Small molecule compound composition and application thereof in glioma treatment

Abstract

The invention provides a small molecule compound combination. The small molecule compound combination is prepared from DAPT, LDN193189, CHIR99021, Ibet-762 and Isx9. The invention also provides application of the small molecule compound composition in treatment of glioma, preparation of drugs for treatment of glioma and preparation of drugs for inducing transdifferentiation of glioma cells into neuron-like cells. The invention also provides a method for inducing transdifferentiation of glioma cells into neuron-like cells. The method comprises the step of applying the small molecule compound combination. The small molecule compound composition can induce transdifferentiation of human glioma cells into neuronal cells, and has an inhibition effect on proliferation, migration, invasion and tumor formation capabilities of tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a small molecule compound combination and its application in the treatment of glioma. Background Art

[0002] Glioma is the most common primary brain tumor of the central nervous system. Its clinical manifestations can be divided into three main types: (1) symptoms such as headache, nausea and papilledema caused by increased intracranial pressure; (2) compression of brain tissue by the growing tumor tissue, resulting in impaired cognitive function; (3) infiltration of brain tissue by glioma and abnormal metabolism often cause secondary epileptic seizures in patients. Surgical resection is the main treatment for glioma. By performing surgical resection on solid tumors and retaining normal neurological function as much as possible, it can effectively reduce intracranial pressure and compression on brain tissue, restore normal cerebrospinal fluid circulation, and relieve the patient's condition. However, due to its strong invasiveness, patients are still prone to recurrence after traditional surgical resection combined with radiotherapy and chemotherapy, and the prognosis is poor. Therefore, there is an urgent need to find alternative treatments for glioma.

[0003] Transdifferentiation, also known as direct reprogramming, refers to the transformation of differentiated cells from one lineage to another without entering the pluripotent cell stage. Direct reprogramming can be performed in three ways: by induction of transcription factors, miRNA, or chemical small molecules. In the field of neural regeneration, many studies have successfully achieved transdifferentiation of cells such as fibroblasts, NG2 glial cells, astrocytes, and glioma cells. This method of using endogenous cells to repair central nervous system damage avoids the risk of causing teratomas in stem cell therapy because it does not go through the pluripotent cell stage; this method can complete the transformation of cell identity in situ in vivo, bypassing the problem of transplantation difficulties in in vitro directed differentiation applications.

[0004] Several studies have found a new method to transdifferentiate glial cells into neurons by overexpressing neural transcription factors, and this method is also effective when applied to glioma cells. Previous studies have shown that overexpressing a single transcription factor, Ascl1, Neurog2, or NeuroD1, can transdifferentiate human glioma cells into neurons. This method can induce proliferating glioma cells into terminally differentiated neurons and inhibit the proliferation of glioma cells, which may be a new way to treat gliomas. Another way to enable cells to achieve transdifferentiation is through chemical induction through a combination of small molecule compounds that act on different signaling pathways. Compared with virus-mediated transdifferentiation, small molecule drugs have low cost, controllable processes, no immunogenicity, and do not integrate into the genome, and have great potential for clinical transformation. Summary of the invention

[0005] One object of the present invention is to provide a small molecule compound combination that can transform glioma cells into neuron-like cells and inhibit their tumor characteristics in response to the above technical problems to be solved.

[0006] Another object of the present invention is to provide an application of the above small molecule compound combination.

[0007] In order to achieve the above objectives, the present invention provides a small molecule compound combination, which includes DAPT, LDN193189, CHIR99021, Ibet-762 and Isx9.

[0008] On the other hand, the present invention also provides the use of the small molecule compound combination in the treatment of glioma.

[0009] On the other hand, the present invention also provides the use of the small molecule compound combination in the preparation of a drug for treating glioma.

[0010] On the other hand, the present invention also provides the use of the small molecule compound combination in the preparation of a drug for inducing the transdifferentiation of glioma cells into neuron-like cells.

[0011] On the other hand, the present invention also provides the use of the small molecule compound combination in inducing glioma cells to transdifferentiate into neuron-like cells.

[0012] On the other hand, the present invention also provides a method for inducing glioma cells to transdifferentiate into neuron-like cells, comprising administering the small molecule compound combination.

[0013] The small molecule compound combination of the present invention can induce human glioma cell lines to transdifferentiate into neuronal cells, and has an inhibitory effect on the proliferation, migration, invasion and tumor formation ability of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram of the experimental technical route for achieving glioma cell transdifferentiation using a combination of small molecule compounds.

[0015] Figure 2 Schematic diagram showing the transdifferentiation process of glioma cells.

[0016] Figure 3The protein expression of glioma cell lines without small molecule induction is shown. (A) Immunofluorescence staining results of proliferation-related proteins of U251 cells. Cell stemness-related protein SOX2, proliferation-related protein KI67 and human nuclear protein HuNu were positively stained. (B) Immunofluorescence staining results of tumor-related proteins of U251 cells. Cells were positive for nestin and vimentin but not PDGFA. (C) Immunofluorescence staining results of glial cell-related proteins of U251 cells. Cells expressed three glial cell marker proteins, GFAP, S100B and SOX9. (D) Immunofluorescence staining results of nerve-related proteins of U251 cells. Cells expressed weak Tuj1 and MAP2; did not express immature neuron marker DCX, mature neuron marker NeuN and GABAergic neuron marker GABA; glutamatergic neuron markers were positive but in the cell nucleus, which was different from the expression position of neurons. (E) Immunofluorescence staining results of tumor-related proteins of U-87MG-Luc cells (U87-Luci). The cells expressed proliferation-related protein KI67 and human nuclear protein HuNu, but did not express stemness-related protein SOX2. (F) Immunofluorescence staining results of glial cell-related proteins in U-87MG-Luc cells. The cells were positive for nestin and vimentin but not PDGFA. (G) Immunofluorescence staining results of proliferation-related proteins in U-87MG-Luc cells. Among the three glial cell proteins, only S100B was positively stained. (H) Immunofluorescence staining results of nerve-related proteins in U-87MG-Luc cells. (Scale bar 50μm)

