Use of a liquid fermentation product of a corticium comosum associated fungus for the preparation of an anticancer drug

Abstract

The application discloses application of a liquid fermentation product of a cryptoporus symbiotic bacteria in preparation of an anticancer drug and belongs to the technical field of cell biology. The cryptoporus symbiotic bacteria is Trichoderma stromaticum, which was preserved in the China General Microbiological Culture Collection Center on November 8, 2023, has a preservation number of CGMCC No. 40914 and a preservation address of No. 3, Institute of Microbiology, Chinese Academy of Sciences, Xiliujiaobei, Chaoyang District, Beijing. The liquid fermentation product of the cryptoporus symbiotic bacteria provided in the application has good biological activity, can inhibit the growth of tumors and has a good anticancer effect on lung cancer cells. The application provides a theoretical basis for the liquid fermentation product of the cryptoporus symbiotic bacteria as an antitumor drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of a product of liquid fermentation of a Cryptoporus mycobiota in preparation of an anticancer drug. BACKGROUND

[0002] Lung cancer is a malignant tumor that seriously endangers human health, with an estimated 2200 million new cases and 179 million deaths each year. In China, the incidence and mortality rates are also the highest, accounting for about 20% of the total number of new cancer cases in China, and the number of deaths caused by cancer accounts for 27%. And 85% of lung cancer patients are of histological type of non-small cell lung cancer (NSCLC). NSCLC is mainly divided into squamous cell carcinoma, adenocarcinoma and large cell carcinoma, of which adenocarcinoma and squamous cell carcinoma are the main types. Tumor metastasis has been considered as one of the main factors leading to the failure of NSCLC treatment, and about 80% to 90% of NSCLC patients die due to metastasis. Therefore, it is urgent to determine the molecular targets for the treatment of metastatic NSCLC and to develop specific and effective drugs.

[0003] Trichoderma Trichoderma Fungi belong to the subdivision Deuteromycotina Deuteromycotina Phycomycetes Hyphomycets Trichiales Hyphomycetales The mycoparasite of Trichoderma is a world-wide distributed fungus. Trichoderma widely exists in soil under different living environmental conditions, and can also exist in plant seeds, rhizosphere, phyllosphere and corms as well as rotten wood. The secondary metabolites of Trichoderma fungi mainly include polyketides, peptides, terpenes and other types, which have various biological activities such as cytotoxicity, antibacterial activity and enzyme inhibition. The specific volatile substance 6-pentyl-2H-pyran-2-one is a natural active substance, which has been reported to have various biological and pharmacological activities and plant antibacterial effects. SUMMARY

[0004] In view of the current treatment of lung cancer, the present application provides application of a product of liquid fermentation of a Cryptoporus mycobiota in preparation of an anticancer drug, which provides more and better optional solutions for the treatment of cancer.

[0005] The technical scheme of the present application is as follows:

[0006] The application of the product of liquid fermentation of the Cryptoporus mycobiota in preparation of an anticancer drug is used for treating lung cancer.

[0007] The Cryptoporus mycobiota is Trichoderma atroviride, which was preserved in the China General Microbiological Culture Collection Center on November 8, 2023, with a preservation number of CGMCC No.40914 and a preservation address of No.3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road 1st Courtyard.

[0008] As preferred, the anticancer drug is used for treating lung adenocarcinoma.

[0009] As further preferred, the anticancer drug is used for treating small cell lung cancer.

[0010] The test results show that the anticancer drug of the present application can be used for inhibiting the proliferation of lung cancer cells, reducing the invasion and metastasis of lung cancer cells, and inducing the apoptosis of lung cancer cells.

[0011] As preferred, the lung cancer cells are HCC827 or H-2227 cells.

[0012] As preferred, other anticancer drugs are also included; the other anticancer drugs are cisplatin, osimertinib or erlotinib.

[0013] As preferred, the other anticancer drug is erlotinib;

[0014] The molar ratio of 6-pentyl-2H-pyran-2-one in the liquid fermentation product of the Cryptomyces amycodermus associated bacteria to erlotinib is 5:0.5~10. As further preferred, the concentration of 6-pentyl-2H-pyran-2-one is 5mM, and the concentration of erlotinib is 0.5~10mM, at which time the combination of 6-pentyl-2H-pyran-2-one and erlotinib can have a moderate or above synergistic effect; as still further preferred, the concentration of 6-pentyl-2H-pyran-2-one is 5mM, and the concentration of erlotinib is 10mM, at which time the combination of 6-pentyl-2H-pyran-2-one and erlotinib has a high synergistic effect.

