CN110664821A

CN110664821A - Application of panaxadiol in preparing medicine for inhibiting expression of PD-L1 and tumor cell proliferation protein

Info

Publication number: CN110664821A
Application number: CN201911086155.0A
Authority: CN
Inventors: 金学军; 马娟; 王喆
Original assignee: Yanbian University
Current assignee: Yanbian University
Priority date: 2019-11-08
Filing date: 2019-11-08
Publication date: 2020-01-10

Abstract

The invention provides application of panaxadiol in preparation of a medicine for inhibiting expression of PD-L1 protein and tumor cell proliferation protein, and belongs to the technical field of natural small molecule medicine application, wherein the panaxadiol can quantitatively inhibit expression of PD-L1 protein, RNA transcription level of PD-L1 and PD-L1 positive cell rate; can also inhibit the expression of hypoxia-induced tumor proliferation-related proteins Cyclin D1, c-Myc and VEGF in a dosage manner; reducing the number of EdU positive cells quantitatively, and inhibiting the proliferation of tumor cells; the activity of T cells in a co-culture system can be obviously improved; effectively inhibiting the volume of a nude mouse transplanted tumor; therefore, the panaxadiol has a remarkable effect as a medicine for inhibiting the colon cancer.

Description

Application of panaxadiol in preparing medicine for inhibiting expression of PD-L1 and tumor cell proliferation protein

Technical Field

The invention belongs to the technical field of natural small molecule drug application, and particularly relates to application of panaxadiol in preparation of drugs for inhibiting expression of PD-L1 protein and tumor cell proliferation protein.

Background

The colon cancer belongs to one of nine common malignant tumors in China, and according to WHO statistics, the incidence rate of the colon cancer is the third common tumor. Most colon cancers progress from focal stealth injury to adenomas to malignancy, a process usually mediated by chronic inflammation resulting from local tissue damage. During the growth, invasion and metastasis of tumors, the tumors need to escape host immune surveillance, so that the body has no immune response to tumor antigens. Under the tumor microenvironment, the immune system is usually in a suppressed state, which not only is beneficial to the invasion and metastasis of colorectal cancer, but also promotes the drug resistance of the tumor, and reduces the killing effect of traditional chemoradiotherapy on tumor cells.

In this case, elimination of the immune escape microenvironment specific to the tumor area, and release of its suppression of the immune system, especially cytotoxic T lymphocytes, are also a new direction for the treatment of colon cancer.

Programmed cell death Ligand 1 (PD-L1) belongs to B7 family member, it is programmed cell death Ligand 1 (PD-1) natural binding Ligand, PD-L1 through binding with PD-1, negatively regulate T cell activity, inhibit the immune response that T cell produces. Under normal conditions, PD-1/PD-L1 signals can maintain the immune tolerance of the body and prevent excessive inflammatory reaction and autoimmune diseases; in the tumor environment, the signal path inhibits the anti-tumor immunity of the organism and reduces the number and functions of T cells in the body of a tumor patient. Blocking the interaction of PD-1/PD-L1 can restore the in vivo anti-tumor immune effect and T cell function, thereby inhibiting tumor growth and metastasis, and the PD-1/PD-L1 signal pathway has become a new target of immunotherapy in recent years.

Clinical sample studies show that PD-L1 is expressed in tumor tissues of colon cancer patients to different degrees, and the expression degree is associated with the tumor malignancy degree and the survival time of the patients, which indicates that PD-L1 is not only one of the prediction indexes for judging the prognosis of the colon cancer patients, and anti-PD-L1 treatment is also an effective means for treating the colon cancer.

At present, the development of the PD-1/PD-L1 inhibitor is mainly focused on the field of monoclonal antibodies, a plurality of medicaments such as nivolumab (nivolumab), pembrolizumab (pembrolizumab) and avilamab (avelumab) are available on the market, and the inhibitor is applied to a plurality of malignant tumors and has a remarkable treatment effect.

Disclosure of Invention

In view of the above, the present invention aims to provide the use of panaxadiol in the preparation of drugs for inhibiting the expression of PD-L1 protein and tumor cell proliferation protein; the panaxadiol can inhibit PD-L1 protein expression and tumor cell proliferation related protein expression in a dose-dependent manner, can enhance the activity of tumor-related cytotoxic T lymphocytes, and can reduce the proliferation capacity of tumor cells.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides application of panaxadiol in preparing a medicament for inhibiting expression of PD-L1 protein.

The invention provides application of panaxadiol in preparing a medicament for inhibiting PD-L1 gene transcription.