[0017] Figure 4 The preliminary screening results of small molecule compound combinations are shown. (A) Schematic diagram of the screening process of small molecule compound combinations in U251 cells. After 2 days of cell plating, when the confluence reaches about 90%, the whole volume is replaced with different small molecule induction medium for incubation, and half of the medium is replaced every 2 days; after 4 days of incubation, the medium is replaced with neural differentiation medium for continued culture to make the transdifferentiated cells more mature. On the 8th day, the cells are fixed and stained, and the number and conversion rate of DCX-positive cells are counted. (B) The results of cell immunofluorescence staining on the 8th day of the initial screening. A large number of DCX-positive cells were found in the combination with the addition of small molecules Isx9, Skl2001, dihydromyricetin, Ibet-151, Ibet-762 and VPA. (Scale bar 300μm) (C) Statistical analysis of the conversion rate of DCX+ cells transformed by different small molecule compound combinations. Small molecules Isx9, Skl2001, dihydromyricetin, Ibet-151, Ibet-762 and VPA can effectively improve the transformation efficiency of U251 cells induced by the core combination. (****P<0.0001, one-way ANOVA with Tukey's multiple comparisons, n=3)

[0018] Figure 5 The necessity of four small molecules in SLCD (SB431542, DAPT, CHIR99021 and LDN193189) for transdifferentiation is shown. (A) Immunofluorescence staining results after removing small molecules SB431542, DAPT, CHIR99021 and LDN193189 respectively. There are almost no DCX-positive cells in the combination without LDN193189. (Scale bar 300μm) (B) Statistical analysis of the conversion rate of DCX+ cells after removing a single small molecule in SLCD on day 8. Removing SB431542 improves the conversion efficiency, and the combination of small molecule compounds almost loses the transdifferentiation effect after removing LDN193189. (*P<0.05, ****P<0.0001, one-way ANOVA with Tukey's multiple comparisons, n=3)

[0019] Figure 6 The DLC79 combination is shown to induce transdifferentiation of glioma cells. (A) The immunofluorescence staining results of glioma cells induced by the small molecule compound combination DLC79 and SLCD (i.e. "core", SB431542, DAPT, CHIR99021 and LDN193189) for 6 days and compared with the control group. The small molecule compound treatment group expressed the immature neuron marker DCX and was not co-labeled with the glial cell marker GFAP and the tumor marker vimentin. (B) The immunofluorescence staining results of glioma cells induced by the small molecule compound combination DLC79 and SLCD for 10 days and compared with the control group. DCX-positive cells continue to mature and are co-labeled with neuronal markers MAP2 and Tuj1. (Scale bar 50μm) (C) Statistical chart of the number of DCX+ cells in the field of view on the 6th and 10th days. The statistical results show that on the 6th and 10th days, the optimized small molecule compound combination induced U251 cell transformation efficiency much higher than the core combination. (****P<0.0001, one-way ANOVA with Tukey's multiple comparisons, n=3)

[0020] Figure 7The effect of each small molecule in the DLC79 combination on transdifferentiation is shown. (A) After subtracting a single small molecule in the combination, immunofluorescence staining shows the induction of transdifferentiation of U251 cells. (Scale bar 500μm) (B) The results of counting the number of DCX+ cells in the field of view show that subtracting DAPT or CHIR99021 will reduce the transdifferentiation efficiency, subtracting LDN193189 or Ibet762 will cause the small molecule compound combination to lose the transdifferentiation effect, and the number of DCX+ cells will increase slightly after subtracting Isx9. (C) The results of counting the total number of cells in the field of view show that the small molecule compound combination will be slightly reduced after subtracting DAPT, while the number of cells in the group subtracting Isx9 or LDN193189 is similar to that of the control group, indicating that the small molecule compound combination may lose the inhibitory effect on glioma cell proliferation, and subtracting CHIR99021 or Ibet762 has no obvious effect on the total number of cells. (D) The statistical graph of cell transformation efficiency within the field of view shows that removing any small molecule in the combination will reduce the transdifferentiation efficiency (*P<0.05, **P<0.01, ****P<0.0001, one-way ANOVA with Tukey's multiple comparisons, n=3)

[0021] Figure 8 The DLC79 combination is shown to inhibit the migration and invasion ability of glioma cells. (A) Representative pictures of U251 cell migration and invasion experiments 4 days after induction. After small molecule treatment, the U251 cell skeleton shrank, the morphology became more slender, and the cells that invaded or migrated to the lower chamber were sparser than those in the control group. (B) Statistical results of U251 cell migration and invasion experiments. After small molecule treatment, the number of cells that invaded or migrated to the lower chamber of U251 cells decreased, and the effect of small molecules in inhibiting the invasive ability of U251 was more obvious than the effect of inhibiting its migration. (C) Representative pictures of U-87MG-Luc cell migration and invasion experiments 4 days after induction. After small molecule treatment, the cells that invaded or migrated to the lower chamber were sparser than those in the DMSO control group. (Scale bar 500μm) (D) Statistical results of U-87MG-Luc cell migration and invasion experiments. After small molecule treatment, the number of U-87MG-Luc cells invading or migrating to the lower chamber decreased, and the effect of small molecules in inhibiting the invasion ability of U-87MG-Luc was more obvious than that in inhibiting its migration. (*P<0.05, **P<0.01, ***P<0.001, Student's test, n=3)

[0022] Fig. 9The effect of a combination of small molecule compounds on cell proliferation is shown. (A) A line graph showing the changes in the OD measurement values ​​of U251 cells at 450nm within 6 days of administration as determined by the CCK8 experiment. The control group continued to grow, but the growth rate slowed down after 2 days. The OD values ​​of the small molecule and temozolomide treatment groups increased in the first two days, but gradually decreased after 2 days, and the effect of inhibiting cell proliferation began to appear. The inhibitory effect was the strongest in the DLC79 group, followed by the DLC7 combination and the temozolomide group. (B) A line graph showing the changes in the OD measurement values ​​of U-87MG-Luc cells at 450nm within 6 days of administration as determined by the CCK8 experiment. The control group and the temozolomide treatment group continued to grow within 6 days, while the OD values ​​of the two small molecule compound treatment groups changed very little. This shows that both small molecule compound combinations can inhibit the proliferation of U-87MG-Luc cells. (**P<0.01, ****P<0.0001, one-way analysis of variance using Tukey's multiple comparisons, n=3)

