JP2023097778A - Cancer therapeutic agent, and method for producing cancer therapeutic agent - Google Patents

Abstract

To provide a retarder of anti-cancer agent resistance that is extracted from Termitomyces sp. being a basidiomycete, and that hardly causes acquired resistance, and a cancer therapeutic agent containing the retarder.SOLUTION: A cancer therapeutic agent contains an active ingredient derived from Termitomyces sp.. The active ingredient may be dry powder of an artificially cultured culture solution of Termitomyces sp.. The active ingredient may be alcohol extract from an artificially cultured culture solution of Termitomyces sp.. The active ingredient may be a fruit body or mycelium of Termitomyces sp. extract from alcohol.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a therapeutic agent for cancer and a method for producing the therapeutic agent for cancer.

Malignant tumors, or cancer, account for the largest number of deaths by disease in Japan, and about 38,000 people died of cancer in 2019. Lung cancer is the most common malignant tumor causing death, and it is reported that about 75,000 people have died.

Current cancer treatments are mainly surgery, irradiation and chemotherapy, and immunotherapy is attracting attention as a fourth treatment method. It has been found that cancer cells put a brake on immune function and inhibit the activation of immune cells. Immunotherapy is a treatment that releases this brake by cancer cells and reactivates immune cells.

Proteins that can induce immunosuppressive signals in the cancer cell mechanisms described above are called "immune checkpoint molecules." PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), CTLA4 receptor and the like are known as immune checkpoint molecules. PD-L1 and PD-L2 are PD-1 receptors on T cells and ligands expressed on cancer cells that are involved in the PD-1 (programmed cell death-1) ligand pathway. The CTLA4 receptor is present on T cells that participate in the CTLA4 (negative co-stimulatory receptor) pathway.

The PD-1 molecule is an immunosuppressive co-signaling receptor belonging to the CD28 (positive co-stimulatory receptor) family, expressed on activated T cells, B cells and myeloid cells, PD-L1 and PD- Binding to L2 is known to suppress T cell activity in an antigen-specific manner. The PD-1 pathway is said to be involved in the suppression of immunity against cancer cells, and anti-PD-1 antibodies and anti-PD-L1 antibodies that target this pathway are expected as cancer therapeutic drug candidates, and antibody drugs is under development.

Patent Document 1 discloses a composition for the treatment of cancer or the treatment of infectious diseases through immunostimulation induced by inhibition of immunosuppressive signals induced by PD-1, PD-L1 or PD-L2. are doing. Patent Document 2 discloses that anti-human PD-1 monoclonal antibodies and anti-mouse PD-1 monoclonal antibodies are useful for treating cancer or infectious diseases. Patent Document 3 discloses a therapeutic agent for cancer containing a PD-1 antibody as an active ingredient.

In addition, it has been found that there are gene mutations specific to cancer types. It has been found that lung cancer has gene mutations such as "ALK fusion gene mutation" and "EGFR gene mutation". EGFR (epidermal growth factor receptor) exists on the surface of cancer cells and plays a role like a switch for cancer cell proliferation. If there is a mutation in a part of the gene (tyrosinase site) that constitutes EGFR, the switch that causes cancer cells to proliferate is always on, and cancer cells proliferate without limit. For lung cancer, “individualized therapy” targeting these gene mutations is becoming possible, and EGFR tyrosinase inhibitors (EGFR-TKI), anti-EGFR antibodies, and the like have been developed.

Activation of AXL, a receptor tyrosine kinase, has been reported to be a novel EGFR-TKI resistance mechanism. Furthermore, the relationship between AXL and epithelial-mesenchymal transition (EMT) has been pointed out, and it has been reported that AXL is involved in worsening prognosis, tumor growth, and metastasis. AXL is known to be overexpressed in various cancers and involved in proliferation and progression. In addition to EGFR-TKI, AXL activation is known to be involved in the acquisition of resistance to various anticancer drugs such as the anticancer drug cisplatin and the HER2 targeting drug lapatinib.

International Publication 2004/004771 JP 2009-286795 A JP 2014-065748 A

Drugs that act to inhibit the immune checkpoint molecules described above, so-called "immune checkpoint inhibitors," are becoming clinically effective. However, treatment with antibody drugs is very expensive and can be an undue burden on the patient.

In addition, long-term administration of immune checkpoint inhibitors has been observed to cause re-aggravation of malignant tumors (also called acquired resistance). This acquired resistance represents a new obstacle in cancer therapy.

Mushrooms have been used in oriental medicine since ancient times, and are believed to be beneficial for the prevention and treatment of malignant tumors by enhancing immunity and inducing apoptosis. However, there are not many reports on immune checkpoint inhibitory effects and AXL inhibitory effects of compounds obtained from natural products such as mushrooms.

An object of the present invention is to provide an anticancer drug resistance suppressor that is extracted from a basidiomycete, Pleurotus pygmaea, and is unlikely to cause acquired resistance, and a therapeutic agent for cancer containing the same.