The invention provides application of panaxadiol in preparing a medicament for inhibiting tumor cell proliferation.

The invention provides application of panaxadiol in preparing a medicament for inhibiting expression of tumor cell proliferation protein.

Preferably, the tumor cell proliferation proteins include Cyclin D1, c-Myc and VEGF.

The invention provides application of panaxadiol in preparation of a medicament for enhancing the activity of cytotoxic T lymphocytes.

Preferably, the cytotoxic T lymphocyte is a cytotoxic T lymphocyte that is inhibited by the tumor PD-L1 pathway.

The invention provides application of panaxadiol in preparation of a medicament for inhibiting the volume of a transplanted tumor of a nude mouse.

The invention has the beneficial effects that: the natural small molecular compound panaxadiol provided by the invention can inhibit the expression level of PD-L1 protein, block a PD-1/PD-L1 signal channel, recover the activity of T cells and inhibit the proliferation of tumor cells; the panaxadiol provided by the invention can be used as a novel micromolecular drug to be applied to the treatment of colon cancer.

According to the description of the embodiment, the panaxadiol can inhibit the expression of PD-L1 protein in a dosage manner, and inhibit the RNA transcription level of PD-L1 in a dosage manner; can inhibit PD-L1 positive cell rate quantitatively. The panaxadiol provided by the invention can also inhibit the expression of hypoxia-induced tumor proliferation related proteins Cyclin D1, c-Myc and VEGF in a dose; the number of EdU positive cells is quantitatively reduced, the proliferation of tumor cells is inhibited, and the panaxadiol can obviously improve the activity of T cells in a co-culture system; the panaxadiol can effectively inhibit the volume of transplanted tumor of nude mice, and has no obvious influence on the body weight of nude mice.

Drawings

FIG. 1 shows that panaxadiol was demonstrated to inhibit PD-L1 protein expression in three human colon cancer cell lines in a dose-dependent manner using the Westernblot method;

FIG. 2 shows that panoxadiol inhibits RNA transcription of PD-L1 in three human colon cancer cell lines in a dose-dependent manner as demonstrated by PCR;

FIG. 3 shows that Panaxadiol was effective in inhibiting the positive rate of PD-L1 on the surface of tumor cells as confirmed by flow cytometry;

FIG. 4 shows that Panaxadiol was able to restore T cell viability inhibited by the tumor PD-L1 pathway as demonstrated by ELISA;

FIG. 5 shows the Westernblot method demonstrating that panaxadiol inhibits proliferation-associated protein expression in tumor cells in a dose-dependent manner;

FIG. 6 shows that the ability of Panaxadiol to inhibit tumor cell proliferation was confirmed by the EdU method;

FIG. 7 shows that panaxadiol can inhibit the proliferation of tumor cells by colony formation;

FIG. 8 shows that Panaxadiol inhibits the volume of nude mice transplanted tumors as demonstrated by nude mice transplanted tumors;

fig. 9 is a solid-beat image of the transplanted tumor volume in nude mice.

Detailed Description

The invention provides application of panaxadiol in preparing a medicament for inhibiting PD-L1 protein expression and application of panaxadiol in preparing a medicament for inhibiting PD-L1 gene transcription.

In the present invention, the panaxadiol can inhibit the transcription of PD-L1 gene and the expression of PD-L1 protein in a dosage manner; preferably can inhibit the transcription of PD-L1 gene and the expression of PD-L1 protein in three stable human colon cancer cell strains HCT116, SW620 and HT 29; and is capable of inhibiting the rate of PD-L1 positive cells in a dose; the panaxadiol can be used as a medicine for inhibiting the transcription of PD-L1 gene and the expression of PD-L1 protein in colon cancer, and further can be used as a medicine for inhibiting colon cancer. The invention has no special limitation on the dosage form and the auxiliary materials of the medicine, and the invention can adopt the conventional dosage form and auxiliary materials in the field.

The invention also provides the application of the panaxadiol in preparing the medicines for inhibiting the proliferation of tumor cells and inhibiting the expression of tumor cell proliferation proteins. The tumor cell proliferation proteins described in the present invention preferably include Cyclin D1, c-Myc and VEGF; the panaxadiol can inhibit the expression of the proteins Cyclin D1, c-Myc and VEGF quantitatively. The invention has no special limitation on the dosage form and the auxiliary materials of the medicine, and the invention can adopt the conventional dosage form and auxiliary materials in the field.