[0015] As preferred, the selected other anticancer drug is cisplatin;

[0016] The molar ratio of 6-pentyl-2H-pyran-2-one in the liquid fermentation product of the Cryptomyces amycodermus associated bacteria to cisplatin is 1~2.5:0.5~10. As further preferred, the concentration of 6-pentyl-2H-pyran-2-one is 1~2mM, and the concentration of cisplatin is 0.5~10mM, at which time the combination of 6-pentyl-2H-pyran-2-one and cisplatin has at least a high synergistic effect; as further preferred, the concentration of 6-pentyl-2H-pyran-2-one is 2.5mM, and the concentration of cisplatin is 0.5~5mM, at which time the combination of 6-pentyl-2H-pyran-2-one and cisplatin has a moderate synergistic effect.

[0017] As preferred, the selected other anticancer drug is osimertinib;

[0018] The molar ratio of 6-pentyl-2H-pyran-2-one to osimertinib in the liquid fermentation product of the *Cryptocorum* consortium is 0.5~1:1~10. As a further preferred embodiment, the concentration of 6-pentyl-2H-pyran-2-one is 0.5 mM, and the concentration of osimertinib is 1~2.5 mM, at which point the combination of 6-pentyl-2H-pyran-2-one and osimertinib exhibits a moderate synergistic effect.

[0019] The beneficial effects of this invention are:

[0020] (1) This invention provides a new anticancer use for the liquid fermentation product of Cryptoporum simulans, which has a good anticancer effect on lung cancer, especially non-small cell lung cancer;

[0021] (2) The present invention further provides a combination of Cryptoporum simulans-associated liquid fermentation product and several other anticancer drugs. The combination of the two can have a synergistic effect and further improve the anticancer effect.

[0022] Biological preservation instructions for Trichoderma cyanobacterium YKBS-T-01:

[0023] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);

[0024] Accession number: CGMCC No. 40914;

[0025] Date of deposit: November 8, 2023;

[0026] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0027] Taxonomic nomenclature: Trichoderma caerulescens . Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments.

[0029] MTT assay: The MTT assay was used to detect the survival and mortality rates of human non-small cell lung cancer (NSCLC) cells. NSCLC cells (4.5 × 10⁻⁶) were used. 3 Cells were seeded per well in 96-well plates and treated with liquid fermentation broth containing Cryptoporum simulans at concentrations of 0, 0.5, 1, 2, and 2.5 mM for 6 h. After 48 h, MTT solution was added at a rate of 25 mL / well, and the plates were incubated at approximately 37 °C in a CO2 incubator for 4 h. Formalazan crystals were dissolved in 150 mL of dimethyl sulfoxide, and the optical density (OD) of the MTT solution was recorded using a microplate reader (490 nm absorbance). Cell viability and IC50 values ​​were calculated using GraphPad Pro Prism 8.0.

[0030] Colony formation assay: Human NSCLC cells (1000 cells / well) were seeded in 6-well plates at 37°C, 5% CO2 atmosphere overnight. Dimethyl sulfoxide (control), Serpula Incarnata associated bacteria liquid fermentation (0.5 mM, 1 mM, 2 mM or 2.5 mM, 5 mM, 10 mM) was added to the cells for 7 days. The medium was replaced with fresh medium every two days to maintain cell growth for 7 days. The colonies were washed with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS for 3 times, stained with crystal violet for 10 min. Each experiment was divided into three times, and a total of three independent experiments were performed.

[0031] EDU experiment: Cells were cultured in medium containing different concentrations of Serpula Incarnata associated bacteria liquid fermentation (0, 0.5, 1, 2 mM), and EdU staining was performed using BeyoClick to study cell proliferation. First, the cells were washed with PBS three times, each for five minutes. A certain amount of EDU was added to the fresh medium. The cells were cultured in a CO2 incubator at 37°C for 1 hour. After incubation, the cells were washed with PBS twice, each for 10 minutes. The cells were fixed with 4% paraformaldehyde at room temperature for 30 min, and then stained with DAPI for 10 min. After washing with PBS again, the cells were observed under an inverted microscope.

[0032] TRANSWELL experiment: According to the manufacturer's instructions, transwell filters (BD Biosciences, USA) were used to study cell migration. The cell density was 1 x 10 3 , cells were added to the upper chamber containing serum-free medium, and medium containing 10% FBS was added to the lower chamber. Different concentrations of Serpula Incarnata associated bacteria liquid fermentation (0, 0.5, 1, 2 mM) were administered according to the preset, and after 48 hours of drug treatment, the cells were fixed with 4% paraformaldehyde, and the uncrushed cells were removed from the upper surface of the filter. The cells on the lower surface of the membrane were stained with 1% crystal violet for 15 minutes at room temperature in the dark. The number of migrated cells was recorded and counted under a light microscope.

