。
图17A-1至图17A-5：利用AnnexinⅤ/PI双染法检测，发现藤黄酰胺可诱导胶质瘤干细胞凋亡。藤黄酰胺在0μM(对照组，图17A-1)、0.1μM(图17A-2)、0.3μM(图17A-3)、1μM(图17A-4)、3μM(图17A-5)浓度下处理GSC2 12小时，AnnexinⅤ/PI双染，流式分析各处理组内细胞的凋亡情况，图17A-1至图17A-5(门：P2)发现藤黄酰胺可明显诱导胶质瘤干细胞的凋亡。
图17B-1至图17B-5：利用AnnexinⅤ/PI双染法检测，发现藤黄酰胺可诱导胶质瘤干细胞凋亡。藤黄酰胺在0μM(对照组，图17B-1)、0.1μM(图17B-2)、0.3μM(图17B-3)、1μM(图17B-4)、3μM(图17B-5)浓度下处理GSC2 24小 时，AnnexinⅤ/PI双染，流式分析各处理组内细胞的凋亡情况，图17B-1至图17B-5中(门：P2)发现藤黄酰胺可明显诱导胶质瘤干细胞的凋亡。
图18：TrkA抑制剂GW44176对藤黄酰胺发挥对胶质瘤干细胞的抑制有部分回复作用。胶质瘤干细胞铺板后用GW44176(800nM)，K252a(30nM)预先处理2小时，而后加入不同浓度下的11#药物共同作用于胶质瘤干细胞株(GSC2)4小时，而后用MTS的方法检测细胞存活率。
具体实施方式
下面将通过下述非限制性实施例进一步说明本发明，本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。
下述实验方法如无特别说明，均为常规方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。
实施例1：MTS法检测细胞存活率
(1)按照产品说明书配制以下溶液：
a、DPBS：依次加入0.2gKCl，8.0gNaCl，0.2gKH2PO4，1.15gNa2HPO4至1L，调pH至7.35，之后加入0.1gMgCl2·6H2O，充分混匀至溶液清澈后，加入0.133gCaCl2·2H2O后，在此充分混匀至溶液清澈。用0.2μM滤器过滤除菌两次后，分装保存在4℃。
b、PMS溶液的配制：使用DPBS配制0.92mg/mL的PMS溶液，用0.2μM滤器过滤除菌两次后，分装于用锡泊纸包裹好的EP管中，避光保存-20℃。
c、MTS溶液的配制：按照1mg MTS粉末用0.5mLDPBS来溶解的比例配制，轻轻混匀，约15min，至MTS完全溶解。测量pH，使其保存在6.0—6.5之间。用0.2μM滤器过滤除菌两次后，分装于用锡泊纸包裹好的EP管中，避光保存-20℃。
d、MTS/PMS溶液的配制：分别融化MTS和PMS，37℃孵育15min，之后按照4.2mg MTS粉末溶于2.1mL DPBS中，使用时加入0.1mL PMS的比例来配制。
(2)对于藤黄酰胺对胶质瘤干细胞细胞活性的影响检测，实验用96孔细胞培养板用100ug/mL多聚赖氨酸包被过夜，次日生理盐水洗两遍，晾干后待用。将细胞消化后重悬为单细胞悬液，细胞计数，铺于96孔细胞培养板内(U87MG SLC(5000个/孔)、U251SLC(10000个/孔)、GSC2(10000个/孔)、GSC5(10000个/孔))，H1P56NSC(10000个/孔)生长过夜。次日加入藤黄酰胺(购自美国MicroSource公司，及SantaCruzBio，sc-221655A，USA)，其化学式如式I所示，其化学分子式为(C38H45NO7)，其结构简式为CC(＝CCCC1(C＝CC2＝C(O1)C(＝C3C(＝C2O)C(＝O)C4＝CC5CC6C4(O3)C(C5＝O)(OC6(C)C)CC＝C(C)C(＝O)N)CC＝C(C)C)C)C。并设置如下浓度梯度：0.03μM、0.1μM、 0.3μM、1μM、3μM、10μM、30μM，每个药物浓度3个复孔，作用48小时。