[0023] Fig.10 The effect of a combination of small molecule compounds on the cloning ability of glioma cells is shown. (A) Representative images of U251 cell cloning. The number of U251 cell clones decreased after small molecule treatment. (B) Representative images of U-87MG-Luc cell cloning. The number of U-87MG-Luc cell clones decreased after small molecule treatment. (C) Quantitative results of the U251 cell cloning experiment. The DLC79 group had the best inhibitory effect on the cloning ability of U251 cells, followed by DLC7 and 50μM temozolomide (Temo). (*P<0.05, **P<0.01, ****P<0.0001, one-way ANOVA with Tukey's multiple comparisons, n=3) (D) Quantitative results of the U-87MG-Luc cell cloning experiment. The cloning ability of U-87MG-Luc cells decreased after 4 days of DLC79 treatment. (*P<0.05, Student's test, n=3)

[0024] Fig.11 The effect of a combination of small molecule compounds on the tumorigenicity of glioma cells in vivo is shown. (A) Schematic diagram of the subcutaneous tumor formation experiment in nude mice. 1.2×10 6U-87MG-Luc cells treated with 0.5% DMSO or a combination of small molecule compounds for 6 days were allowed to form tumors subcutaneously to observe the tumor-forming ability of the cells. (B) Representative images of in vivo fluorescence imaging of nude mice on day 12 (left: DMSO-treated group; right: small molecule compound-treated group). The fluorescence intensity of the tumor in the control group was higher than that in the small molecule compound-treated group. (*P<0.05, two-way ANOVA with Sidak's multiple comparisons, n=4) (C) Quantitative results of fluorescence intensity of in vivo imaging of nude mice. On day 2, there was little difference in the fluorescence intensity of the tumors in the two groups. As the tumor formation time increased, the fluorescence intensity of the tumors in the two groups gradually increased, and the difference in fluorescence intensity between the two groups also increased. During the 12 days of tumor bearing, the fluorescence intensity of the tumor in the control group was always higher than that in the small molecule compound-treated group. (D) Images of the size of the tumor masses in each group when the samples were collected on day 12 (left: small molecule compound-treated group; right: DMSO-treated group). The tumor masses in the control group were larger than those in the small molecule compound-treated group when the samples were collected. (E) Quantitative results of the weight of tumor masses in each group after sampling. The tumor weight of the control group was greater than that of the small molecule compound treatment group at the time of sampling. (*P<0.05, Student's test, n=4) DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. If it is not specifically pointed out, the reagents, culture media, experimental instruments, and biomaterials used in the present invention are all conventional reagents, culture media, experimental instruments, and biomaterials known to those skilled in the art.

[0026] 1. Reagents and Materials

[0027] The common reagents are shown in Table 1.

[0028] Table 1: List of common reagents

[0029] Reagents company Part Number PBS powder Boster Biological AR0030 95% saline Jiangxi Kelun Pharmaceutical none Paraformaldehyde (PFA) LEAGENE DF0135 Triton x-100 Solarbio T8200 Tween-20 Solarbio T8220 BSA powder Sigma WXBC7017V Trizol Thermo Fisher 15596018 Chloroform Guangzhou Chemical Reagent Factory none Isopropyl alcohol Guangzhou Chemical Reagent Factory none Anhydrous ethanol Guangzhou Chemical Reagent Factory none Mounting medium Sigma 10981-100ML Crystal violet Blue Sky C0121 D-Luciferin potassium salt Abmole M8873 NaOH Sigma 71690 <![CDATA[NaH2PO4]]> Aladin C1525004 <![CDATA[Na2HPO4·12H2O]]> Aladin J1811180 DAPI Electron Microscopy Sciences none Tribromoethanol Sigma T48402 tert-Amyl alcohol Sigma 152463

[0030] The components and concentrations of glioma cell culture medium are shown in Table 2.

[0031] Table 2: Glioma cell culture medium components and concentrations

[0032] name company Part Number concentration DMEM Medium Gibco C11995500BT - FBS Gibco 1099-141C 10％

[0033] The components and concentrations of the small molecule compound combination induction medium are shown in Table 3.

[0034] Table 3: Composition and concentration of small molecule compound combination induction medium

[0035] name company Part Number concentration DMEM / F12 Medium Gibco C11330500BT - B27supplement Gibco 17504-044 0.4％ N2 supplement Gibco 17502-048 0.8％ SB431542 Selleck S1067 2.5μM DAPT Selleck S2215 2.5μM CHIR99021 Selleck S1263 1.5μM LDN193189 Selleck S7507 0.25-1μM Ibet-151 Selleck S278003 1μM Ibet-762 Selleck S718905 1μM Isx9 Selleck S791401 5-20μM

[0036] The components and concentrations of the glioma cell neural differentiation medium are shown in Table 4.

[0037] Table 4: Components and concentrations of glioma cell neural differentiation medium

[0038] name company Part Number concentration DMEM / F12 Medium Gibco C11330500BT - B27supplement Gibco 17504-044 0.4％ N2 supplement Gibco 17502-048 0.8％ FBS Gibco 1099-141C 0.5％ Penicillin & Streptomycin Gibco 15140122 0.2％ Vc Sigma 134032 5 μg / mL NT-3 Peprotech 45003 10 ng / mL IGF1 Peprotech 10011 20 ng / mL BDNF Peprotech 45002 10 ng / mL GDNF Peprotech 45010 10 ng / mL FqV Sigma 50919 10μM Uridine Cayman chemical 58-96-8 10μM Forskolin Selleck S244907 10μM Dorsomorphine Cayman chemical 86605643 1μM

[0039] Other reagents required for cell culture are listed in Table 5.

[0040] Table 5: Other reagents required for cell culture

[0041] name company Part Number DPBS Gibco C14190500BT 0.05%Trypsin-EDTA Gibco 25300-054 Matrigel BD 356234 Gelatin from porcine skin Sigma 48722-500G-F

[0042] The antibodies used are shown in Table 6.