[0033] Scratch experiment: Cells (5 x 10 5 were seeded in 6-well plates and allowed to adhere overnight. At 80-90% confluence, a sterile 10 mL pipette tip was used to draw a "reference line" on the bottom of the plate. After washing with PBS three times, the cells that no longer adhered were washed away, and cell migration was checked in the absence of cell growth. The blank control group was not added with any drug, and the test group was added with different concentrations of Serpula Incarnata associated bacteria liquid fermentation (0, 0.5, 1, 2 mM), respectively. After 0, 24, 48 hours of treatment, the cell migration micrographs that crossed the reference line were photographed by digital camera in different areas. Compared with the control group, the migration rate was quantified by the distance of cell migration from the reference line to the center. All experiments were repeated 3 times.

[0034] Apoptosis experiment: HCC827 cells were seeded in 6-well culture medium and grown to 80% confluence in complete medium. Cells were then treated with dimethyl sulfoxide (control), Serpula Incarnata associated bacteria liquid fermentation (0.5 mM, 1 mM, 2 mM) for 48 hours to evaluate the effect of these compounds on apoptosis. Cells were collected, washed twice in ice-cold PBS, and then resuspended in binding buffer according to the instructions of the apoptosis kit. Treated cells (as described above) were incubated with fluorescein-labeled annexin V and PI simultaneously. Annexin V binding buffer was then added to the mixture. Fluorescence was then measured on a FACSCalibur (BD Biosciences, Baltimore, MD, USA). Flowjo software was used to analyze the data.

[0035] WB experiment: Cancer cells were seeded in 6-well plates at a density of 500000 cells / well and then cultured overnight. Dimethyl sulfoxide (control), Serpula Incarnata associated bacteria liquid fermentation (0.5, 1, 2 mM or 2.5, 5, 10 mM) was added to the 6-well plates. Treated cells were washed with ice-cold PBS and lysed in RIPA lysis buffer (total protein extraction kit) with 1% phenylmethylsulfonyl fluoride. After high-speed centrifugation to obtain the supernatant, the protein was collected and quantified with an enhanced BCA protein assay kit. Subsequently, about 60 mg of protein was loaded onto an 8%, 10%, or 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and separated. They were then transferred to a PVDF membrane. The membrane was washed three times with TBST and blocked with 5% fresh skim milk at room temperature for 1.5 hours. The blot was incubated with specific primary antibodies overnight at 4°C. After washing with TBST, the membrane was incubated with the relevant secondary antibody for 60 minutes at room temperature. The immunoreactive bands were observed using a 0mni ECL femtosecond photochemical luminescence kit (EpiZyme, Shanghai, China). The visualization of the bands was analyzed using ImageJ computer software.

[0036] Example 1

[0037] The method for preparing the liquid fermentation product of Serpula Incarnata associated bacteria comprises the following steps:

[0038] 1) The blue Trichoderma strain is inoculated into PDA culture medium and cultured in the dark to prepare a spore suspension;

[0039] 2) The spore suspension of step (1) is inoculated into PD culture medium, and after culture, the supernatant is obtained by filtering the spores and mycelium;

[0040] 3) The fermentation supernatant of step (2) is mixed with PDA and ready for use.

[0041] Example 2

[0042] This example demonstrates that the liquid fermentation product of the Ceriporiopsis subvermispora associated fungus has dose-dependent and time-dependent cytotoxicity and anti-proliferation effects on NSCLC cells by MTT assay, colony formation assay and EDU assay. The effect of the liquid fermentation product of the Ceriporiopsis subvermispora associated fungus on the growth of NSCLC cells was detected by EDU and colony formation experiments. EDU cell proliferation detection suggests that the liquid fermentation product of the Ceriporiopsis subvermispora associated fungus can dose-dependently inhibit NSCLC cell proliferation; the results of the colony formation experiment show that the liquid fermentation product of the Ceriporiopsis subvermispora associated fungus significantly inhibits the number of HCC827 and H-2227 cell colonies. These results indicate that the liquid fermentation product of the Ceriporiopsis subvermispora associated fungus can inhibit NSCLC cell proliferation.

[0043] Example 3

[0044] This example further observes the effect of the liquid fermentation product of the Ceriporiopsis subvermispora associated fungus on NSCLC invasion. In the TRANSWELL experiment, the addition of the drug greatly reduces the invasion and metastasis ability of HCC827 and H-2227 cells. The results of the scratch healing experiment of HCC827 cells further support the role of the liquid fermentation product of the Ceriporiopsis subvermispora associated fungus in metastasis. In addition, we further detected the invasion and migration proteins, which suggest that the liquid fermentation product of the Ceriporiopsis subvermispora associated fungus can induce a decrease in the expression of proteins SNAIL and MMP2.