The present invention has the following aspects.
[1] A therapeutic agent for cancer, containing an active ingredient derived from Pleurotus fungus.
[2] The therapeutic agent for cancer according to [1], wherein the active ingredient is a dry powder of a culture medium of P. pumilatus that has been artificially cultured.
[3] The therapeutic agent for cancer according to [1], wherein the active ingredient is an alcoholic extract from a culture medium of P. pumilatus that has been artificially cultured.
[4] The therapeutic agent for cancer according to [1], wherein the active ingredient is an alcoholic extract from the fruiting body or mycelium of Pleurotus fungus.
[5] The therapeutic agent for cancer according to any one of [1] to [4], wherein the active ingredient inhibits AXL expression.
[6] The therapeutic agent for cancer according to any one of [1] to [5], wherein the active ingredient inhibits the expression of immune checkpoint molecules.
[7] The cancer therapeutic agent of [6], wherein the immune checkpoint molecule is at least one of PD-L1 and PD-L2.
[8] The termite fungus Termitomyces sp. T-120 strain, which is a basidiomycete deposited at the Patent Microorganism Depositary of the National Institute of Technology and Evaluation under the accession number NITE P-03541. The cancer therapeutic agent according to any one of [1] to [7].
[9] The therapeutic agent for cancer according to [8], wherein the therapeutic agent for cancer is at least one of a therapeutic agent for lung cancer and a therapeutic agent for skin cancer.
[10] The termite fungus Termitomyces sp. T-108 strain, which is a basidiomycete deposited at the Patent Microorganism Depositary of the National Institute of Technology and Evaluation under the accession number NITE P-03542. The cancer therapeutic agent according to any one of [1] to [7].
[11] The therapeutic agent for cancer according to [10], wherein the therapeutic agent for cancer is a therapeutic agent for skin cancer.
[12] The therapeutic agent for cancer according to any one of [1] to [11], further comprising at least one of an anticancer agent, an antibiotic, an anti-inflammatory agent, an antipyretic and an analgesic.
[13] a step of liquid culturing P. aeruginosa; a step of filtering the culture of P. aeruginosa obtained by the liquid culture to separate the culture solution and a residue; and pulverizing the dried and solidified product to obtain a dry powder.
[14] The method for producing a cancer therapeutic agent according to [13], further comprising the steps of: shaking the dry powder with alcohol; and recovering the alcohol and concentrating it to dryness to obtain an alcohol extract. .
[15] a step of liquid culturing P. aeruginosa; a step of filtering the culture of P. aeruginosa obtained by the liquid culture to separate it into a culture solution and a residue; a step of drying the residue; A method for producing a therapeutic agent for cancer, comprising the steps of pulverizing the residue, shaking the pulverized residue with alcohol, and recovering the alcohol and concentrating and drying to obtain an alcohol extract. .
[16] The Pleurotus versicolor Termitomyces sp. T-108 strain, or an independent Any of [13] to [15], which is a basidiomycete Termitomyces sp. 1. A method for producing a cancer therapeutic agent according to 1 above.

According to the above aspect, it is possible to provide an anticancer drug resistance suppressor that is extracted from a basidiomycete, Pleurotus pallidum, and is unlikely to cause acquired resistance, and a cancer therapeutic agent containing the same.

1 is a graph showing the effect of suppressing AXL expression in human lung cancer cell line A549 of Examples 1 to 3, which is one aspect of the present invention. 1 is a graph showing the inhibitory effect on PD-L1 expression in human lung cancer cell line A549 of Examples 1 to 3, which is one aspect of the present invention. 1 is a graph showing the inhibitory effect on PD-L1 expression in human lung cancer cell line A549 of Examples 1 to 3, which is one aspect of the present invention. Mouse skin cancer cell line B16. Fig. 3 is a graph showing the AXL expression inhibitory effect of F101. Mouse skin cancer cell line B16. FIG. 10 is a graph showing the inhibitory effect of F101 on PD-L1 expression. FIG. Mouse skin cancer cell line B16. Fig. 3 is a graph showing the inhibitory effect of F101 on PD-L2 expression. FIG. 2 shows the apoptosis-inducing effect of the extract of Example 1, which is one aspect of the present invention, on human lung cancer cell line A549. 1 is a graph showing the apoptosis-inducing effect of the extract of Example 1, which is one aspect of the present invention, on human lung cancer cell line A549.

One aspect of the present invention is a therapeutic agent for cancer containing an active ingredient derived from Porphyra moritake. Further, another aspect of the present invention includes the steps of liquid culturing P. aeruginosa, filtering the culture of P. aeruginosa obtained by the liquid culture to separate the culture solution and a residue, and the culture solution A method for producing a cancer therapeutic agent, comprising the steps of drying to obtain a dry solidified product, and pulverizing the dry solidified product.