Figure PCTCN2017077350-appb-000001
(3)对于三株胶质瘤细胞株(U251(购自美国ATCC)、U87MG(购自美国ATCC)、N3(从病人肿瘤组织分离得到的原代胶质瘤细胞，获赠于天坛医院江涛老师实验室))、则无需包被实验用96孔细胞进行包被。其细胞铺板浓度均为：5000个/孔、5000个/孔、5000个/孔、10000个/孔。其它操作如上。
(4)藤黄酰胺作用48小时后，根据需要配制相应体积的MTS/PMS检测液，然后按照检测液：培养基＝1:5的比例加入无血清培养基。最终每孔体系120μL，37℃，5％CO2孵育2小时。根据MTS还原后产生的甲臜产物的吸光度光谱，在吸收峰490nm读取数据。并减去630nm所读取的数据，用以减去细胞碎片等造成的噪音值，以及其它非特异性吸光度值。
(5)根据吸光值A计算细胞存活率，细胞存活率＝(实验组A/对照组)×100％。计算IC50值，并绘制剂量反应曲线。
结果见图1，藤黄酰胺特异性抑制胶质瘤干细胞的细胞活性；
图7，藤黄酰胺在低浓度(0.1μM、0.3μM)下对胶质瘤干细胞作用4小时对其细胞活性影响较小，仅高浓度(1μM、3μM)下对胶质瘤干细胞的细胞活性具有一定影响；
以及图9，藤黄酰胺对胶质瘤干细胞的影响在一定程度上是不可逆的。
实施例2：真核细胞慢病毒感染
具体步骤如下：
(1)感染前将GSC2、U87MG SLC、HA细胞消化为单细胞悬液，计数后以相应密度(1-3×105个/mL)接种于6孔细胞培养板内，体系为2mL，让GSC2、U87MG SLC自然生长2小时，HA细胞自然生长直至细胞贴壁(大约4小时)；
(2)分别往GSC2细胞培养基中加入带有绿色荧光的含LV3载体(带嘌呤霉素筛选标记)，往U87MG SLC细胞培养基中加入带有绿色荧光的含pLenti6载体(带杀稻瘟素(Blasticidin)筛选标记，购自Promega公司)的慢病毒2uL，往HA细胞培养基中加入带有红色荧光的慢病毒(不带筛选标记)2uL，充分混匀，过夜培养；
(3)次日上午，移去含病毒培养基，各加入新鲜培养基2mL，让其自然生长；
(4)48小时～72小时，荧光显微镜下观察细胞，检测是否有相应荧光表达，以确定是否感染成功；
(5)确定有荧光表达后，用相应浓度的杀稻瘟素筛选稳定表达绿色荧光的U87MG SLC细胞，用相应浓度的嘌呤霉素筛选稳定表达绿色荧光的GSC2细胞，并通过挑选单克隆的方法筛选稳定表达红色荧光的HA细胞。
实施例3：流式细胞分析
预先用100ug/mL的多聚赖氨酸包被6孔及96孔细胞培养板，37℃过夜，生理盐水洗两遍，晾干。将稳定表达绿色荧光的GSC2-GFP细胞及稳定表达红色荧光的HA细胞，消化为单细胞，新鲜培养基重悬，细胞计数，均稀释为5×104个/mL，两种细胞1:1充分混匀，分为五管，分别加入藤黄酰胺，混匀，使其浓度分别为0μM、0.1μM、0.3μM、1μM、3μM。分别加入6孔细胞培养板(2mL)及96孔细胞培养板(100uL，每组3个复孔)，分别待其作用4小时、12小时、24小时、48小时，MTS方法检测96孔细胞培养板中各处理组细胞活性，并分别收集6孔细胞培养板中细胞，PBS洗两遍，各用100uL重悬细胞，流式分析各处理组活细胞中GSC2-GFP、HA-RFP所占比例。并结合共培养模型经药物处理后细胞活性变化，统计分析共培养模型中各细胞成分的相对细胞存活率。共培养模型——U87MG SLC-GFP+HA-RFP的操作如上；
结果见图2A至图3C，相对于人正常星形胶质细胞，藤黄酰胺可特异性的抑制共培养模型中胶质瘤干细胞的细胞活性。
实施例4：肿瘤球形成实验
