Application of equivalent component group combined protein sensitizer of Sangui decoction in tumor drug resistance

Abstract

The invention discloses application of an equivalent component group combined protein sensitizer of Sangui decoction in tumor drug resistance, and belongs to the technical field of tumor treatment. The equivalent component group of the three-component scutellaria baicalensis decoction comprises baicalin and sophocarpidine, and the protein sensitizer is StcE mucoprotease. The StcE mucoprotease has rapid and remarkable growth inhibition and apoptosis promotion activity on cancer cells, the dosage of chemotherapeutic drugs in tumor cells can be greatly reduced under the extremely low and non-toxic dosage, baicalin and sophocarpidine have various pharmacological effects of resisting inflammation, resisting oxidation, resisting tumors and the like and have huge potential in the aspect of drug resistance reversion, and the application has a good application prospect. The two are combined for reversing tumor drug resistance, so that the killing effect of chemical drugs can be promoted, the use amount of the chemical drugs is effectively reduced, and the killing effect of the chemical drugs on cells is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor treatment, and particularly relates to the application of the equivalent component group of Sanwu Huangqin Decoction combined with a protein sensitizer in tumor drug resistance. Background Art

[0002] Sanwu Huangqin Decoction is a classic traditional Chinese medicine prescription composed of three Chinese herbs, namely Scutellaria baicalensis, Sophora flavescens, and Rehmannia glutinosa, and is mainly used for treating symptoms such as fever and restlessness in the limbs and headache caused by blood deficiency and yin deficiency after childbirth and the invasion of wind pathogens into the interior and transformation into heat. In addition, it is also clinically used for treating cancer pain and fever, restlessness in spring and summer, erythematous limb pain and other diseases. In traditional Chinese medicine theory, the occurrence and development of tumors are related to "deficiency of healthy qi and excess of pathogenic factors", that is, deficiency of healthy qi and accumulation of pathogenic factors. Drug resistance can be regarded as a further pathological change after the stubbornness of pathogenic factors and the damage of healthy qi. Scutellaria baicalensis and Sophora flavescens in Sanwu Huangqin Decoction clear heat and dry dampness, detoxify and dispel pathogenic factors, and inhibit the growth of tumors to achieve the effect of strengthening healthy qi and dispelling pathogenic factors. Moreover, Scutellaria baicalensis is cold in nature and Sophora flavescens is bitter and cold in nature. The combination of the two can clear heat and detoxify, and this effect of regulating yin and yang helps to restore the balance of the internal environment of the body, thereby counteracting tumor drug resistance. In addition, Sophora flavescens has the effects of expelling wind and dampness, dredging collaterals and relieving pain, and can dredge the meridians and improve the state of qi and blood stasis in the local area of the tumor, thereby enhancing the penetration and efficacy of drugs.

[0003] In addition, modern research shows that in drug-resistant tumor cells, baicalin can down-regulate the expression of drug resistance-related genes (such as c-met), thereby enhancing the sensitivity of chemotherapy drugs, and matrine can reverse the drug resistance of tumor cells through multiple mechanisms (such as inhibiting the expression of drug resistance proteins). Baicalin and matrine can act synergistically through multiple targets, and respectively play roles from multiple angles by regulating cell signaling pathways, immune functions, and drug resistance gene expressions.

[0004] StcE mucinase is an engineered bacterial protease that can selectively degrade mucins on the surface of cancer cells. Mucins are usually overexpressed in tumor cells, and they promote tumor growth and drug resistance by regulating cell signaling pathways, cell-cell interactions, and immune escape. By degrading these mucins, StcE disrupts the protective mechanism of tumor cells, making them more sensitive to chemotherapy drugs or immunotherapy. Summary of the Invention

[0005] Object of the Invention: To solve the problems existing in the prior art, the object of the present invention is to provide the application of the equivalent component group of Sanwu Huangqin Decoction combined with a protein sensitizer in tumor drug resistance.

[0006] Technical Solution: The present invention provides the application of the equivalent component group of Sanwu Huangqin Decoction combined with a protein sensitizer in tumor drug resistance.

[0007] Furthermore, the equivalent component group of Sanwu Huangqin Decoction includes baicalin and matrine.

[0008] Furthermore, the molar concentration ratio of baicalin to matrine is 2.5 - 5:1, preferably 3:1.

[0009] Furthermore, the protein sensitizer is mucinase, preferably StcE mucinase.

[0010] Furthermore, the molar concentration ratio of StcE mucinase to baicalin is 50 - 250 nM: 200 μM.

[0011] Furthermore, the addition concentration of StcE mucinase is preferably 200 - 250 nM.

[0012] Furthermore, the tumors include breast cancer and melanoma.

[0013] Furthermore, the chemotherapeutic drugs for tumors include doxorubicin and paclitaxel.

