Mulberry leaf deoxynojirimycin pharmaceutical composition for treating cancer

Abstract

The invention belongs to the technical field of biological medicines, and discloses a mulberry leaf deoxynojirimycin pharmaceutical composition for treating cancers. The pharmaceutical composition comprises an IFITM3 protein monoclonal antibody and mulberry leaf deoxynojirimycin, the amino acid sequence of an antibody light chain variable region of the IFITM3 protein monoclonal antibody is as shown in SEQ ID NO.3, and the amino acid sequence of an antibody heavy chain variable region of the IFITM3 protein monoclonal antibody is as shown in SEQ ID NO.4. In the monoclonal antibody and mulberry leaf deoxynojirimycin composition provided by the invention, the IFITM3 protein monoclonal antibody can be produced in batches, can be specifically combined with human IFITM3 protein, and has the effect of reducing the activity of gastric cancer cells; the mulberry leaf deoxynojirimycin is simple in extraction step and contains few impurities; after the IFITM3 protein monoclonal antibody and the mulberry leaf deoxynojirimycin are used together, the growth and migration ability of gastric cancer cells can be effectively inhibited within a short time, the medicine can be positioned to gastric cancer tissues after entering a human body, the gastric cancer can be treated and relapse can be prevented by inhibiting self-renewal and migration of the cancer cells, and the medicine has a relatively good medical application prospect.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a mulberry leaf deoxynojirimycin pharmaceutical composition for treating cancer. Background Art

[0002] Gastric cancer is one of the most common malignant tumors of the digestive tract and ranks fourth among the most common malignant tumors in the world. The incidence of gastric cancer varies from place to place. Gastric cancer is a chronic proliferative disease with long-term multifactorial effects and multigene involvement. Systematic studies in molecular pathology over the past decade have revealed many molecular mechanisms in the occurrence and development of gastric cancer, involving many genetic and non-genetic changes, including oncogenes, tumor suppressor genes, cell cycle regulators, cell adhesion molecules, metastasis-related genes, changes in DNA repair genes, telomerase activation, gene instability, DNA methylation, etc., which can cause uncontrolled cell proliferation and lead to malignant transformation and metastasis. Since chemotherapy and radiotherapy are generally insensitive to gastric cancer and there is a lack of effective alternative treatment options, the development of new gastric cancer treatment methods and drugs has become a hot topic in basic and clinical research in recent years.

[0003] Human interferon-induced transmembrane proteins (IFITMs) are products of interferon-stimulated genes (ISGs), which are usually expressed by Immune microenvironment Interferon (IFN) induces a significant increase in expression. The human IFITM family includes five genes, namely IFITM1, IFITM3, IFITM3, IFITM5 and IFITM10, all located on chromosome 11. IFITMs have multiple functions, including controlling cell proliferation, regulating germ cell development, promoting homotypic cell adhesion, preventing viral infection and bone matrix mineralization and maturation. In recent years, researchers have found that some IFITMs play a role in cancer progression, such as IFITM3, which promotes cell proliferation, induces cell cycle arrest and subsequent p53-dependent cell apoptosis. IFITM3 in prostate cancer cells promotes tumor progression and bone metastasis by activating the TGFβ pathway; colorectal cancer cells with high expression of IFITM3 promote Treg chemotaxis in vitro and infiltration in vivo; IFITM3 phosphorylation leads to PI3K signal transduction amplification, which can promote the malignant transformation of B cells. Therefore, IFITM3 can be used as one of the potential targets for cancer treatment. Compared with traditional chemotherapy, radiotherapy and traditional Chinese medicine, IFITM3 monoclonal antibody therapy has more obvious effects, longer efficacy and fewer toxic side effects.

[0004] 1-Deoxynojirimycin (1-DNJ) is a piperidine polyhydroxy alkaloid with multiple biological activities such as hypoglycemic, antiviral, and anti-tumor metastasis. However, there are no reports on the combined treatment of gastric cancer with 1-DNJ and monoclonal antibodies. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, one of the objects of the present invention is to provide a mulberry leaf deoxynojirimycin pharmaceutical composition for treating cancer, which can target cancer cells overexpressing IFITM3 protein and inhibit the vitality, proliferation and migration of cancer cells.