[0045] Example 4

[0046] This example further analyzes the effect of the liquid fermentation product of the Ceriporiopsis subvermispora associated fungus on NSCLC cell apoptosis using flow cytometry. Annexin V-FITC is used as a marker for cell apoptosis, and propidium iodide (PI) is used as a marker for cell viability. The results show that the liquid fermentation product of the Ceriporiopsis subvermispora associated fungus can promote H-2227 cell apoptosis, and further detection of apoptosis proteins suggests that the liquid fermentation product of the Ceriporiopsis subvermispora associated fungus can induce an increase in the expression of apoptosis proteins C1-PARP, BAX and C1-Caspase3, while inhibiting the expression of the anti-apoptotic protein Bc1-2.

[0047] Example 5

[0048] 6-Pentyl-2H-pyran-2-one measurement method: the crude extract of fermentation broth was diluted 50 times with chromatographic methanol, and passed through a 0.22 μm organic microporous filter for HPLC analysis. The chromatographic column was ZORBAX SBC18 column (250 mm x 4.6 mm, 5 μm), the mobile phase was 100% acetonitrile, the detection wavelength was 254 nm, the column temperature was 35 °C, the flow rate was 1 mL / min, and the injection volume was 10 μL. 5 mg of 6-pentyl-2H-pyran-2-one standard was accurately weighed, dissolved and diluted to 10 mL with chromatographic pure methanol, and passed through a 0.22 μm organic microporous filter to obtain a 0.5 mg / mL 6-pentyl-2H-pyran-2-one standard solution. Then, the 6-pentyl-2H-pyran-2-one standard working solution was diluted with methanol to 0.4, 0.3, 0.2, and 0.1 mg / mL, respectively. The content of the sample to be tested was determined under the above conditions, and the standard curve was drawn with the mass concentration of 6-pentyl-2H-pyran-2-one as the abscissa and the corresponding peak area as the ordinate.

[0049] For the synergistic experiment of 6-pentyl-2H-pyran-2-one and cisplatin, osimertinib and erlotinib in the liquid fermentation product of Ceriporiopsis subvermispora associated bacteria, the interaction between the two can be judged according to the CI value. For HCC827 cells, the combination of 5 mM 6-pentyl-2H-pyran-2-one and 10 mM erlotinib had a drug combination index of 0.585, indicating a high synergistic effect. The combination of 5 mM 6-pentyl-2H-pyran-2-one and 0.5 mM, 1 mM, 2.5 mM, and 5 mM osimertinib had drug combination indexes of 0.625, 0.587, 0.663, and 0.728, respectively, indicating a moderate synergistic effect. For H-2227 cells, 6-pentyl-2H-pyran-2-one and cisplatin showed a strong synergistic effect. For HCC827 cells, 6-pentyl-2H-pyran-2-one and cisplatin showed a high synergistic effect.

[0050] The combination index (CI) can be used to judge the interaction between the two. When 0.9 ≤ CI ≤ 1.1, it indicates that the two have an additive effect; when 0.8 ≤ CI ≤ 0.9, it indicates that the two have a low synergistic effect; when 0.6 ≤ CI ≤ 0.8, it indicates that the two have a moderate synergistic effect; when 0.4 ≤ CI ≤ 0.6, it indicates that the two have a high synergistic effect; and when 0.2 ≤ CI ≤ 0.4, it indicates that the two have a strong synergistic effect.

Claims

1. The application of liquid fermentation products of Cryptoporum simulans combined with other anticancer drugs in the preparation of drugs for treating lung cancer, characterized in that, The other anticancer drugs mentioned are osimertinib or erlotinib; The associated fungus of Cryptoporum is Trichoderma cyanobacterium, which was deposited on November 8, 2023 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40914. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The liquid fermentation product of the Cryptoporum synergistum is 6-pentyl-2H-pyran-2-one.

2. The application according to claim 1, characterized in that, The aforementioned anticancer drug is used to treat lung cancer and lung adenocarcinoma.

3. The application according to claim 1, characterized in that, The aforementioned anticancer drug is used to treat small cell lung cancer.

4. The application according to any one of claims 1-3, characterized in that, The aforementioned anticancer drug is used to inhibit the proliferation of lung cancer cells, reduce the invasion and transformation of lung cancer cells, and induce apoptosis of lung cancer cells.

5. The application according to claim 4, characterized in that, The lung cancer cells mentioned are HCC827 or H-2227 cells.

6. The application according to any one of claims 1-3, characterized in that, The other anticancer drug mentioned is erlotinib; The molar ratio of 6-pentyl-2H-pyran-2-one to erlotinib in the liquid fermentation product of the Cryptoporum simulans associated with the bacteria is 5:0.5~10.

7. The application according to any one of claims 1-3, characterized in that, The other anticancer drug mentioned is osimertinib; The molar ratio of 6-pentyl-2H-pyran-2-one to osimertinib in the liquid fermentation product of Cryptoporum simulans is 0.5~1:1~10.