[0014] Furthermore, the concentration of the chemotherapeutic drug is 100 nM - 10 μM.

[0015] Principle of the invention: In the present invention, the protein sensitizer is StcE mucinase, which is naturally derived from enterohemorrhagic Escherichia coli and prepared by the engineering bacterium Escherichia coli BL21, using the vector PET - 28b, and the protein sequence refers to US6872559B. StcE mucinase is an engineered bacterial protease that can selectively degrade mucins on the surface of cancer cells. Mucins are usually overexpressed on the surface of tumor cells, and they promote tumor growth and drug resistance by regulating cell signaling pathways, cell - cell interactions, and immune escape. StcE degrades these mucins, disrupting the protective mechanism of tumor cells and making them more sensitive to chemotherapeutic drugs or immunotherapy. At extremely low and non - toxic usage doses, it can significantly reduce the dosage of chemotherapeutic drugs in tumor cells. In the present invention, according to the special role of mucinase in cell signaling pathways and its effect on multidrug resistance reversal, when combined with the chemotherapeutic drug doxorubicin, it is found that StcE mucinase can effectively promote the killing effect of chemical drugs, effectively reduce the usage amount of chemical drugs, and increase the killing effect of chemical drugs on cells, and it is an effective sensitizer for tumor chemotherapy.

[0016] Baicalin and matrine are the main anti - tumor active components in the Three - Herb Scutellaria Decoction. In traditional Chinese medicine theory, Scutellaria baicalensis has the effects of clearing away heat and dampness, purging fire and detoxifying, and is commonly used to treat febrile diseases, damp - heat jaundice and other diseases. Sophora flavescens has the functions of clearing away heat and dampness, killing insects and promoting diuresis. Modern pharmacological research shows that baicalin and matrine have various pharmacological effects such as anti - inflammation, anti - oxidation, and anti - tumor, and show great potential in multidrug resistance reversal.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following remarkable effects: On the one hand, StcE mucinase selectively degrades the mucin on the surface of cancer cells, enhances the uptake and absorption of drugs by cancer cells, and strengthens the efficacy of baicalin and matrine in the equivalent component group of the Three-Herb Scutellaria Decoction; on the other hand, the use of StcE mucinase reduces the drug resistance of cancer cells and makes cancer cells more sensitive to chemotherapy drugs. Brief Description of the Drawings

[0018] Figure 1 It is the SDS-PAGE diagram of the successful expression of mucinase;

[0019] Figure 2 It is the SDS-PAGE diagram of the successful purification of mucinase;

[0020] Figure 3 It is the diagram of the successful cleavage of C1-INH enzyme by mucinase;

[0021] Figure 4 It is the test diagram of mucinase on breast cancer drug-resistant cells;

[0022] Figure 5 It is the cytotoxicity test diagram of low-concentration DOX (100 nM) combined with each component;

[0023] Figure 6 It is the cytotoxicity test diagram of medium-concentration DOX (1 μM) combined with each component;

[0024] Figure 7 It is the cytotoxicity test diagram of high-concentration DOX (10 μM) combined with each component;

[0025] Figure 8 It is the morphological change diagram of 4T1 / DOX drug-resistant strain after the combined administration of StcE and DOX. Detailed Embodiments

[0026] The present invention will be further described in detail below in conjunction with the embodiments and the drawings.

[0027] Embodiment 1: The application of the equivalent component group of the Three-Herb Scutellaria Decoction combined with a protein sensitizer in tumor drug resistance provided in this embodiment is as follows:

[0028] 1.1 Experimental Materials and Instruments

[0029] 1.1.1 Experimental Materials

[0030] (1) Compounds

[0031] Doxorubicin hydrochloride (adriamycin), with a purity of ≥98%, was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Before use, the compound powder was dissolved in dimethyl sulfoxide to prepare a stock solution with a concentration of 10 mM, and the above stock solution was stored in a -20°C refrigerator. When used, it was diluted to the required concentration with the corresponding cell culture medium.

[0032] Baicalin and matrine, with a purity of ≥98%, were purchased from Bidepharm.

[0033] (2) Cell line

[0034] The doxorubicin-resistant strain of mouse breast cancer cells (4T1 / DOX) was selected and donated by the research group of Professor Liurunhui at East China University of Science and Technology.

[0035] (3) Cell culture reagents

[0036] RPMI 1640 medium, high-glucose DMEM medium, fetal bovine serum (FBS), and penicillin-streptomycin mixture (100×) were all purchased from Servicebio.

[0037] (4) Kits for detecting related indicators

[0038] CellCountingKit-8 (CCK8) was purchased from APExBIO, stored in a -20°C refrigerator, and dissolved at room temperature in advance before use.