The invention also provides application of the panaxadiol in preparation of a medicament for enhancing the activity of cytotoxic T lymphocytes. In the present invention, the cytotoxic T lymphocyte is preferably a cytotoxic T lymphocyte that is inhibited by the tumor PD-L1 pathway; the panaxadiol can remarkably improve the activity of T cells in a tumor cell/T cell co-culture system. The invention has no special limitation on the dosage form and the auxiliary materials of the medicine, and the invention can adopt the conventional dosage form and auxiliary materials in the field.

The invention also provides application of panaxadiol in preparation of a medicament for inhibiting the volume of transplanted tumor of a nude mouse. In the invention, the volume of the transplanted tumor of the nude mouse can be effectively inhibited, and the body weight of the nude mouse is not obviously influenced. The invention has no special limitation on the dosage form and the auxiliary materials of the medicine, and the invention can adopt the conventional dosage form and auxiliary materials in the field.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Cell culture technique

Selecting three stable human colon cancer cell strains HCT116, SW620 and HT29 (purchased from American bacteria)Seed collection ATCC) and, after the cells entered the logarithmic growth phase, they were digested with 0.25% trypsin at 5 × 10 cells by number⁴The density was equally divided into 6 cell culture dishes of 6cm diameter, and 3mL of RPMI-1640 medium (containing inactivated 10% calf serum, 100U/L of penicillin-streptomycin) was added to each dish, and the mixture was placed in a constant temperature incubator (37 ℃ C., 5% CO)₂Normal oxygen concentration and saturation humidity), and an anoxic environment consisting of an anoxic incubator (1% O)₂，94％N₂，5％CO₂) And (6) simulating. After the cells are completely attached to the wall, the cells are divided into a control group and a drug concentration gradient group (the acting doses of panaxadiol are respectively 1 mu M, 3 mu M and 10 mu M), and the cells are cultured for 12 hours for standby.

Western blotting method

(1) Preparation of protein samples: cells from control and Panaxadiol-treated groups (applied at 1. mu.M, 3. mu.M, 10. mu.M), respectively, were harvested and total protein extracted by lysing the cells with total protein cell lysate (purchased from Thermo Fisher Scientific, USA).

(2) Protein denaturation: and (3) sampling 15 mu L of the protein, respectively adding the sampled protein into corresponding centrifuge tubes, adding 5 mu L of sample buffer solution, and boiling for 5 min.

(3) The boiled protein samples were run on SDS-PAGE gels.

(4) After the electrophoresis was stopped, the protein sample on the gel was transferred to a methanol-soaked PVDF membrane.

(5) The PVDF membrane is put into 5% skimmed milk and sealed for 1h at room temperature.

(6) Primary antibody hybridization: primary antibody was formulated with PBS-T20 at a ratio of 1:1000, incubated with the transferred PVDF membrane for 12 hours at 4 ℃ and then washed in PBS-T20 for 15 min.

(7) And (3) hybridization of a second antibody: secondary antibodies corresponding to the primary antibodies were formulated at a ratio of 1:2000 and incubated for 1 hour at room temperature, followed by 15min washes in PBS-T20.

(8) ECL luminescence kit (Millipore, usa) for detection.

PD-L1 antibodies were purchased from Novus Biologicals, Cyclin D1, c-Myc, VEGF and Tubulin antibodies, USA from Santa Cruz Biotechnology.

The experimental results are shown in fig. 1 and fig. 5, and the results show that the panaxadiol can quantitatively inhibit the expression of PD-L1 protein and can quantitatively inhibit the expression of hypoxia-induced tumor proliferation-related proteins Cyclin D1, c-Myc and VEGF.

Example 2

PCR method for determining influence of panaxadiol on PD-L1 gene transcription

(1) Total RNA extraction:

the cells of the control group and the Panaxadiol-treated group (action dose was 1. mu.M, 3. mu.M, 10. mu.M, respectively) were washed three times with PBS buffer, and 1ml of LTtizol was added thereto and repeatedly blown up to completely lyse them. Add 200. mu.L CCl₄The solution was separated and centrifuged (4 ℃ C.; 12000 rpm; 15 min). The upper layer was put into a new centrifuge tube, 500. mu.L of isopropanol was added, mixed well, and centrifuged (4 ℃ C.; 12000 rpm; 10 min). The supernatant was carefully discarded, and 1mL of 75% ethanol was added to the tube containing RNA, shaken, and centrifuged (4 ℃; 12000 rpm; 5 min). Discarding the supernatant, and placing a super clean bench for ventilation drying. After thorough drying, 30. mu.L of DEPC-DDW was added and allowed to dissolve completely.