96孔细胞培养皿包被后每孔加入100uL含5000个GSC2单细胞悬浮液，自然生长4天后，吸去原培养基，每孔加入各浓度藤黄酰胺的100uL新鲜培养基，药物浓度分别为：0.03μM、0.1μM、0.3μM、1μM、3μM、10μM、30μM，药物作用时间为处理48小时后镜下观察各处理组肿瘤球的形态变化，并同时统计各处理组肿瘤球数目。
结果见图4A至图4F和图5，利用该体外实验模拟临床上胶质瘤干细胞形成肿瘤后检测藤黄酰胺的抑制效果，发现藤黄酰胺可显著减小肿瘤球的体积、数量，并使肿瘤球边缘细胞死亡直至整个肿瘤球。
实施例5：有限稀释实验
步骤如下：
(1)将悬浮培养的GSCs肿瘤球细胞收集到15mL离心管中，加入0.5mL ACCUTASE酶(购自sigma公司)37℃消化5mins，将细胞吹成单细胞，离心去 上清；
(2)加入新鲜的Neurobasal培养基(购自gibco公司)，并取部分细胞台盼蓝染色后细胞计数；
(3)计算所需细胞液的体积，设置细胞浓度梯度，200，100，50，25个/孔，每种浓度设置10个重复孔。设置DMSO对照组，及藤黄酰胺0.1μM药物处理组；
(4)将细胞接种于96孔板中，100uL/孔，37℃，5％CO2，孵箱中培养，2周后统计无肿瘤球孔的比例；
结果见图6，藤黄酰胺在低浓度0.1μM浓度下可显著抑制胶质瘤干细胞克隆形成，即能明显抑制胶质瘤干细胞的自我更新能力；及图10，藤黄酰胺在0.1μM，0.3μM，1μM，3μM浓度下处理GSC2细胞4小时，去药物后也可明显抑制胶质瘤干细胞自我更新能力。
实施例6：二次(Secondly,2nd)肿瘤球形成实验
藤黄酰胺在0.1μM、0.3μM、1μM、3μM浓度下处理GSC2细胞4小时，离心收集细胞去带药物培养基，PBS洗两次并用新鲜培养基重悬细胞，分别取部分细胞台盼蓝染色后细胞计数，各处理组细胞铺于96孔板，每组3个复孔，每孔5000个活细胞，4天后光镜下观察各处理组肿瘤球形成情况，并统计各处理组肿瘤球的数目。
结果见图8A、8B和图8C-1至图8C-5，藤黄酰胺处理胶质瘤干细胞4小时，撤药后，其肿瘤球形成能力也明显被抑制。
实施例7：三次(Thirdly，3rd)肿瘤球形成实验
藤黄酰胺在0.1μM、0.3μM、1μM、3μM浓度下处理GSC2细胞4小时，离心收集细胞去带药物培养基，PBS洗两次并用新鲜培养基重悬细胞，使细胞自然生长至可传代，分别取部分细胞台盼蓝染色后细胞计数，各处理组细胞铺于96孔板，每组3个复孔，每孔5000个活细胞，4天后光镜下观察各处理组肿瘤球形成情况，并统计各处理组肿瘤球的数目。
结果见图11A、11B和图11C-1至图11C-3，藤黄酰胺处理胶质瘤干细胞4小时，撤药后，细胞传一代，仍然可明显抑制肿瘤球形成能力。
实施例8：回复试验(Recovery assay)
(1)用100ug/mL多聚赖氨酸包被96孔细胞培养板
(2)ACCUTASE酶将GSCs消化为单细胞，新鲜Neurobasal培养基重悬，将细胞铺于预先包被的96孔细胞培养板，过夜；
(3)加入藤黄酰胺，使其浓度为0.03μM、0.1μM、0.3μM、1μM、3μM、10μM、30μM，每个浓度9个复孔；
(4)作用24小时后，往每个浓度的3个复孔中加入20uL MTS/PMS混合试剂，检测其细胞活性。吸去其它6个复孔加药培养基，往其中3个孔中加入新鲜培养基，另外3个复孔中加入相同浓度的含藤黄酰胺培养基；
(5)继续培养48小时后，MTS检测细胞活性，药物作用48小时，而后检测恢复48小时的情况，操作如上；
结果见图12A和图12B，藤黄酰胺在0.3μM及以上浓度，对细胞作用24小时，48小时后撤药，GSC2的细胞活性将无法回复。
实施例9：裸鼠原位成瘤
收集经藤黄酰胺1μM浓度下预处理24小时后细胞，PBS洗两遍，部分细胞台盼蓝染色，细胞计数。取细胞原位注射于5～7周龄Balb/C裸鼠颅内纹状体，105个细胞/只，每组5只，并设置DMSO对照组。