In the present invention, the term "cancer therapeutic agent" refers to a substance that exhibits effective effects in cancer treatment. Effective effects in cancer therapy include mechanisms that can act to develop resistance to anticancer drug therapy, such as increased expression of immune checkpoint molecules and effects of suppressing AXL activation.

<Active Ingredients Derived from Pleurotus fungus and Method for Producing the Same>
Termitomyces sp. used in the present invention is a tropical edible basidiomycete belonging to the genus Termitomyces. It grows naturally in Asia and Africa, and in Japan, Ishigaki Island and Iriomote Island in Okinawa Prefecture can be mentioned as a natural habitat.

The P. terminus-derived active ingredient of the present invention may be prepared from a P. terminus culture. More preferably, it is prepared from a culture of the T-120 strain of P. pallens or T-108 of P. pallens. The T-120 and T-108 strains of Pleurotus fungus were discovered on Ishigaki Island, Okinawa Prefecture, and were first isolated in 2021 by the inventor of the present application. The T-120 strain is dated December 21, 2021 as accession number NITE P-03541, and the T-108 strain is dated December 21, 2021 as accession number NITE P-03542. It has been deposited at the Patent Microorganisms Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan).

The fungus can be collected as a fruiting body or cultured as a mycelium. From the standpoint of increasing productivity, it is preferable to culture P. melanoma as a mycelium.

The following three are the active ingredients of Pleurotus fungus.
1. Dried powder of the culture solution of Pleurotus fungus2. Alcoholic extract from the culture of Porphyrinus 3. Alcoholic extract from the fruiting body or mycelium of Pleurotus fungus

1. The active ingredient of Porphyra spp. In the case of a dried powder of the culture solution of Pleurotus fungi, the method for producing a cancer therapeutic agent in one aspect of the present invention includes the following steps.

First, a fungus strain of Pleurotus fungus is inoculated into a liquid medium and cultured in a liquid medium. A fungal strain of P. melanoma may be a self-growing strain or a subcultured strain. The medium is not particularly limited as long as it is a medium in which Pleurotus fungus grows, but examples include a medium containing at least one of potato dextrose broth (PDB, manufactured by Difco), yeast extract, malt extract and polypeptone. be done. The pH of the culture solution is preferably pH 5.0 to 7.0, more preferably pH 5.5 to 6.0. The culture temperature is preferably 20-30°C, more preferably 24-27°C. The humidity during cultivation is preferably 40 to 80% RH, more preferably 50 to 60% RH. The shaking speed during culture is preferably 0 to 100 rpm, more preferably 0 to 50 rpm. The liquid medium may be aerated, and the aeration gas preferably contains oxygen. The culturing period is not particularly limited as long as the fungus can grow sufficiently, but in the case of culturing as mycelium, it may be 30 to 120 days.

The culture of Pteris angustica obtained by the above-described method is separated into mycelia and a culture medium by filtration. The resulting culture can be freeze-dried and then ground to obtain a dry powder.

2. The active ingredient of Pleurotus fungus. In the case of the alcoholic extract from the fruiting body or mycelium of Porcosus moritake, the method for producing a therapeutic agent for cancer in one aspect of the present invention includes the following steps.

The dry powder obtained by the above method is added to alcohol and shaken for a day and night to extract the active ingredient contained in the dry powder. The alcohol is recovered and the process of adding fresh alcohol and shaking overnight is repeated. This extraction step may be performed multiple times, for example 2 to 4 times. All of the recovered alcohol is concentrated to dryness using an evaporator, and freeze-dried again in a nitrogen gas atmosphere to obtain an alcohol extract from the culture solution after culturing P. rustis.

Alcohols include, for example, monohydric alcohols such as methanol, ethanol and isopropanol; and dihydric alcohols such as ethylene glycol, 1,2-propanediol and 1,3-propanediol. The alcohol is preferably a monohydric alcohol, more preferably methanol, ethanol, or isopropanol.

An aqueous alcohol solution may be used instead of alcohol. An aqueous alcohol solution is an aqueous solution comprising one or more of the aforementioned alcohols. The total content of the alcohol with respect to the total mass of the alcohol aqueous solution is preferably 50 to 99% by mass, more preferably 60 to 95% by mass, more preferably 70 to 90% by mass, from the viewpoint of increasing the extraction efficiency of the active ingredient. preferable.

The temperature of the alcohol or the alcohol aqueous solution in the extraction step is, for example, 4 to 40°C. The mass ratio of the pulverized product and the alcohol or the alcohol aqueous solution at the time of extraction may be appropriately adjusted, for example, within the range of 10:1 to 1:10.

3. The active ingredient of Pleurotus fungus. In the case of the alcohol extract from the culture broth of P. pumilatus, the method for producing a therapeutic agent for cancer in one aspect of the present invention includes the following steps.