[0006] The second object of the present invention is to provide the application of the mulberry leaf deoxynojirimycin pharmaceutical composition.

[0007] One of the purposes of the present invention is achieved by the following technical solution:

[0008] A deoxynojirimycin pharmaceutical composition for treating cancer comprises an IFITM3 protein monoclonal antibody and deoxynojirimycin.

[0009] Furthermore, the amino acid sequence of the light chain variable region of the IFITM3 protein monoclonal antibody is shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.4.

[0010] Furthermore, the method for preparing the monoclonal antibody of the IFITM3 protein comprises the following steps: constructing an expression vector comprising an IFITM3 protein encoding gene, transforming Escherichia coli, inducing expression of the IFITM3 protein, immunizing mice with the IFITM3 protein, isolating spleen cells, preparing, screening and cloning hybridoma cells, and isolating the monoclonal antibody from the hybridoma cell culture fluid.

[0011] Furthermore, the extraction method of mulberry leaf deoxynojirimycin is as follows: dehydrate and crush the mulberry leaves, add water for microwave extraction, then centrifuge to remove mulberry leaf residues, add ethanol to the aqueous solution, centrifuge to remove precipitation, and reduce pressure to dry the supernatant to obtain an intermediate product; dissolve the intermediate product in water again, remove impurities with an ultrafiltration membrane, and concentrate and dry to obtain a 1-deoxynojirimycin product.

[0012] Furthermore, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

[0013] The second object of the present invention is achieved by adopting the following technical solution:

[0014] The pharmaceutical composition is used in preparing medicine for treating cancer.

[0015] Furthermore, the cancer is gastric cancer.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] In the monoclonal antibody and deoxynojirimycin composition provided by the present invention, the IFITM3 protein monoclonal antibody can be mass-produced, can specifically bind to human IFITM3 protein, and has the effect of reducing the vitality of gastric cancer cells; the extraction step of deoxynojirimycin is simple and contains few impurities; after the IFITM3 protein monoclonal antibody and deoxynojirimycin are used together, the growth and migration ability of gastric cancer cells can be effectively inhibited in a short time; the drug can be located in gastric cancer tissue after entering the human body, and can treat gastric cancer and prevent recurrence by inhibiting the self-renewal and migration of cancer cells, and has good medical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 For specific identification of the monoclonal antibody against IFITM3 protein of the present invention;

[0019] Figure 2 IFITM3 protein monoclonal antibody reduces the viability of human gastric cancer SGC-7901 cells;

[0020] Figure 3 The monoclonal antibody against IFITM3 protein reduces the invasion ability of human gastric cancer SGC-7901 cells. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0022] Example 1

[0023] Preparation of IFITM3 protein monoclonal antibody:

[0024] (1) Preparation of IFITM3 protein antigen: Using normal gastric tissue cDNA as a template, specific primers were designed according to the coding nucleotide sequence of IFITM3 protein, and BamHI and SalI restriction sites were inserted into both ends of the primers:

[0025] Upstream primer: 5′-ggatccATGAATCACACTGTCCAAACCT-3′ (SEQ ID NO. 1)

[0026] Downstream primer: 5′-CTATCCATAGGCCTGGAAGATcaggtc-3′ (SEQ ID NO. 2)

[0027] The IFITM3 gene was amplified by PCR (PCR parameters: 95℃5min; 95℃30s, 55℃30s, 72℃60s, 40 cycles; 72℃10min), and then double-digested with BamHI and SalI and connected to the pGEX-4T-1 vector, chemically transformed into BL21(DE3) competent cells, and single clones were picked to extract plasmids for sequencing to verify the sequence. The positive bacteria with correct sequencing were selected and induced to express for 4 hours with 1mmol / LIPTG. After induction, the bacteria were collected, ultrasonically broken and centrifuged, and the supernatant and precipitate were subjected to SDS-PAGE electrophoresis respectively. The bands were cut for MALDI-TOF / TOF identification, confirming that the expression product was IFITM3. The IFITM3 protein concentration was adjusted to 2mg / mL with PBS solution.