结果见表1、图13A和图13B，藤黄酰胺预处理胶质瘤干细胞GSC2，可显著抑制其裸鼠原位成瘤能力。
表1
#细胞(GSC2) 对照 藤黄酰胺-1μM
1*105 5/5 1/5
实施例10：斑马鱼胶质瘤移植模型给药实验
往48小时斑马鱼胚胎的卵黄囊内注射3000个带有绿色荧光标记的U87MG SLC-GFP，让其自然生长48小时，利用荧光显微镜检测斑马鱼体内肿瘤形成情况，而后往斑马鱼孵化液中加入藤黄酰胺，使其浓度为1μM，药物处理48小时，荧光显微镜检测斑马鱼卵黄囊部位绿色荧光的强弱，用以判断藤黄酰胺对斑马鱼移植瘤模型体内肿瘤的抑制效果；
结果见图14A和图14B，藤黄酰胺可显著抑制斑马鱼移植瘤模型体内肿瘤的生长。
实施例11：裸鼠腋下皮下成瘤
将离心消化后的重悬于PBS的GSC2单细胞接种于15只7周大的Balb/C裸鼠腋下皮下，接种数目为5*104个/只，记录裸鼠的肿瘤大小(裸鼠肿瘤体积＝长*宽2/2)，当裸鼠皮下肿瘤长至约100mm3时，将其分为DMSO组、及藤黄酰胺药物给药组，给药剂量为2mg/kg，腹腔注射给药，给药时间为11天，期间记录两组裸鼠肿瘤的体积及体重变化。
结果见图15A至图15C，藤黄酰胺可显著抑制斑裸鼠移植瘤模型的肿瘤生长，且其副作用较小。
实施例12：蛋白质印记(Western Blotting)
以10％分离胶进行SDS-PAGE电泳，然后凝胶小心转移至电转缓冲液中转硝酸纤维素膜，封闭非特异性结合位点后加入一抗抗体(PARP、Caspase3、Cleaved caspase3)、细胞周期相关抗体(p27、p21)抗体购自Cell signaling公司，货号分别为：#9542L；#9665S；#9661L；#3686S；#2947S，4℃孵育过夜，然后用辣根过氧化物酶标记的二抗：山羊抗兔抗体(购自中山金桥)室温结合1-2h。然后用ECL法进行显色。
结果见图16A、图16B，藤黄酰胺可诱导胶质瘤干细胞凋亡，对胶质瘤干细胞的细胞周期影响不大。
实施例13：AnnexinⅤ/PI双染检测细胞凋亡
具体步骤如下；
(1)藤黄酰胺在0.1μM、0.3μM、1μM、3μM浓度下处理GSC2 12小时或24小时；
(2)收集细胞，PBS洗两遍；
(3)取50uL 1*结合缓冲液(binding buffer)重悬细胞，使细胞密度约为1*106个/mL；
(4)加入2.5uL FTIC AnnexinⅤ和2.5uL PI；
(5)避光情况下，室温孵育15min；
(6)往体系中加入200uL1*结合缓冲液(binding buffer)，各处理管分别过细胞筛；
(7)流式细胞仪分析；
所用AnnexinⅤ/PI双染试剂盒购自BD pharmingen，产品批号为556547。
结果见图17A-1至图17B-5，藤黄酰胺可诱导胶质瘤干细胞细胞凋亡。
实施例14：拯救实验
具体步骤如下：
(1)收集GSC2细胞，将细胞消化后重悬为单细胞悬液，细胞计数；
(2)将细胞稀释为100000个细胞/mL；
(3)分别往稀释后的GSC2细胞液中加入GW44176，K252a，使其浓度分别人800nM，30nM；
(4)将分别加有GW44176(Selleck公司，800nM)，K252a(abcam公司，30nM)的细胞液加入96孔细胞培养板内，每孔100uL；
(5)放入细胞培养箱内，培养2小时；
(6)而后分别往每种药物添加组内加入藤黄酰胺，并使其浓度为0.1uM,0.3uM,1uM,3uM；
(7)继续培养4小时后，MTS方法检测细胞存活率。
结果见图18，TrkA抑制剂GW44176对藤黄酰胺发挥对胶质瘤干细胞的抑制有部分回复作用。