The fruiting body or mycelium of P. melanoma is freeze-dried and then ground. The mycelium may be a mycelium obtained by cultivating P. moritake by the method described above and filtering the culture. The pulverized material is added to alcohol and shaken all day and night to extract the active ingredient contained in the pulverized material. As the alcohol to be used, the one described above can be used, and instead of the alcohol, the alcohol aqueous solution described above may be used. The alcohol is recovered and the process of adding fresh alcohol and shaking overnight is repeated. This extraction step may be performed multiple times, for example 2 to 4 times. All of the recovered alcohol is concentrated to dryness using an evaporator, and freeze-dried again in a nitrogen gas atmosphere to obtain an alcohol extract.

<Immune checkpoint molecule and AXL>
In one aspect of the present invention, the active ingredient derived from P. pumila can inhibit the expression of immune checkpoint molecules. In one aspect of the present invention, immune checkpoint molecules whose expression is inhibited include PD-L1 and PD-L2.

PD-L1 (also called CD274) and PD-L2 (also called CD273) are known ligands for PD-1, a 55 kD type I membrane protein belonging to the immunoglobulin family. GenBank accession No. AF233516 (Gene ID: 29126 in the NCBI database, etc.) or NM_014143 for human PD-L1 cDNA nucleotide sequence information, etc., and GenBank accession No. NM_025239 for human PD-L2 cDNA nucleotide sequence information, etc. Gene ID: 80380).

In one aspect of the present invention, the active ingredient derived from Pteris moritake can inhibit the expression of AXL. AXL is a member of the receptor tyrosine kinase family. AXL is involved in complex signaling networks involved in the control of cell proliferation and differentiation, and is also known to be involved in carcinogenesis. The nucleotide sequence information of human AXL cDNA and the like can be obtained from the NCBI database, etc. as GenBank Accession No. NM_001699 (Gene ID: 558).

<Evaluation of inhibition of immune checkpoint molecule and AXL expression>
Inhibition of PD-L1, PD-L2 and AXL expression can be confirmed in in vitro or in vivo systems containing cells expressing these molecules. As an in vitro system, a system comprising target cells expressing PD-L1, PD-L2 and AXL can be used. Target cells include human cancer cells. Examples of human cancer cells include stomach cancer cells, lung cancer cells, breast cancer cells, colon cancer cells, ovarian cancer cells, uterine cancer cells, prostate cancer cells, pancreatic cancer cells, liver cancer cells, myeloma cells and skin cancer cells. be done. As an in vivo system, for example, it can be confirmed using an animal model in which cancer develops.

Inhibition of gene expression in vitro and in vivo is not particularly limited, but is carried out, for example, by the following methods. Using the in vitro or in vivo system described above, mRNA is extracted from a sample to which an active ingredient derived from Pteris moritake in one aspect of the present invention is added or administered and a sample to which it is not added or administered, and cDNA is obtained by reverse transcription reaction. become After amplifying a DNA fragment using cDNA as a template by PCR using primers prepared based on the nucleotide sequence information of the polynucleotide encoding the gene of interest, the expression level can be confirmed. Primers that are commercially available as primer sets can also be used. As is commonly practiced in the art, the expression of PD-L1, PD-L2 and AXL as a ratio to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) whose expression level is considered to be constant It can represent quantity. Whether or not the expression of these molecules is inhibited by the extract of Porphyromatus rotundus in one aspect of the present invention can be determined by comparison with the case where the extract of P. perforatum in one aspect of the present invention is not added or administered. can.

When the expression level of at least one of PD-L1 and PD-L2 is confirmed to be low by the above method, it is judged in the present specification to have an immune checkpoint inhibitory effect. In addition, when it is confirmed that the expression level of AXL is low by the above method, it has an effect as a cancer therapeutic agent, for example, an effect of suppressing at least one of cancer cell proliferation, invasion and metastasis, and acquisition of an anticancer drug. Judging that it is effective in suppressing resistance.

<Evaluation of apoptosis-inducing action>
In an in vitro system, target cells were cultured in the presence of the P. pharris moritake extract of one embodiment of the present invention, and the apoptosis of the target cells was evaluated by flow cytometry to induce the apoptosis of the P. pylori mushroom extract. effects can be evaluated. A large apoptosis-inducing action means that the proliferation of target cells, ie, cancer cells can be suppressed.

In one aspect of the present invention, the active ingredient derived from P. pumilatus can suppress the expression of PD-L1 and PD-L2, which are immune checkpoint molecules. In addition, the extract of Pteris moritake in one embodiment of the present invention can suppress the expression of AXL. Therefore, a cancer therapeutic agent containing an extract of Porphyrinus pallidum has an immune checkpoint inhibitory effect. In addition, a cancer therapeutic agent containing an extract of P. pallens has the effect of suppressing at least one of proliferation, invasion and metastasis of cancer cells, and is less likely to develop acquired resistance.