(2) cDNA is synthesized by reverse transcription with RNA as a template.

The system is as follows:

RT-PCR reaction conditions:

42℃ 20min

99℃ 5min；

1 cycle

(3) And (3) PCR reaction: double-stranded DNA was synthesized from the cDNA.

The system is as follows:

PCR reaction procedure:

the PCR conditions are 30-40 cycles of 94 ℃, 58 ℃ and 30s, and 72 ℃ and 30s, and the PCR conditions are PD-L1: 38 cycles, primer sequence: 5'-CATCAGCTATTTGCGTGTGAGGA-3' (positive), 5'-AGCAATTC ATCTGTGCTTTCATGTC-3' (negative), GAPDH: 30 cycles, primer sequence: 5'-ACCACAGTCCATGC CATCAC-3' (positive), 5'-TCCACCCCCTGTTGCTGTA-3' (negative).

The results are shown in FIG. 2, and indicate that panaxadiol can inhibit RNA transcription level of PD-L1 quantitatively.

Example 3

Flow cytometry surface staining method for determining influence of panaxadiol on PD-L1 positive cell rate in human colon cancer cells

(1) Cells in the log phase of growth were taken and plated in 6cm dishes.

(2) When the cells grow to 50-80%, the cells are collected, mixed in PBS buffer, and collected in a 1.5mL centrifuge tube.

(3) Centrifuged (4 ℃ C.; 1000 rpm; 5min) and the cells resuspended in PBS buffer to a concentration of 1X 10⁶one/mL.

(4) mu.L of PD-L1 antibody (Novus Biologicals, USA) was added to each sample and incubated on ice for 1h in the absence of light.

(5) The mixture was centrifuged (4 ℃ C.; 1000 rpm; 5min) and the supernatant was discarded, and 1mL of PBS buffer was added again and mixed well.

(6) And (5) repeating.

(7) Adding fluorescent secondary antibody, and incubating for 15min on ice in dark.

(8) After centrifugation (4 ℃ C.; 1000 rpm; 5min), the cells were washed twice with PBS buffer, resuspended in 500. mu.L of PBS buffer, and analyzed by flow cytometry.

The results are shown in FIG. 3, which indicates that panaxadiol can inhibit PD-L1 positive cell rate quantitatively.

Example 4

ELISA (enzyme Linked immunosorbent assay) experiment for detecting influence of panaxadiol on T cell activity

The following procedures were performed in accordance with the instructions provided in ELISA kits, and TNF-. alpha.kits and IFN-. gamma.kits were purchased from Shanghai enzyme-linked Biotechnology Ltd.

(1) The kit was equilibrated at room temperature for half an hour.

(2) 50 μ L of each standard was added to the specific wells and incubated at room temperature for 1 h.

(3) Cleaning: the supernatant was discarded and washed five times with 300. mu.L of washing solution; after the final wash, the 96-well plate was inverted on filter paper to remove residual liquid.

(4) And adding 50 mu L of biotin antigen working solution into each hole, slightly shaking and uniformly mixing, and incubating for 1h at room temperature.

(5) Cleaning: the supernatant was discarded and washed five times with 300. mu.L of washing solution; after the final wash, the 96-well plate was inverted on filter paper to remove residual liquid.

(6) Add 50 μ L of avidin-HRP into each well, mix them by gentle shaking, incubate for 15min at room temperature in the dark.

(7) 50 mul of stop solution is added into each hole to stop the reaction, and the absorbance is detected at 450nm by a microplate reader within 30 min.

The results are shown in fig. 4, and indicate that panaxadiol can significantly improve T cell viability in the co-culture system.

Example 5

EdU cell proliferation assay

The following procedures were performed in accordance with the procedures of the instructions provided in the EdU cell proliferation kit (Ruibo Biotech, Inc., Guangzhou)

(1) Uniformly dividing cells in logarithmic growth phase into 96-well plates, adding drugs with different concentrations for treatment for 12h after the cells adhere to the walls

(2) Taking a cell culture medium according to a ratio of 3000: the EdU solution was diluted at a ratio of 1, and 100. mu.L of the diluted EdU solution was added to each well and incubated for 6 h.

(3) The medium was discarded and the cells were washed 2 times for 5min in PBS buffer.

(4) Add 50. mu.L of 4% paraformaldehyde to each well, incubate at room temperature for 30min, and discard paraformaldehyde.

(5) Add 50. mu.L of 2mg/mL glycine per well, incubate for 5min on a shaker, and discard glycine.