[0043] Table 6: Antibodies

[0044] Antibody Species company Part Number concentration GFAP Rat Invitrogen 13-0300 1:1000 DCX Rabbit Abcam Ab18723 1:2000 Tuj1 Mouse Sigma T8660 1:1000 MAP2 Chicken Abcam Ab5392 1:2000 GABA Rabbit Sigma A2052 1:1000 Mash1 Rabbit Abcam ab211327 1:1000 Neurog2 Rabbit Invitrogen PA5-78556 1:1000 NeuroD1 Rabbit Abcam ab205300 1:1000 vGlut1 Rabbit SYSY 135302 1:1000 Ki67 Rat Invitrogen 14-5698-82 1:1000 SOX2 Rabbit Millipore Ab5603 1:1000 SOX9 Rabbit Millipore Ab5535 1:1000 S100B Mouse Sigma S2532 1:1000 PDGFRα Rabbit Santa Cruz Sc-338 1:500 NeuN Rabbit Millipore ABN78 1:1000 HuNu Mouse Millipore MAB1281 1:10000 Alexa Fluor 488 Donkey anti mouse Life A21202 1:1000 Alexa Fluor 488 Goat anti chicken Life A11039 1:1000 Alexa Fluor 555 Donkey anti rabbit Life A31572 1:1000 Alexa Fluor 546 Goat anti chicken Life A11040 1:1000 Alexa Fluor 647 Donkey anti mouse Life A31571 1:1000 Alexa Fluor 647 Goat anti rat Life A21247 1:1000

[0045] 2. Conventional reagent configuration

[0046] 2.1 PBS buffer (0.01M)

[0047] Pour a pack of PBS powder into a 2L beaker, add 1900ml of ultrapure water and place the large beaker on a magnetic stirrer. After the powder in the beaker is completely dissolved, add ultrapure water to make the solution 2L, and transfer it to a bottle for storage.

[0048] 2.2 1.25% tribromoethanol solution

[0049] Weigh 1.25g of tribromoethanol powder and pour it into a 100ml glass bottle wrapped in tin foil, add 2.5ml of tert-amyl alcohol and 90ml of deionized water, place the glass bottle on a magnetic stirrer and stir overnight to fully dissolve it. The next day, dilute to 100ml with deionized water. Filter and sterilize the prepared solution with a 0.22um microporous filter in a clean bench. Store the liquid in a 4 degree refrigerator away from light.

[0050] 2.3D-Luciferase Potassium Salt

[0051] Accurately weigh 30 mg of D-luciferase potassium salt and dilute it with 2 ml of sterile DPBS. Prepare it as soon as possible and store the excess solution in a -20℃ refrigerator.

[0052] 2.4 0.1% Gelatin

[0053] Weigh 0.2g of gelatin particles into a small beaker, add 200ml of DPBS and place in an autoclave at 121℃ for 30 minutes to swell and sterilize the gelatin. Mix the liquid and filter it with a 0.22μm filter membrane in a clean bench and transfer it into a sterile bottle and store it in a 4℃ refrigerator.

[0054] 3. Experimental Methods

[0055] 3.1 Culture of human glioma cell lines

[0056] The human U251 glioblastoma cell line was purchased from Wuhan Pronocell Life Science Co., Ltd., and the human U-87MG-Luc cell line was donated by Suzhou Shenxixingsheng Biopharmaceutical Co., Ltd. Both glioma cell lines were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum.

[0057] The cells were subcultured when they reached 90% confluence in the culture dish. The culture medium was aspirated, 1 mL of DPBS was added along the wall of the culture dish, and the culture dish was gently shaken to wash away the cell debris and residual culture medium. 1 mL of 0.05% Trypsin was added to the dish and digested in a 37°C incubator for 2 minutes. When most of the cells shrank and became round, and the intercellular gaps became larger, and the remaining cells fell off by tapping the culture dish, complete culture medium containing 10% serum was immediately added to terminate the digestion. After gently blowing and mixing with a 1 mL pipette, the cell suspension was aspirated into a centrifuge tube and centrifuged at 1200 rpm for 3 minutes. The supernatant was discarded, and the cells were resuspended in 1 mL of culture medium. After subculturing at 1:5, the cells were placed in a 37°C CO2 incubator for culture.

[0058] 3.2 Chemical small molecule drug combination induced glioma cells

[0059] After the round coverslips were placed in a 24-well plate in advance, 300 μL of 0.1% gelatin was added to each well and incubated overnight for coating. The gelatin was aspirated the next day, and U251 cells were seeded at a density of 10,000 cells per slide after washing with DPBS. When the cell confluence reached about 80%, the culture medium was replaced with an induction medium containing small molecules or 0.5% DMSO solvent for 6 days of induction, and then replaced with a neuronal differentiation medium for longer culture.

[0060] 3.3 Cell immunofluorescence staining and imaging

[0061] Aspirate the culture medium in the culture plate, wash it with PBS once, and then add 300μL of paraformaldehyde solution to each well for 15 minutes. After removing the waste paraformaldehyde solution, place the cells on a shaker and wash them three times with PBS, each time for 5 minutes. Add blocking solution (0.2% Triton X-100 and 3% BSA in 0.01M PBS) and incubate at room temperature for 45 minutes. Add the primary antibody to the blocking solution according to the dilution ratio. After the blocking is completed, aspirate the blocking solution, add 250μL of primary antibody to each well and incubate it in a 4℃ refrigerator overnight.

[0062] The next day, take the cells out of the refrigerator, and after rewarming, wash them three times on a shaker with 0.2% PBST (0.2% Tween-20 added to 0.01M PBS), 5 minutes each time. After washing, add the secondary antibody diluted in 0.01M PBS and incubate at room temperature in the dark for 1 hour. After the incubation, aspirate the secondary antibody and wash it three times with PBS, 5 minutes each time. Use pointed tweezers to carefully pick out the glass slide and dry it. Add a drop of sealing agent on the slide, and gently turn the slide upside down on the slide, taking care not to produce bubbles that affect subsequent imaging. After sealing, store the sample in a 4°C refrigerator in the dark.