In one aspect of the present invention, the active ingredient derived from Pteriotus fungus may be the active ingredient derived from the T-120 strain. As shown in Examples below, the active ingredient derived from the T-120 strain can suppress the expression of PD-L1, PD-L2 and AXL at least in lung cancer cells and skin cancer cells. A therapeutic agent for cancer containing an active ingredient derived from the T-120 strain can be used as a therapeutic agent for lung cancer and a therapeutic agent for skin cancer. The active ingredient derived from the T-120 strain is preferably at least one of an alcoholic extract from a culture medium after culturing P. aurantica and a culture medium after culturing P. auricularia.

In one aspect of the present invention, the active ingredient derived from Pterioscyllium may be the active ingredient derived from the T-108 strain. As shown in Examples below, the active ingredient derived from the T-108 strain can suppress the expression of PD-L1. More specifically, the active ingredient derived from the T-108 strain can suppress the expression of PD-L1 in at least skin cancer cells. A therapeutic agent for cancer containing an active ingredient derived from the T-108 strain can be used as a therapeutic agent for skin cancer. It is preferable that the active ingredient derived from the T-108 strain is an alcohol extract from the culture medium after cultivating P. moritake.

<Other active ingredients>
In one aspect of the present invention, the therapeutic agent for cancer may contain, in addition to the active ingredient derived from Pleurotus fungus, further active ingredients such as at least one of anticancer agents, antibiotics, anti-inflammatory agents, antipyretics and analgesics. However, the invention is not so limited.

Diseases targeted by the therapeutic agent for cancer in one aspect of the present invention include gastric cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, uterine cancer, prostate cancer, pancreatic cancer, liver cancer, leukemia, lymphoma, myeloma, skin cancer, and the like. are mentioned, but are not particularly limited. Mechanisms involving immune checkpoint molecules and mechanisms of acquired resistance are found in immune responses to all cancer cells. Therefore, the therapeutic agent for cancer according to one aspect of the present invention is capable of enhancing the immune response against cancer cells, which are target cells, by inhibiting the expression of immune-suppressing proteins such as immune checkpoint molecules and proteins associated with acquired resistance. can.

The active ingredient and the further active ingredient derived from Pistolus vulgaris in one aspect of the present invention can be administered separately or can be combined together, eg, in a single pharmaceutical composition. The form of the pharmaceutical composition, administration route and the like are not particularly limited, and the form and administration route commonly used in the art can be appropriately selected, but oral administration or intravenous injection is preferred. When formulated as a pharmaceutical composition, at least one of excipients, fillers, disintegrants, preservatives, colorants, sweeteners, flavoring agents, film-forming agents, and the like, which are commonly used in pharmaceutical formulations, are appropriately blended. be able to. In one aspect of the present invention, the active ingredient derived from Pleurotus fungus can also be administered as an injection. When administered as an injection, it is possible to use a solubilizing agent or the like, or to use an emulsion form.

When administered as a therapeutic agent for cancer, the dosage can be appropriately set in consideration of various factors such as administration route, patient's age, body weight and symptoms. As an example, a dose of 0.2-8 mg/kg body weight per day, preferably 0.4-4 mg/kg body weight per day can be used. The number and frequency of administration can be appropriately adjusted according to the disease expected to be treated or prevented and the physical condition of the subject.

In one aspect of the present invention, the active ingredient derived from Pleurotus fungus can be used as foods and drinks such as foods with health claims and foods for sick people, or as supplements. When used as a food, drink or supplement, the recommended intake for humans of the active ingredient derived from Pleurotus fungus is preferably in the range of 1 mg to 5 g, more preferably 100 mg to 2 g per day. The form of the food, drink or supplement may be any form such as powder, tablet, capsule, liquid, etc., and the present invention is not limited to these. The food, drink or supplement appropriately contains at least one of commonly used excipients, fillers, disintegrants, preservatives, coloring agents, sweeteners, flavoring agents, film-forming agents, and the like, similarly to pharmaceutical compositions. be able to. Moreover, the active ingredient derived from Pleurotus fungus may be in a form for ingestion with general foods, or may be mixed in foods.

EXAMPLES The present invention will be further described with reference to examples below, but the present invention should not be construed as being limited to these examples.

[Lung cancer cell test]
Human lung cancer cell line A549 (the American Type Culture Collection (Rockville, MD, USA) CCL185) was precultured in DMEM medium containing 10% FBS (manufactured by Thermo Fisher Scientific) at 37°C under 5% CO ₂ , The cells were seeded on a 12-well plate at a cell density of 1×10 ⁶ /well. After that, an active ingredient derived from Pteriophyllalis fungi obtained by the method described later was added to each plate so as to be 20 μg/ml. Cultured at 37° C., 5% CO ₂ for 24 hours. The cultured cells were trypsinized, subjected to centrifugation at 1500 rpm, 4° C. for 5 minutes, and the A549 cell pellet was collected.