(6) Add 100. mu.L of PBS buffer to each well, wash the cells on a shaker for 5min, and discard the PBS buffer.

(7) mu.L of 0.5% Triton-100 was added to each well and the cells were permeabilized for 10min on a shaker.

(8) Add 10. mu.L of PBS buffer to each well, wash the cells on a shaker for 5min, and discard the PBS buffer.

(9) Add 100 μ LApollo staining solution to each well, incubate on shaker for 30min at room temperature in the dark, discard staining solution.

(10) The Hoechst33342 reaction solution was diluted with triple distilled water at a ratio of 100: 1. Add 100u LHoechst33342 reaction solution into each hole, incubate for 30min on the shaker at room temperature in the dark, discard the reaction solution.

(11) Cells were washed 3 times for 2min each time by adding 100. mu.L of PBS buffer to each well and photographed with a fluorescent microscope.

The results are shown in FIG. 6, which indicates that Panaxadiol can decrease the number of EdU positive cells and inhibit tumor cell proliferation.

Example 6

Cell colony formation assay

(1) Taking a trace of cells in the logarithmic growth phase, and uniformly distributing the cells into 6-well plates.

(2) After the cells are attached to the wall, the cells are observed under a microscope from time to time, when the cells show small colonies, the culture medium is replaced after the cells are treated for 12 hours by using panaxadiol (0 mu M, 1 mu M, 3 mu M and 10 mu M) with different concentrations, and the cell replacement culture medium is observed periodically.

(3) Crystal violet staining was performed when both colony number and size were significantly increased.

(4) The medium was discarded, washed three times with 1mL of PBS buffer, and fixed with 10% formaldehyde (500. mu.L) for 5min after washing. After fixation, the plates were stained with 500. mu.L of 1% crystal violet for 30s, blotted off and washed three times with 1mL of PBS buffer to remove excess dye.

(5) And (5) air-drying, photographing and storing.

The results are shown in FIG. 7, and show that the size and number of tumor cell colonies are inhibited as the concentration of panaxadiol is increased.

Example 7

Nude mouse transplantation tumor experiment

(1) 15 nude mice (purchased from Beijing Huafukang Biotechnology GmbH) of 18-22g, which are 4-5 weeks old, are randomly divided into three groups of 5 mice each, and after adaptive feeding for one week, the nude mice are transplanted with subcutaneous tumors.

(2) The cells were collected with physiological saline and resuspended to 1X 10⁸At one ml, 200. mu.L of the cell suspension was injected subcutaneously into mice using a syringe.

(3) After solid tumors had formed, they were administered by gavage for 30 days, and the body weight of the mice was measured every 3 days, and the tumor size was measured by a vernier caliper, according to the formula (length x (width)²) The tumor volume of nude mice was calculated.

The results are shown in fig. 8 and 9, and indicate that panaxadiol can effectively inhibit the volume of transplanted tumor in nude mice, and has no obvious influence on the body weight of nude mice.

From the above examples, it is known that panaxadiol can inhibit the expression of PD-L1 protein, the RNA transcription level of PD-L1, and the PD-L1 positive cell rate in a dose; can also inhibit the expression of hypoxia-induced tumor proliferation-related proteins Cyclin D1, c-Myc and VEGF in a dosage manner; reducing the number of EdU positive cells quantitatively, and inhibiting the proliferation of tumor cells; the activity of T cells in a co-culture system can be obviously improved; effectively inhibiting the volume of a nude mouse transplanted tumor; therefore, the panaxadiol has a remarkable effect as a medicine for inhibiting the colon cancer.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. Application of panaxadiol in preparing medicine for inhibiting PD-L1 protein expression is provided.

2. Application of panaxadiol in preparing medicine for inhibiting PD-L1 gene transcription is provided.

3. Application of panaxadiol in preparing medicine for inhibiting tumor cell proliferation is provided.

4. The application of panaxadiol in preparing medicine for inhibiting tumor cell proliferation protein expression is provided.

5. The use of claim 4, wherein the tumor cell proliferation proteins comprise Cyclin D1, c-Myc and VEGF.

6. Use of panaxadiol in preparing medicine for enhancing cytotoxic T lymphocyte activity is provided.

7. The use of claim 6, wherein said cytotoxic T lymphocytes are cytotoxic T lymphocytes inhibited by the tumor PD-L1 pathway.

8. Application of panaxadiol in preparing medicine for inhibiting the volume of transplanted tumor in nude mouse is disclosed.