[0063] Immunofluorescence staining results were imaged using a Zeiss fluorescence upright microscope and confocal microscope.

[0064] 3.4 Cell proliferation ability assay

[0065] U251 or U-87MG-Luc cells were resuspended in DMEM medium containing 10% FBS, evenly plated in a 96-well plate at a density of 4000 cells per well, 100 μL per well, and placed in a 37°C incubator for 24 hours until the cells adhered. The culture medium was replaced with a culture medium containing DLC79, 100 μM Temozolomide or a 0.05% DMSO control group for the required time, with 5 replicates per group. During the test, 10 μL of cck8 reagent was added to each well and transferred to a 37°C incubator containing 5% CO2 for incubation for 1 hour. The absorbance at 450 nm was measured using an ELISA reader and plotted.

[0066] 3.5 Cell transwell assay

[0067] Cell invasion experiment: Dilute the matrix gel with pre-cooled DMEM medium at a ratio of 1:8, and add 60 μL of matrix gel to each transwell chamber. The process is carried out on ice. After adding, transfer the well plate to a carbon dioxide incubator and incubate at 37°C for 3 hours to wait for gelation. After the bottom layer is gelled, aspirate the upper layer of liquid and add 100 μL of DMEM medium to each well and transfer to an incubator at 37°C for another 30 minutes to hydrate the matrix gel. Digest the cells treated with small molecules or DMSO for 4 days and gently centrifuge to harvest the cells. Resuspend the cells in the culture medium and adjust the U251 cell density to 6.0*10 5 cells / mL, U-87MG-Luc cell density is 3.0*10 5 Then 100 μL of cell suspension was transferred to the upper chamber, and 500 μL of culture medium containing 5% FBS was added to the lower chamber, and U251 cells were incubated for 24 hours, and U-87MG-Luc cells were incubated for 16 hours.

[0068] Cell migration assay: After digestion of cells treated with small molecules or DMSO for 4 days, the cells were gently centrifuged and harvested. The cells were resuspended in culture medium and the U251 cell density was adjusted to 2.0*10 5 cells / mL, U-87MG-Luc cell density is 2.0*10 5 Then 100 μL of cell suspension was transferred to the upper chamber, and 500 μL of culture medium containing 5% FBS was added to the lower chamber, and U251 cells were incubated for 24 hours, and U-87MG-Luc cells were incubated for 12 hours.

[0069] After the cells migrated to the lower chamber membrane, 500 μL of 4% paraformaldehyde solution was added to each well to fix the cells for 15 minutes, washed once with DPBS, stained with 500 μL of crystal violet solution for 15 minutes, and washed 3 times with DPBS. The cells that did not penetrate the upper chamber membrane were wiped off, and the membrane was carefully cut off after drying. After sealing with neutral gum, the film was taken with a Zeiss microscope under white light. The number of cells was counted and statistical graphs were made using ImageJ1.54 version software, and each experiment was repeated three times.

[0070] 3.6 Clone formation assay

[0071] U251 or U-87MG-Luc cells were made into a single cell suspension using DMEM medium containing 10% FBS, and the cells were evenly plated in a 6-well plate at a density of 300 cells per well, and placed in a 37°C incubator for 24 hours to allow the cells to adhere to the wall. The culture medium was replaced with a culture medium containing half a dose of DLC79 or a 0.05% DMSO control group for 6 days, and then the full amount was replaced with 0.05% DMSO for 4 days, with 3 replicates for each group. After the formation of visible clones in the well plate, the culture medium was discarded, and 4% paraformaldehyde solution was added after rinsing with PBS once for 15 minutes. After fixation, the 4% paraformaldehyde solution was discarded, and after rinsing with PBS twice, a crystal violet solution was added to stain in the dark for 10 minutes. After the cells were stained purple, the crystal violet solution was recovered, washed with pure water 3 times to remove the excess crystal violet solution, and then dried and photographed, and the number of clones formed was counted and plotted.

[0072] 3.7 Establishment of nude mouse subcutaneous glioma model

[0073] The nude mice used in the present invention were purchased from Guangdong Jicui Pharmaceutical. The experimental animals were kept in an environment with a temperature of about 25°C, a relative humidity of 40%-60%, and a 12-hour light / dark cycle, while ensuring that the animals had clean and sufficient water and food. The animal experiments involved in the present invention were approved by the Animal Experiment Ethics Committee of Jinan University, with the ethics approval number 20221227-05. During the experiment, the principles of animal welfare were followed to minimize the suffering of the experimental animals. The long diameter of the subcutaneous tumor did not exceed 15mm, and the volume of a single tumor did not exceed 2000mm 3 At the same time, experiments should be arranged scientifically and rationally to minimize the use of experimental animals.

[0074] First, the U-87MG-Luc cells in the small molecule compound treatment group and the control group were digested with 0.05% trypsin for 2 minutes, the cells were collected and centrifuged; after removing the supernatant, the cells were resuspended with DPBS and counted. The cell concentration was adjusted to 1.2*10 7 cells / mL and place the cell suspension on ice until use.

[0075] After anesthetizing 5-7 week old female nude mice with isoflurane, place them on the operating table, and disinfect the skin near the back and hind legs of the nude mice with iodine. Use a 1ml syringe to draw 100μL of cell suspension, insert the needle on the dorsal side, and slowly push it subcutaneously. The needle should be inserted about 1cm subcutaneously to prevent leakage. Move the needle left and right before injection. If no resistance is felt, it proves that it is subcutaneous and can be injected. Otherwise, it may be in the skin or muscle. After the injection, an obvious bulge can be seen. After staying for a while, rotate and slowly withdraw the needle to prevent the cell suspension from flowing out of the needle port. After the injection is completed. Put the nude mice back into the cage and continue to raise them and continue to observe their condition.

[0076] 3.8 In vivo fluorescence imaging of nude mice

[0077] D-luciferase potassium salt solution was injected into the peritoneal cavity of nude mice at a dose of 150 mg / kg. After 7 minutes, tribromoethanol (250 mg / kg) was injected into the peritoneal cavity to anesthetize the nude mice. After the nude mice were anesthetized for 3 minutes, they were placed in a small animal in vivo imager for imaging with an exposure time of 1 second. Imaging was performed every 3-4 days until the sample was obtained. The software measured the fluorescence intensity of the tumor area and statistically plotted it.