[0039] 1.1.2 Experimental instruments

[0040] VS-1300L-U clean bench (Suzhou Antai Air Technology Co., Ltd.), carbon dioxide incubator (Thermo Scientific), Invitrogen hemocytometer, inverted fluorescence microscope (Leica, Germany), BioTek microplate reader, BIO-RAD gel imager, BIO-RAD electrophoresis tank.

[0041] 1.2 Experimental methods

[0042] 1.2.1 Expression of mucinase

[0043] BL21 Escherichia coli cells were transformed with the plasmid confirmed by sequencing and grown in LB liquid medium (37°C and 200 rpm) until the optical density reached 0.4 - 0.8. Protein expression was induced with 0.3 mM isopropyl-β-D-1-thiogalactopyranoside, and the culture was incubated overnight at 20°C and 200 rpm.

[0044] 1.2.2 Purification of mucinase

[0045] Centrifuge the bacteria at 6000 r / min for 10 minutes and lyse them using the lysis buffer in 20 mM HEPES (pH 7.5) and 500 mM NaCl. Clarify the lysate by centrifuging at 11000 r / min for 10 minutes and filter it through a low-protein-binding 0.22 μm polyethersulfone membrane vacuum filtration bottle. Add the lysate to 3 - 4 ml of Ni-NTA agarose per liter of bacterial culture, wash with 200 ml of 20 mM HEPES (pH 7.5), 500 mM NaCl, and 20 mM imidazole, and elute with 20 ml of 20 mM HEPES (pH 7.5), 500 mM NaCl, and 250 mM imidazole. Exchange the buffer of the purified protein into cold PBS.

[0046] 1.2.3 SDS-PAGE Gel Electrophoresis

[0047] Separation gel preparation: In the fume hood, prepare a 12% separation gel, calculate the required reagent amounts (acrylamide and N,N'-methylenebisacrylamide mixture, Tris-HCl buffer, 10% SDS, distilled water, etc.), add them to a beaker in sequence, and finally add 10% ammonium persulfate and TEMED. Mix quickly and evenly and immediately pour it into the gel-making mold of the vertical electrophoresis tank, add an appropriate amount of water to cap it, and let it stand at room temperature to solidify. When a distinct interface appears at the junction of the gel and water, it indicates that the separation gel has solidified. Pour off the water capping layer.

[0048] Concentrating gel preparation: Similarly, in the fume hood, prepare a 5% concentrating gel solution, calculate the required reagent amounts (acrylamide and N,N'-methylenebisacrylamide mixture, Tris-HCl buffer, 10% SDS, distilled water, etc.), add them to a beaker in sequence, and finally add 10% ammonium persulfate and TEMED. Mix quickly and evenly and immediately cover the solidified separation gel, insert the comb, and let it stand at room temperature to solidify. The solidification time is generally 15 - 30 minutes. After solidification, carefully pull out the comb to make the gel pores clear and not broken.

[0049] Protein denaturation treatment. Take 20 μL of the protein sample, add an equal volume of 5× loading buffer, mix well, heat in a 100 °C water bath for 5 - 10 minutes to fully denature the protein, then quickly cool to room temperature, mix well, and then it can be loaded.

[0050] Fix the prepared gel in the vertical electrophoresis tank, add 1× SDS-PAGE electrophoresis buffer to ensure that the gel is completely immersed in the buffer. Use a micropipette to add the treated sample and the standard molecular weight marker to the gel wells respectively, at a voltage of 120 V for 2 h. Carefully take out the electrophoresed gel, put it into the Coomassie Brilliant Blue R-250 staining solution, stain at room temperature for 20 min, decolorize overnight, and take a photo using a BIO-RAD gel imager.

[0051] 1.2.4 Activity assay of mucoproteinase

[0052] C1-INH and StcE were placed in a 96-well plate and incubated overnight at 37 °C. The reaction was terminated by adding EDTA (50 μl, 50 mM solution), and the mixture was transferred to a 96-well enzyme-linked immunosorbent assay (ELISA) plate and incubated overnight at 4 °C. The wells were emptied and blocked with a PBS solution containing 1% bovine serum albumin (BSA) for 1 hour at room temperature. The wells were emptied and washed 3 times with PBS containing 0.1% Tween 20 for 1 minute each time. The wells were probed with the 3C7 monoclonal antibody that does not recognize StcE-cleaved C1-INH. The wells were washed again and incubated with goat anti-mouse antibody conjugated to horseradish peroxidase (HRP) (1:3000) for 30 minutes. Detection was performed using a microplate reader at a wavelength of 450 nm.

[0053] 1.2.5 Cytotoxicity assay

[0054] Cell viability assay: The CCK8 kit was used to detect the viability of cells, and the OD value at 450 nM was measured with a microplate reader. Cell viability (%) = [(experimental wells - blank wells) / (control wells - blank wells)] × 100%; Graphpad Prism 8.0 software was used for plotting.