[0028] (2) Animal immunization: IFITM3 protein was used as an antigen. After emulsification with an equal volume of Freund's complete adjuvant, it was injected into the abdominal cavity and back of healthy female BALB / c mice aged 8 to 12 weeks at multiple points, 100 μL per mouse, and a total of 10 mice were injected. Two weeks later, IFITM3 protein antigen was emulsified with an equal volume of Freund's incomplete adjuvant, and Balb / c female mice were immunized by multiple subcutaneous injections for booster immunization, which was repeated 3 times. Blood was collected from the mouse eye sockets 7 days after the third booster immunization, and the serum was collected by centrifugation. The mouse serum antibody titer was detected by indirect ELISA. Mice with a titer >1:30,000 were selected for shock immunization. 100 μg protein + saline to 100 μL were injected into the tail vein for shock immunization, and fusion was allowed to occur 3 days later.

[0029] (3) Cell fusion and cloning: Mice with serum titers that meet the standard are killed by cervical dislocation and immersed in 75% alcohol for 5 minutes for disinfection. The mouse abdomen is cut open to lift and separate the spleen, and the spleen is punctured and ground with a sterile syringe to release spleen cells. The ground cell suspension is filtered through a 70μm filter, centrifuged, resuspended in HAT medium, counted, and evenly distributed into a 96-well plate. Spleen cells and SP2 / 0 cells are mixed in a ratio of 10:1, fused with PEG, and then the effect of PEG is terminated with incomplete culture medium. The fused cells are resuspended in HAT medium and plated in a 96-well plate at 37°C and 5% CO 2 After 10 days of HAT screening culture, the supernatant was taken and the monoclonal hybridoma cell line with the highest titer (titer of 1:256000) of monoclonal antibody that stably secreted anti-IFITM3 protein was screened by indirect non-competitive ELISA method for subcloning, and the subcloning was repeated 3 times.

[0030] (4) Preparation and purification of monoclonal antibodies: Mouse hybridoma cell lines were cultured in Gibco serum-free medium at 37°C and 5% carbon dioxide. When the cell density reached 1×106 / mL, centrifuge and collect the culture supernatant, and the IFITM3 protein monoclonal antibody is present in the culture supernatant. Pass the supernatant of mouse hybridoma cell culture through the ProteinG affinity column (according to the instructions), and wash the column with 10 times the column volume of PBS (pH7.4). Elute the IFITM3 protein monoclonal antibody on the chromatography column with 0.1M glycine (pH2.5). Adjust the antibody concentration to 5mg / mL for later use.

[0031] Total RNA was extracted from hybridoma cell lines using Qiagen's RNeasy kit, and mRNA was reverse transcribed into a cDNA library of IFITM3 protein monoclonal antibody using Invitrogen's SuperScript III First-Strand kit. The light and heavy chains of the antibody were cloned using Takara's SMARTer RACE cDNA Amplification kit, followed by DNA sequencing and amino acid polypeptide sequence translation, to obtain the light chain variable region sequence of the monoclonal antibody as shown in SEQ ID NO.3, and the heavy chain variable region sequence as shown in SEQ ID NO.4.

[0032] DITCSQSPVNASASVGETVTLNCRASSVANQSNYFLAWYQTKQGRTCNALVYNASTR APGVMTRFSATGSGTDFTLKITTVNPEDYANFYCHFYNNTPPTFGGGTKIDLK(SEQ ID NO.3)

[0033] EVKLDESGAELVRACATVKPTCKANGNTFSGAQMLHWAKQKCAQGLEFLGVINDYYI DQKSYNEVKGKVTVNSDKSSNNAYCDASGLTSEDSAIFWCTMMDYFFNGYWGQGTNVAV SS(SEQ ID NO.4)

[0034] Example 2

[0035] Specificity identification of monoclonal antibodies against IFITM3 protein:

[0036] The IFITM3 recombinant protein, BSA protein, growth factor EGF, and histone H3 prepared in step (1) of Example 1 were added to the reducing SDS-PAGE loading buffer, denatured at 100°C for 10 min, and 3 μL was taken for 125 g / L SDS-PAGE. The gel protein band was transferred to a PVDF membrane in a Bio-Rad electrotransfer system according to a conventional method. After blocking with 50 g / L skim milk powder blocking solution for 1 hour, the IFITM3 protein monoclonal antibody was added and incubated at room temperature for 2 hours to bind to the protein on the membrane. The membrane was rinsed with TBST to remove unbound antibodies and other impurities, and then HRP-goat anti-mouse IgG (1:5000 dilution) was added to further amplify the signal. After incubation at room temperature for 1 hour, the membrane was washed with TBST buffer, and the results were developed and observed to determine whether the monoclonal antibody has the ability to specifically recognize the IFITM3 protein.