Total RNA was extracted from the collected cells, reverse transcriptase Revertra Ace (manufactured by Toyobo Co., Ltd.) was used to synthesize DNA using the RNA as a template, and PCR was performed using the DNA as a template. The abundance of mRNA was then examined using primers corresponding to PD-L1, PD-L2 and AXL, respectively. PCR was performed with forward primer 1 (SEQ ID NO: 1) and reverse primer 1 (SEQ ID NO: 2) for PD-L1 mRNA and forward primer 2 (SEQ ID NO: 3) and reverse primer 2 (SEQ ID NO: 4) for PD-L2 mRNA. , forward primer 3 (SEQ ID NO: 5) and reverse primer 3 (SEQ ID NO: 6) for AXL mRNA, forward primer 4 (SEQ ID NO: 7) and reverse primer (SEQ ID NO: 8) for GAPDH mRNA used as a control set.

[Skin cancer cell test]
mouse melanoma cells B16. F10 (JCRB1675) was precultured in 10% FBS-containing DMEM medium (manufactured by Thermo Fisher Scientific) at 37°C under 5% CO ₂ , and then plated on a 12-well plate at a cell density of 1 × 10 ⁶ /well. sown. After that, an active ingredient derived from Pteriophyllalis fungi obtained by the method described later was added to each plate so as to be 20 μg/ml. Cultured at 37° C., 5% CO ₂ for 24 hours. Cultured cells were trypsinized and subjected to centrifugation at 1500 rpm, 4° C. for 5 minutes and B16. A pellet of F10 cells was collected.

Total RNA was extracted from the collected cells, reverse transcriptase Revertra Ace (manufactured by Toyobo Co., Ltd.) was used to synthesize DNA using the RNA as a template, and PCR was performed using the DNA as a template. The abundance of mRNA was then examined using the primers described above for PD-L1, PD-L2 and AXL, respectively.

[Apoptosis-inducing action]
Human lung cancer cell line A549 was cultured by the method described in [Cancer cell test] above, and seeded on a 12-well plate at a cell density of 1×10 ⁶ /well. After that, an extract of Porphyra angustifolia obtained by the method described later was added to each plate so as to have a concentration of 20 μg/ml. Cultured at 37° C., 5% CO ₂ for 24 hours. The cultured cells were trypsinized, subjected to centrifugation at 1500 rpm, 4° C. for 5 minutes, and the A549 cell pellet was collected. Cells were stained with Annexin V and propidium iodide (PI) using an apoptosis detection kit (FITC Annexin V Apoptosis Detection Kit with PI, Biolegend, San Diego, Calif.). Annexin V-FITC/PI was then measured by flow cytometry (FACScan, BD Biosciences, Oxford, UK).

(Example 1)
Potato dextrose broth (PDB, manufactured by Difco) 10 mL medium, inoculated with Pleurotus fungus strain (T-120 strain, accession number NITE P-03541) native to Ishigaki Island and Iriomote Island, Okinawa Prefecture, and allowed to stand at 25 ° C. cultured. After about one month, the mycelium and the culture medium were separated by filtration. The resulting culture was freeze-dried and then pulverized to obtain a dry powder (0.5 g). Hereinafter, this dry powder may be referred to as the active ingredient 1 derived from Pleurotus fungus.

(Example 2)
The T-120 strain was cultured in the same manner as in Example 1, and filtered to separate the mycelium and the culture broth. The isolated mycelium was lyophilized and then ground to obtain a dry powder (0.1 g). 40 mL of methanol was added to 100 mg of the obtained dry powder, and the mixture was shaken for a whole day and night. After that, the residue and methanol were separated and recovered, and the process of adding fresh methanol to the residue and shaking the mixture overnight was repeated twice, and then all of the recovered methanol extract was concentrated to dryness using an evaporator. After that, nitrogen gas was added and freeze-dried again to obtain an alcohol extract. Hereinafter, this alcohol extract may be referred to as the active ingredient 2 derived from Pleurotus fungus.

(Example 3)
40 mL of methanol was added to 100 mg of the dry powder obtained in Example 1, and the mixture was shaken for a whole day and night. After that, the residue and methanol were separated and recovered, and the process of adding fresh methanol to the residue and shaking the mixture overnight was repeated twice, and then all of the recovered methanol extract was concentrated to dryness using an evaporator. After that, nitrogen gas was added and freeze-dried again to obtain an alcohol extract. Hereinafter, this alcohol extract may be referred to as the active ingredient 3 derived from Pleurotus fungus.

(Example 4)
10 mL of potato dextrose broth (PDB, manufactured by Difco) medium was inoculated with a fungus strain (T-108 strain, NITE P-03542) native to Okinawa Ishigaki Island and Iriomote Island, and statically cultured at 25°C. After about one month, the mycelium and the culture medium were separated by filtration. The resulting mycelium was freeze-dried and then ground to obtain a dry powder (0.1 g).