[0078] 3.9 Tissue sampling

[0079] The nude mice were anesthetized by intraperitoneal injection of tribromoethanol (250 mg / kg), and the samples were collected by perfusion after the pinch reflex disappeared. After the nude mice were fully anesthetized, their limbs were fixed on the operating table, and the upper end of the abdominal cavity was cut horizontally to open the chest cavity and expose the heart. A flat needle was inserted into the left ventricle and entered the aorta. The needle was fixed and physiological saline was perfused at a flow rate of 5-10 ml / min. The perfusion was stopped after the liver turned white. The skin of the nude mice was cut open, and the subcutaneous tumor was carefully separated and photographed, and its size was measured and weighed and recorded.

[0080] 3.10 Data processing and statistical analysis

[0081] The immunohistochemical images were analyzed and processed using ZEN 2.3 blue edition, and the data were analyzed using Graphpad Prism 8.2.1 software. Student's test and one-way ANOVA were used to check whether there were significant differences between the data. When P < 0.05, the difference between the groups was considered significant.

[0082] 3.11 Experimental Design

[0083] First, small molecules that can induce the production of more DCX+ cells were screened on the U251 human glioma cell line, and then unnecessary small molecules were removed and the concentrations of each component in the combination were adjusted. After the combination was determined, the inhibitory effect of the combination treatment on the tumor characteristics of the glioma cell line was verified through experiments such as transwell experiments, CCK8 experiments, clone formation experiments, and tumor formation experiments in nude mice. The schematic diagram of the experimental technical route is shown in the figure. Figure 1 shown.

[0084] 4. Experimental Results

[0085] 4.1 Protein expression in glioma cell lines

[0086] To characterize the two human glioma cell lines used in the experiment, immunofluorescence staining was performed to determine the characteristics of U251 and U-87MG-Luc without small molecule medium induction.

[0087] The U251 and U-87MG-Luc cell lines used in the staining results identification experiment are both human cell lines, expressing the human nuclear marker Human Nuclei. In order to determine the proliferation characteristics of the two cell lines, the expression of KI67, a conventional indicator for measuring brain tumor proliferation, and SOX2, a protein involved in glioma stemness and chemotherapy resistance, were detected. The results showed that both cell lines expressed high levels of KI67, indicating that they have strong proliferation ability; however, the expression of SOX2 was not detected in the U-87MG-Luc cell line ( Figure 3 , A, E).

[0088] Next, three proteins that are often highly expressed in tumor tissues (platelet-derived growth factor receptor α (PDGFRA), nestin, and vimentin) were selected for staining. Nestin and vimentin proteins are expressed in the early stages of neural differentiation in normal brain tissue, but are no longer expressed after maturation; however, in tumor tissue, the expression of nestin and vimentin is higher than that in normal tissue, and is positively correlated with the invasiveness and infiltration of tumor cells. The expression of nestin and vimentin proteins was detected in both cell lines, but the expression of PDGFRA was not found ( Figure 3 , B, F), indicating that both cell lines have strong migration and invasion abilities.

[0089] Among the glial cell-related genes, both cell lines expressed S100B, but only the U251 cell line expressed GFAP and SOX9, while the U-87MG-Luc cell line did not express them ( Figure 3 , C, G).

[0090] Among neuronal markers, both cell lines weakly expressed TUBB3 (Tuj1) and microtubule-associated protein 2 (MAP2), and did not express immature neuron marker DCX, mature neuron marker NeuN, and GABAergic neuron marker GABA. The U251 cell line expressed vesicular glutamate transporter receptor (vGlut1) protein, while the U-87MG-Luc did not; however, unlike neurons expressing it in glutamatergic nerve terminals, the vGlut1 expression in U251 was concentrated in the cell nucleus ( Figure 3 , D, H).

[0091] The differences in protein expression between the two glioma cell lines show the different differentiation characteristics of the two glioma cell lines.

[0092] 4.2 Screening of small molecule compound combinations to transdifferentiate glioma cells into neurons

[0093] A combination of four small molecules, SB431542, DAPT, CHIR99021 and LDN193189, was selected as the basis (the combination was called "core"), and some other small molecules related to the transdifferentiation effect of transcription factors and used for transdifferentiation or tumor inhibition were added to conduct preliminary screening of glioma transdifferentiation small molecule compound combinations (Table 7).

[0094] Table 7: Screened small molecules and their pathways of action

[0095]

[0096] First, the glioma cells were induced with a combination of small molecule compounds for 4 days, and then the medium was replaced with neural differentiation medium for 4 days to make the transformed cells more mature. After that, the cells were fixed with PFA and immunofluorescence staining was performed ( Figure 4 , A). It was found that after 8 days of culture, a certain number of DCX+ cells could be induced. DCX is a microtubule-associated protein that is highly expressed in immature neurons and neuroblasts during development. This suggests that these cells have a strong tendency to continue to develop into neurons, while no DCX-positive cells were found in the control group ( Figure 4 , B). Existing studies have shown that overexpression of DCX also has the effect of inhibiting the proliferation and invasion of glioma cells. Therefore, the efficiency of inducing DCX+ cells on day 8 was used as a standard for preliminary small molecule screening. Statistics show that the small molecules Isx9, Skl2001, dihydromyricetin, Ibet-151, Ibet-762 and VPA can effectively improve the conversion efficiency of U251 cells induced by the core combination, and the conversion efficiency increased from 0.9% to 3.3%, 2.8%, 2.5%, 5.8% and 2.2% respectively.

[0097] It was found that among the small molecules that can improve transdifferentiation efficiency, Skl2001 and dihydromyricetin are agonists of the Wnt pathway, just like the small molecule CHIR99021 in the core combination; Ibet-151 and Ibet-762 are BET protein inhibitors, and like VPA, they have the function of regulating cell epigenetics; Isx9 has a neural induction effect, which can cause neural progenitor cells to differentiate into neurons. Considering that Skl2001 and dihydromyricetin have relatively repeated functions with CHIR99021, and the high concentration of VPA may not be conducive to drug development, the subsequent experimental screening is more focused on the three small molecules of Ibet-151, Ibet-762 and Isx9.