[0055] Low concentration group: By the CCK8 method. Specifically, 4T1 / DOX cells (8×10 3 -1.2×10 4 cells / well) were seeded into a 96-well plate and incubated overnight for 12 h. The supernatant was discarded, and then cell treatments with final concentrations of 100 nM DOX + 200 μM baicalin, 100 nM DOX + 66.7 μM matrine, 100 nM DOX + 200 μM baicalin and 66.7 μM matrine, 100 nM DOX, 100 nM DOX + 200 nM mucoproteinase, 100 nM DOX + 200 μM baicalin and 66.7 μM matrine + 200 nM mucoproteinase were performed for 72 h. The supernatant was discarded, CCK8 reagent was added, and incubation was continued for 0.5 - 1.5 h. The absorbance at 450 nm was measured with a microplate reader. Cell viability was expressed as a percentage of the control (untreated cells).

[0056] Medium concentration group: By the CCK8 method. Specifically, 4T1 / DOX cells (8×10 3 -1.2×10 4Cells (at a density of 8×10

[0057] High concentration group: By CCK8 method. Specifically, 4T1 / DOX cells (8×10 3 -1.2×10 4 cells / well) were seeded into 96-well plates and incubated overnight for 12 h. The supernatant was discarded, and then solutions with final concentrations of 1 μM DOX + 200 μM baicalin, 1 μM DOX + 66.7 μM matrine, 1 μM DOX + 200 μM baicalin and 66.7 μM matrine, 1 μM DOX, 1 μM DOX + 200 nM mucinase, 1 μM DOX + 200 μM baicalin and 66.7 μM matrine + 200 nM mucinase were prepared to treat the cells for 72 h. The supernatant was discarded, CCK8 reagent was added, and the cells were incubated for another 0.5 - 1.5 h. The absorbance at 450 nm was measured using a microplate reader. Cell viability was expressed as a percentage of the control (untreated cells).

[0058] 1.2.6 Cell morphology

[0059] 4T1 / DOX cells (8×10 3 -1.2×10 4 cells / well) were seeded into 96-well plates and incubated overnight for 12 h. The supernatant was discarded, and the cells were treated with 400 nM mucinase and 100 nM DOX for 72 h, and then observed and photographed under a microscope.

[0060] The test results are shown in Figures 1 - 8 .

[0061] As Figure 1 shown, StcE is a protein with a molecular weight of approximately 98 KD, and SDS-PAGE indicates the successful expression of this protein.

[0062] As Figure 2 shown, StcE is a protein with a molecular weight of approximately 98 KD, and SDS-PAGE indicates the successful purification of this protein.

[0063] As Figure 3As shown, C1-INH is the substrate of StcE mucinase. The Elisa kit indicates that the expressed enzyme can cleave C1-INH, suggesting that the enzyme is active.

[0064] As Figure 4 shown, StcE mucinase can significantly reduce the IC50 of breast cancer drug-resistant cells, change their drug resistance level, and enhance the drug uptake of cells.

[0065] As Figures 5 - 6 shown, at low (100 nM DOX usage) and medium (1 μM DOX usage) concentrations of chemotherapeutic drugs, the combination group of StcE mucinase and the equivalent component group of Sanwu Huangqin Decoction showed strong killing effects on cancer cells.

[0066] As Figure 7 shown, at high (10 μM DOX usage) concentration of chemotherapeutic drugs, all groups showed strong killing effects on cancer cells. As Figure 8 shown, obvious changes occurred in the cell morphology before and after administration of StcE mucinase.

Claims

1. Application of the equivalent component group of the Three-Herb Scutellaria Decoction combined with a protein sensitizer in tumor drug resistance.

2. The application according to claim 1, wherein The equivalent component group of the Three-Herb Scutellaria Decoction includes baicalin and matrine.

3. The application according to claim 2, wherein The molar concentration ratio of baicalin to matrine is 2.5 - 5:

1.

4. The application according to claim 2, characterized in that, The protein sensitizer is mucinase.

5. The application according to claim 4, wherein The mucinase is StcE mucinase.

6. The application according to claim 5, wherein The molar concentration ratio of StcE mucinase to baicalin is 50 - 250 nM: 200 μM.

7. The application according to claim 6, characterized in that, The added concentration of StcE mucinase is 200 - 250 nM.

8. The application according to claim 1, characterized in that, The tumors include breast cancer and melanoma.

9. The application according to claim 8, wherein The chemotherapeutic drugs for the tumors include doxorubicin and paclitaxel.

10. The application according to claim 9, wherein The concentration of the chemotherapeutic drugs is 100 nM - 10 μM.

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