40 mL of methanol was added to 100 mg of the obtained dry powder, and the mixture was shaken for a whole day and night. After that, the residue and methanol were separated and recovered, and the process of adding fresh methanol to the residue and shaking the mixture overnight was repeated twice, and then all of the recovered methanol extract was concentrated to dryness using an evaporator. After that, nitrogen gas was added and freeze-dried again to obtain an alcohol extract. Hereinafter, this alcohol extract may be referred to as the active ingredient 4 derived from Pleurotus fungus.

(Example 5)
The T-108 strain was cultured in the same manner as in Example 4, and filtered to separate the mycelium and the culture medium. The separated culture was freeze-dried and then ground to obtain a dry powder (0.5 g). 40 mL of methanol was added to 100 mg of the obtained dry powder, and the mixture was shaken for a whole day and night. After that, the residue and methanol were separated and recovered, and the process of adding fresh methanol to the residue and shaking the mixture overnight was repeated twice, and then all of the recovered methanol extract was concentrated to dryness using an evaporator. After that, nitrogen gas was added and freeze-dried again to obtain an alcohol extract. Hereinafter, this alcohol extract may be referred to as the active ingredient 5 derived from Pleurotus fungus.

The above-described [Lung cancer cell test] was carried out using the obtained P. terminus-derived active ingredients 1 to 5. In addition, the above-mentioned [skin cancer cell test] was performed using the active ingredients 2 to 5 of Pleurotus fungus. Furthermore, [apoptosis-inducing action] was performed using the active ingredient 1 derived from Porphyra angustifolia.

(Comparative example 1)
Potato dextrose broth was used in place of the active ingredient derived from Pleurotus fungi, and the above [lung cancer cell test] and [skin cancer cell test] were performed.

(Comparative example 2)
100 g of dried fruiting bodies of Agaricus blazei (Iwade 101 strain) obtained from Iwade Mycological Laboratory Co., Ltd. were pulverized to a size of about 0.3 mm to 3.0 mm. The pulverized material was extracted with hot water at about 90°C for 180 minutes to obtain 44.2 g of hot water extract. The unextracted residue (55 g) was extracted with about 5% NaOH aqueous solution at about 30° C. for 24 hours, and the resulting filtrate fraction was dried to obtain 41.5 g of an alkaline extract of Agaricus blazei.

The above-mentioned [lung cancer cell test], [skin cancer cell test], and [apoptosis-inducing action] were performed using an alkaline extract of Agaricus blazei in place of the active ingredient derived from Porphyra chinensis.

(Comparative Example 3)
The above [lung cancer cell test], [skin cancer cell test] and [apoptosis-inducing action] were carried out using physiological saline instead of the active ingredient derived from Pteriophyllum fungi.

The results of PD-L1, PD-L2 and AXL in the [lung cancer cell test] of Examples 1-3 and Comparative Examples 1-3 are shown in FIGS. 1-3, respectively. 4 to 6 show the results of PD-L1, PD-L2 and AXL in the [skin cancer cell test] of Examples 2 to 5 and Comparative Examples 1 to 3, respectively. The number of data N in Examples 1 to 5 and Comparative Examples 1 to 3 is 3. The amount of mRNA for PD-L1, PD-L2, and AXL was calculated as a ratio to the expression level of GAPDH, which is a subject, and is described as a value when the average value of Comparative Example 3, which is a negative control, is set to 1.0. ing.

The Agaricus blazei extract used in Comparative Example 2 is known to have the effect of reducing the expression of PD-L1, PD-L2 and AXL. Therefore, Comparative Example 2 was used as a positive control.

In the [lung cancer cell test], it was confirmed that the amount of AXL-related mRNA in Examples 1-3 was significantly decreased compared to Comparative Examples 1 and 3. In particular, in Example 1, it was confirmed that the amount of mRNA was smaller than in Comparative Example 2.

In [Lung cancer cell test], it was confirmed that the amount of PD-L1-related mRNA in Examples 2 and 3 tended to decrease compared to Comparative Examples 1 and 3. Especially in Example 3, it was found that there was an effect comparable to that of Comparative Example 2.

In the [lung cancer cell test], it was confirmed that the amount of PD-L2 mRNA in Examples 1 to 3 was inferior to that in Comparative Example 2, but significantly decreased compared to Comparative Examples 1 and 3.

In [skin cancer cell test], it was confirmed that the amount of AXL mRNA in Example 3 was significantly decreased compared to Comparative Examples 1 and 3.

In the [skin cancer cell test], it was confirmed that the amount of PD-L1-related mRNA in Examples 2-3 and 5 tended to decrease compared to Comparative Examples 1 and 3. Especially in Example 3, it was confirmed that the amount of mRNA was smaller than in Comparative Example 2.

In the [skin cancer cell test], it was confirmed that the amount of PD-L2-related mRNA in Examples 2 and 3 was significantly decreased as compared with Comparative Examples 1 and 3. Especially in Example 3, it was found that there was an effect comparable to that of Comparative Example 2.