[0037] The results are as follows Figure 1 As shown, the IFITM3 protein monoclonal antibody can only specifically bind to the IFITM3 protein, but has no obvious binding characteristics with the other proteins, indicating that the IFITM3 protein monoclonal antibody prepared by the present invention has good specificity.

[0038] Example 3

[0039] IFITM3 protein monoclonal antibody inhibits gastric cancer cell viability:

[0040] Human gastric cancer SGC-7901 cells in logarithmic growth phase (purchased from Guangzhou Yuanjing Biotechnology, catalog number YC-C055) were cultured at 3×10 4 100 μL of DMSO was added to each well and incubated for 4 hours. After 12 hours of culture with SGC-7901 cell-specific culture medium, IFITM3 protein monoclonal antibodies were added at concentrations of 0, 10 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. Five replicate wells were set up for each concentration group. After 72 hours of culture, the culture medium was aspirated, and 100 μL of DMEM culture medium containing MTT was added to each well for further incubation for 4 hours. The culture medium was discarded and 150 μL of DMSO was added to each well. After oscillation for 10 minutes, the absorbance of each well at a wavelength of 490 nm was measured with an enzyme-linked immunosorbent assay to calculate the cell viability. Cell viability = average absorbance value / absorbance value of the blank control group × 100%.

[0041] The results are as follows Figure 2 As shown, the IFITM3 protein monoclonal antibody of the present invention has the effect of reducing the viability of human gastric cancer SGC-7901 cells, thereby affecting cell proliferation.

[0042] Example 4

[0043] Preparation of mulberry leaf deoxynojirimycin:

[0044] Take 10kg of fresh mulberry leaves, wash them and place them on a bamboo rack in a ventilated environment at 22±1℃ for 12h, cut them into 3cm-4cm mulberry leaf strips by a strip cutter, and dry them at 45℃ to dehydrate about 75%. Pulverize the mulberry leaves to a diameter of 40-60 mesh, put them into a microwave extractor, operate the microwave extractor at a working frequency of 2300MHz, and continuously add 5Bv of water, the water temperature is 50℃, and stir and extract twice, each time for 20min. After the extraction is completed, centrifuge at 3000rpm to remove the mulberry leaf residue, add 1Bv of 92% ethanol to the aqueous solution obtained after centrifugation, stir for 10min, and obtain partial precipitation at this time. Put the solution containing the precipitation into a disc centrifuge and centrifuge it again at 8000rpm to remove the precipitation, and reduce the pressure to dry the supernatant to obtain the intermediate product and recover the ethanol. The intermediate product is dissolved in water again, and the macromolecular impurities are removed by a 20KDa ultrafiltration membrane, and then some small molecular impurities are removed by a nanofiltration membrane with a molecular weight cutoff of 300Da, and then concentrated by a reverse osmosis membrane, and finally the concentrated liquid product is dried to obtain the 1-deoxynojirimycin product.

[0045] Example 5

[0046] Monoclonal antibody combined with deoxynojirimycin from mulberry leaves inhibits the growth of gastric cancer cells:

[0047] Human gastric cancer SGC-7901 cells in the logarithmic growth phase were gently dispersed into single cells and diluted to 5×10 4 / mL, 100 μL / well was inoculated into a 96-well plate (i.e. 5×10 3 After culturing with SGC-7901 cell-specific culture medium for 12 hours, equal volumes of complete culture medium containing different concentrations of IFITM3 protein monoclonal antibody (0, 50 μg / mL, 100 μg / mL, 200 μg / mL) and different concentrations of mori leaf deoxynojirimycin (0, 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 8.0 μg / mL) were added. Four parallel wells were set for each drug concentration in each group of cells and placed at 37°C, 5% CO 2 The cells were cultured in an incubator, and the OD450 value was detected by CCK-8 method after 7 days to calculate the inhibition rate. Inhibition rate = [1-(Ax-A) / (Ao-A)] × 100%, where Ax is the OD450 value of the drug-added well, A is the OD450 value of the negative control well, and Ao is the OD450 value of the cell-free well.