[0098] In order to verify whether the four small molecules in the core combination are all necessary for U251 cell transdifferentiation, we subtracted any small molecule in SLCD from the combination of SLCD+1μM Ibet-151 with higher transdifferentiation efficiency, and counted the number and proportion of DCX-positive cells on day 8 after induction of the small molecule combination for 4 days and continued to be cultured in differentiation medium for 4 days. The statistical results showed that more DCX-positive cells could be generated after removing the small molecule SB431542, and its efficiency increased from 2.3% to 5.4% ( Figure 5 , B); however, after removing LDN193189, almost no DCX-positive cells were generated ( Figure 5 , A). This suggests that LDN193189 may be a key small molecule for transdifferentiation of U251 cells into neuron-like cells, while the presence of small molecule SB431542 may be detrimental to inducing transdifferentiation of U251 cells. Therefore, in the next experiment, small molecule SB431542 was removed from the combination and the concentration of LDN193189 was increased.

[0099] According to the above experimental results, and considering the drugability and toxicity of drug combinations to normal cells, we tried to select several small molecules with low and similar concentrations for combination and adjusted their concentrations. The final combination selected was DLC79 (i.e. 5μM DAPT+1μM LDN193189+1.5μM CHIR99021+1μM Ibet-762+5μM Isx9). Subsequent attempts to extend the induction time of this combination found that on the sixth day of induction with the small molecule compound combination, the cultured cells were observed to be in good condition and the induced DCX-positive cells were not co-labeled with the glial cell marker GFAP and the tumor marker vimentin ( Figure 6 , A). Considering that longer induction time may be beneficial to further improve the transdifferentiation efficiency and the maturity of neuronal cells, the treatment time of the small molecule compound combination was extended to 6 days.

[0100] After 6 days of induction with the combination of small molecule compounds, the culture medium was replaced with neural differentiation medium to continue differentiation for 4 days to make the induced neuron-like cells more mature. It was found that more DCX-positive neuron-like cells could be transdifferentiated on the 10th day, and these cells were co-labeled with neuronal markers Tuj1 and MAP2 ( Figure 6 , B).

[0101] After the optimal combination was determined, the statistical results showed that the number of DCX-positive cells induced by small molecules in U251 cells was significantly higher than that of the original core combination: the number of DCX-positive cells per square millimeter increased from 5 to 95.5 after 6 days of small molecule induction; after 4 days of culture in differentiation medium, the number of DCX-positive cells per square millimeter increased from the original 19.8 to 108.25 ( Figure 6 , C).

[0102] In order to verify the effect of each small molecule in the selected combination again, we subtracted a single small molecule from the DLC79 combination and statistically analyzed the transdifferentiation of DCX+ cells. It was found that removing the BMP pathway inhibitor LDN193189 and the BET protein inhibitor Ibet-762 from the small molecules in the combination would cause the combination to lose its induction effect on U251 cell transdifferentiation ( Figure 7 , A, B). Removing Isx9 may not inhibit the proliferation of tumor cells. The total number of cells after treatment is nearly twice that of the optimal combination, which is not much different from the number of cells in the control group. At the same time, the small molecule LDN193189 also has a significant inhibitory effect on the number of U251 cells ( Figure 7 , C). Removing DAPT or CHIR99021 also significantly reduced the number of DCX-positive cells and reduced the induction efficiency to 4.9% and 2.2%, respectively ( Figure 7 , D). Therefore, it was finally determined that the optimal combination DLC79 was used, and the transdifferentiation efficiency of this combination could reach about 7% ( Figure 7 , D). Considering both the transdifferentiation effect and the inhibitory effect on glioma cells, the small molecules in this combination are indispensable.

[0103] Figure 2 A schematic diagram of the transdifferentiation process of glioma cells is shown. In the figure, glioma cells are induced into neuron-like cells under the combined action of five small molecule compounds of DLC79 (DAPT, LDN193189, CHIR99021, Ibet-762, Isx9).

[0104] 4.3 Inhibitory effects of small molecule combinations on cell tumor characteristics

[0105] 4.3.1 Inhibition of glioma cell migration and invasion by small molecule compound combinations

[0106] The transwell assay was used to determine the inhibitory effects of the small molecule compound combination on the migration and invasion of two glioma cell lines, U251 and U-87MG-Luc. After small molecule treatment, the morphology of U251 cells changed significantly, from a relatively flat fibroblast-like morphology to a more elongated ( Figure 8, A); U-87MG-Luc cells themselves have a relatively elongated epithelial cell-like morphology, and the changes are not obvious ( Figure 8 , C). During cell migration, the cells in the upper chamber change their cytoskeleton morphology under the chemotactic effect of the serum in the lower chamber and migrate through the small holes on the membrane to the lower chamber; in the cell invasion experiment, due to the presence of matrix gel, the cells need to secrete matrix metalloproteinases to degrade the matrix gel before entering the lower chamber. Statistical results show that the treatment of the small molecule compound combination inhibits the migration and invasion of the two glioma cell lines. The number of U251 cells that migrated to the lower chamber in each field of view decreased from 156.6 in the control group to 120.5; the number of cells that migrated decreased from 209.5 to 103.8 cells ( Figure 8 , B). For U-87MG-Luc cells, the number of cells that migrated to the lower chamber in each field of view decreased from 139.3 in the control group to 87.7; the number of cells that migrated decreased from 194.4 to 82.6 cells ( Figure 8 , D).

[0107] It was found that the inhibitory effect of small molecule induction on the invasion ability of the two glioma cells was more significant than that on migration. It is speculated that this may be because the neuron-like cells obtained by transdifferentiation at this time are not mature and are still in an early stage of development. The DCX expressed in these cells induced by a combination of small molecule compounds is a microtubule-associated protein that is often expressed in neurons during migration and plays a vital role in the normal migration of human neurons. It is believed that the cells induced by small molecules at this time may be similar to those neurons in the early stages of neurogenesis, and still have a certain migration ability but do not have the ability to secrete matrix metalloproteinases to degrade matrix glue, resulting in a more obvious inhibitory effect on cell invasion ability than migration after small molecule treatment.