综上，本发明在细胞水平证实藤黄酰胺相对于胶质瘤细胞(U251、U87MG、N3(从病人肿瘤组织分离得到的原代胶质瘤细胞))、人正常星形胶质细胞(HA)，人神经干细胞(H1P56NSC)对实验室分离得到的四株胶质瘤干细胞(U87MG SLC、U251SLC、GSC2、GSC5)具有明显的特异性抑制效果(48小时作用的IC50值为：0.01～0.64uM)。并进一步通过克隆形成实验，肿瘤球抑制实验，2nd肿瘤球形成实验，3rd肿瘤球形成实验，原位成瘤实验等证实藤黄酰胺可明显抑制胶质瘤干细胞的自我更新，肿瘤球形成及在体肿瘤形成能力。而后本发明利用斑马鱼胶质瘤移植瘤模型、裸鼠胶质瘤皮下移植瘤模型，在体水平证明了藤黄酰胺对胶质瘤的治疗作用。且本发明发现作为人神经营养因子NGF类似物，藤黄酰胺对胶质瘤干细胞的抑制作用可被TrkA抑制剂部分回复，在一定程度上解释了藤黄酰胺发挥对胶质瘤干细胞抑制作用及其发挥对胶质瘤抑瘤效果的机制。
因此，本发明发现了小分子化合物藤黄酰胺可通过激活TrkA及其下游信号通路，抑制人神经胶质瘤干细胞从而达到治疗人神经胶质瘤的目的。众所周知，小分子化合物在体内发挥功能都需倚靠相关的关键基团，而在此基础上其它基团的改变虽能产生不同的化合物，但也能发挥其目的功能。因此，任何在此基础上对藤黄酰胺的修饰，修改而后达到与本发明相同效果的研究均用于本发明。此外，如人神经营养因子NGF，或利用高通量筛选技术从化合物库中小分子化合物库中筛选获得特异性激活TrkA及其下游信号通路，进而达到抑制胶质瘤干细胞，并达到治疗胶质瘤的目的的其它化合物，也落入本发明的范围。

Claims (9)

  1. 藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途。
  2. 根据权利要求1所述的藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途，其中所述药物通过抑制胶质瘤干细胞的细胞活性以治疗所述人神经胶质瘤。
  3. 根据权利要求1所述的藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途，其中所述药物通过抑制胶质瘤干细胞的自我更新、肿瘤球形成，体内肿瘤形成的能力，及体内肿瘤的生长以治疗所述人神经胶质瘤。
  4. 根据权利要求1所述的藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途，其中所述药物通过诱导胶质瘤干细胞凋亡以治疗所述人神经胶质瘤。
  5. 根据权利要求1至4中任一项所述的藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途，其中，所述药物对胶质瘤干细胞的抑制效果可被TrkA抑制剂部分回复。
  6. 根据权利要求1至4中任一项所述的藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途，其中，所述胶质瘤干细胞包括但不限于：U87MG SLC、U251SLC、GSC2、GSC5。
  7. 根据权利要求1所述的藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途，其中所述药物还通过抑制胶质瘤细胞以治疗所述人神经胶质瘤。
  8. 根据权利要求7所述的藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途，其中，所述胶质瘤细胞包括但不限于：U251、U87MG、N3。
  9. 根据权利要求1所述的藤黄酰胺用于制备治疗人神经胶质瘤的药物的用途，其中，所述人神经胶质瘤处于I到IV级。
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