[0048] Table 1 Inhibition rate of gastric cancer cells treated with different concentrations of IFITM3 protein monoclonal antibody and mulberry leaf deoxynojirimycin

[0049]

[0050] The results are shown in Table 1. After 7 days of treatment with IFITM3 protein monoclonal antibody, mulberry leaf deoxynojirimycin, IFITM3 monoclonal antibody and dry mulberry leaf deoxynojirimycin, the cell growth was significantly reduced. The effect of inhibiting cancer cell growth increased with the increase of the concentration of IFITM3 monoclonal antibody and dry mulberry leaf deoxynojirimycin. The combination of 100 μg / mL IFITM3 monoclonal antibody and 4.0 μg / mL dry mulberry leaf deoxynojirimycin had a better inhibitory effect on SGC-7901 cells and the highest cost-effectiveness.

[0051] Example 6

[0052] Monoclonal antibody combined with deoxynojirimycin from mulberry leaves inhibits gastric cancer cell invasion:

[0053] Gently blow the gastric cancer SGC-7901 cells cultured in serum-free suspension into single cells and dilute them with serum-free medium. Dilute Matrigel to 1 μg / μL with serum-free medium on ice, add 100 μL of diluted gel to the upper chamber of Transwell, solidify at room temperature for 1 hour, discard the diluent, and wash twice with medium. Add 600 μL of 10% FBS medium to the lower chamber of Transwell, and add 100 μL of cell suspension (1×10 4 The culture medium contained 100 μg / mL IFITM3 protein monoclonal antibody and different concentrations of mori leaf deoxynojirimycin (0, 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 8.0 μg / mL) without serum, which were respectively called experimental groups 1 to 5. The serum-free culture medium without any additives was the negative control group. Each group had 3 replicate wells and was incubated at 37°C and 5% CO 2 Continue to culture for 24 hours. Remove the upper chamber, wipe off the cells in the upper chamber with a dry cotton ball, and fix with methanol at room temperature for 10 minutes. Wash twice with PBS, cut the membrane with a blade, seal the membrane with 1μg / mL DAPI and 50% glycerol in PBS, and take pictures and count with a fluorescence microscope.

[0054] The results are as follows Figure 3 As shown, the combination of IFITM3 protein monoclonal antibody, IFITM3 monoclonal antibody and dry mulberry leaf deoxynojirimycin significantly inhibited the invasive ability of gastric cancer cells, and the number of invading gastric cancer cells within 24 hours was significantly reduced, which was statistically different from the negative control group.

[0055] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A mulberry leaf deoxynojirimycin pharmaceutical composition for treating cancer, characterized in that: The pharmaceutical composition comprises IFITM3 protein monoclonal antibody and deoxynojirimycin.

2. The pharmaceutical composition according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the IFITM3 protein monoclonal antibody is shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

4.

3. The pharmaceutical composition according to claim 1, characterized in that The method for preparing the monoclonal antibody of the IFITM3 protein comprises the following steps: constructing an expression vector containing an IFITM3 protein encoding gene, transforming Escherichia coli, inducing expression of the IFITM3 protein, immunizing mice with the IFITM3 protein, isolating spleen cells, preparing, screening and cloning hybridoma cells, and isolating the monoclonal antibody from the hybridoma cell culture fluid.

4. The pharmaceutical composition according to claim 1, characterized in that The extraction method of mulberry leaf deoxynojirimycin comprises the following steps: dehydrating and crushing the mulberry leaves, adding water for microwave extraction, centrifuging to remove mulberry leaf residues, adding ethanol to the aqueous solution, centrifuging to remove precipitation, and drying the supernatant under reduced pressure to obtain an intermediate product; dissolving the intermediate product in water again, removing impurities through an ultrafiltration membrane, and concentrating and drying to obtain a 1-deoxynojirimycin product.

5. The pharmaceutical composition according to claim 1, characterized in that One or more pharmaceutically acceptable carriers or excipients are also included.

6. Use of the pharmaceutical composition according to any one of claims 1 to 5, characterized in that: Used in preparing medicines for treating cancer.

7. The use of the pharmaceutical composition according to claim 7, characterized in that: The cancer is gastric cancer.

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