The results of [apoptosis-inducing action] of Example 1 and Comparative Examples 2 and 3 are shown in FIGS. Of the four divided regions in each graph of FIG. 7, the lower left region is the viable cell region, the lower right region is the early apoptosis region, and the upper right region is the late apoptosis region. The more dots plotted in the early apoptosis region and the late apoptosis region, the stronger the apoptosis-inducing effect. Figure 8 shows the percentage of cells stained with Annexin V ⁺ .

It was confirmed that the percentage of cells stained with Annexin V ⁺ in Example 1 was significantly higher than that in Comparative Example 3 and was comparable to that in Comparative Example 2.

Based on the above results, it was confirmed that the P. pumila extract suppresses the expression of PD-L1, PD-L2 and AXL. The T120 strain was confirmed to suppress the expression of PD-L1, PD-L2 and AXL in lung cancer cells and skin cancer cells. It was confirmed that the T108 strain has an inhibitory effect on the expression of PD-L1 on skin cancer cells.

Moreover, in Examples 1 to 5, the active ingredient from the artificially cultured mycelium or the culture medium is used instead of the fruiting body of P. moritake. From the results of Examples 1 to 5, it was confirmed that even the active ingredients from artificially cultured mycelia or culture solutions suppress the expression of PD-L1, PD-L2 and AXL.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an anticancer drug resistance suppressor that is extracted from a basidiomycete, Pleurotus pallidum, and that is unlikely to cause acquired resistance, and a therapeutic agent for cancer containing the same.

Claims (16)

A therapeutic agent for cancer, containing an active ingredient derived from Pleurotus fungus. 2. The therapeutic agent for cancer according to claim 1, wherein the active ingredient is a dry powder of a culture solution of P. pallidum fungus artificially cultured. 2. The therapeutic agent for cancer according to claim 1, wherein the active ingredient is an alcoholic extract from the culture broth of P. pylorisis artificially cultured. 2. The therapeutic agent for cancer according to claim 1, wherein the active ingredient is an alcoholic extract from the fruiting body or mycelium of P. pumila. The cancer therapeutic agent according to any one of claims 1 to 4, wherein the active ingredient inhibits AXL expression. The cancer therapeutic agent according to any one of claims 1 to 5, wherein the active ingredient inhibits expression of an immune checkpoint molecule. 7. The therapeutic agent for cancer according to claim 6, wherein the immune checkpoint molecule is at least one of PD-L1 and PD-L2. Claim 1, wherein the termite fungus is Termitomyces sp. T-120 strain, which is a basidiomycete deposited at the National Institute of Technology and Evaluation Patent Microorganism Depositary under the accession number NITE P-03541. 8. The cancer therapeutic agent according to any one of -7. 9. The therapeutic agent for cancer according to claim 8, wherein the therapeutic agent for cancer is at least one of a therapeutic agent for lung cancer and a therapeutic agent for skin cancer. Claim 1, wherein the termite fungus is Termitomyces sp. T-108 strain, which is a basidiomycete deposited at the National Institute of Technology and Evaluation Patent Microorganism Depositary under the accession number NITE P-03542. 8. The cancer therapeutic agent according to any one of -7. 11. The therapeutic agent for cancer according to claim 10, wherein the therapeutic agent for cancer is a therapeutic agent for skin cancer. The therapeutic agent for cancer according to any one of claims 1 to 11, further comprising at least one of an anticancer agent, an antibiotic, an anti-inflammatory agent, an antipyretic and an analgesic. a step of liquid-cultivating P. aeruginosa; a step of filtering the culture of P. aeruginosa obtained by the liquid culture to separate the culture solution and a residue; and a step of drying the culture solution to obtain a dry solidified product. and a step of pulverizing the dried and solidified product to obtain a dry powder. 14. The method for producing a cancer therapeutic agent according to claim 13, further comprising the steps of: shaking said dry powder with alcohol; and recovering and concentrating said alcohol to dryness to obtain an alcoholic extract. a step of liquid culturing P. aeruginosa; a step of filtering the culture of P. aeruginosa obtained by said liquid culture to separate the culture solution and a residue; a step of drying said residue; and a step of drying said residue. a step of shaking the pulverized residue with alcohol; and a step of recovering the alcohol and concentrating and drying to obtain an alcohol extract. The termite fungus Termitomyces sp. T-108 strain, which is a basidiomycete deposited at the National Institute of Technology and Evaluation Patent Microorganism Depositary under the accession number NITE P-03542, or an independent administrative agency product The T-120 strain of Termitomyces sp. T-120, which is a basidiomycete deposited at the Patent Microorganisms Depositary Center of the Evaluation Technology Base Organization under the accession number NITE P-03541, according to any one of claims 13-15. A method for producing a cancer therapeutic agent.

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