[0108] 4.3.2 Inhibition of glioma cell proliferation by small molecule combination

[0109] In order to determine the effect of small molecule compound combinations on the proliferation ability of glioma cells, the CCK8 assay was used to detect the process of 1-6 days of small molecule treatment.

[0110] The experimental results showed that in the U251 cell line, within 48 hours, the cells in both the experimental group and the control group continued to proliferate and the proliferation rate was faster, and the number of cells in the experimental group was less than that in the control group. After 48 hours, the number of cells in the experimental group decreased day by day, while the control group maintained a slow proliferation. This shows that the combination of small molecule compounds has a significant inhibitory effect on cell proliferation in the U251 glioma cell line, and the inhibitory effect is stronger than that of the positive control group of 100μM temozolomide ( Fig. 9, A). It is speculated that the slowdown in the proliferation rate of U251 cells in the control group at 48 hours may be related to contact inhibition between cells, which is consistent with the result that the inhibitory effect of MKI67 gene expression on U251 cells was most obvious on the second day when the small molecule compound combination was treated.

[0111] In the U-87MG-Luc cell line, cells in the control group and the 100 μM temozolomide group continued to proliferate within 6 days, and the OD value increased rapidly from 0.6 on the first day to 1.6 on the sixth day; while the cells in the small molecule compound treatment group did not proliferate further, and the OD value remained around 0.5 ( Fig. 9 , B), showing that the small molecule compound combination also has an inhibitory effect on the proliferation of the U-87MG-Luc cell line. Compared with the U251 control group, the U-87MG-Luc cells treated with 0.5% DMSO have maintained a strong proliferation ability, which may be because the U-87MG-Luc cells are more malignant and more difficult to treat.

[0112] 4.3.3 Inhibition of glioma cell clone formation ability by small molecule compound combination

[0113] The clone formation test can be used to indicate the ability of a single cell to survive independently. This method was chosen to test the ability of U251 and U-87MG-Luc, which were diluted to a single cell density, to proliferate again in a six-well plate and form a clone after being treated with a half-concentration of DLC79 combination. The results showed that the clone formation ability of U251 cells was inhibited by using half-concentration of DLC79, DLC7 and the positive control group temozolomide compared with the DMSO group ( Fig.10 , A). At the same time, statistics show that the average number of clones formed after DLC79 combination treatment was 25, less than a quarter of the control group (134); and the inhibitory effect of removing Isx9 from the small molecule compound combination on the cell clone formation ability was equivalent to 50μM temozolomide, and the number of clones formed by them was 58.7 and 55, respectively, which was about half of the number of clones formed by the control group ( Fig.10 , C).

[0114] Similarly, in the U-87MG-Luc cell line, it was found that the combination of small molecule compounds at half the concentration could also inhibit its ability to form clones ( Fig.10 , B), the average number of clones formed was 25.7. The average number of clones formed in the control group was 45, and the number of clones formed by tumor cells after small molecule treatment was about half of that in the control group ( Fig.10 , D).

[0115] 4.4 Evaluation of the tumorigenicity of tumor cells induced by small molecule compound combinations in vivo

[0116] In order to detect whether the small molecule compound combination treatment has an inhibitory effect on tumor cells in vivo, the U-87MG-Luc cells treated with the small molecule compound combination or 0.5% DMSO for 6 days were injected subcutaneously into the right and left hind legs of nude mice to form tumors ( Fig.11 , A). The cells carry the Luci gene and can express firefly luciferase. After modeling, the substrate D-luciferin potassium salt is injected into the abdominal cavity of nude mice to produce yellow-green autofluorescence and is detected by a live imaging device. The activity of the subcutaneous tumor in nude mice can be determined by measuring the fluorescence intensity of the tumor area; the tumor mass size can be measured to determine the tumor-forming ability of U-87MG-Luc cells.

[0117] On the 6th day after cell transplantation, the subcutaneous tumors of nude mice reached a size visible to the naked eye. The fluorescence intensity of cells treated with small molecules in the tumors of nude mice was lower than that of the control group ( Fig.11 , B). According to the statistical chart of the changes of the fluorescence intensity of the tumors in the two groups of small animal live imaging with the number of days of tumor formation, it can be observed that with the increase of the time of subcutaneous cell transplantation, the growth rate of the tumor also accelerates; with the extension of the tumor formation time, the difference in fluorescence intensity between the two groups is gradually increasing until the sampling. On the 12th day, the average fluorescence intensity of the tumor in the small molecule compound treatment group was 4.3*10 9 [p / s] was significantly lower than that of the control group 9.9*10 9 [p / s]( Fig.11 , C). The size of the tumor mass after sampling ( Fig.11 , D) and average weight were also slightly smaller than those of the control group ( Fig.11 , E, the average tumor weight of the control group was 0.38g, and the tumor weight of the small molecule compound treatment group was 0.20g). These results can prove that the ability of glioma cells to form tumors in nude mice is reduced after being treated with a combination of small molecule compounds, but the growth of tumors cannot be completely stopped.

[0118] In summary, the small molecule compound combination used in the present invention can induce the transdifferentiation of human glioma cell lines into neuronal cells in vitro, and has an inhibitory effect on the proliferation, migration, invasion and tumorigenicity of tumors.

Claims

1. A small molecule compound combination, characterized in that Including DAPT, LDN193189, CHIR99021, Ibet-762 and Isx9.

2. Use of the small molecule compound combination according to claim 1 in the treatment of glioma.

3. Use of the small molecule compound combination according to claim 1 in the preparation of a drug for treating glioma.

4. Use of the small molecule compound combination according to claim 1 in the preparation of a drug for inducing transdifferentiation of glioma cells into neuron-like cells.

5. Use of the small molecule compound combination according to claim 1 in inducing transdifferentiation of glioma cells into neuron-like cells.

6. A method for inducing glioma cells to transdifferentiate into neuron-like cells, characterized in that The method comprises administering the small molecule compound combination according to claim 1.

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