Tumor immunomodulator and application thereof
阅读说明：本技术 一种肿瘤免疫调节剂及其用途 (Tumor immunomodulator and application thereof ) 是由 尹汉维 曾慧慧 于 2018-08-06 设计创作，主要内容包括：本发明属于肿瘤治疗技术领域,公开了一种肿瘤免疫调节剂及其用途。所述免疫调节剂至少包括苯并异硒唑衍生物,优选包括苯并异硒唑衍生物和铂类抗癌药物,所述苯并异硒唑衍生物与铂类抗癌药物的摩尔比为(1～99):(1～99)。该免疫调节剂能够用于肿瘤治疗,例如早期肿瘤免疫调节和术后肿瘤复发免疫调节。BS本身具有的凋亡诱导+免疫调节的协调作用,使BS能够作为长期使用的、且使患者机体受益的术后肿瘤复发控制调节剂。进一步的,BS与顺铂联合应用则会放大该协调作用,从而使临床更多患者受益。(The invention belongs to the technical field of tumor treatment, and discloses a tumor immunomodulator and application thereof. The immunomodulator at least comprises a benzisoselenazole derivative, preferably comprises a benzisoselenazole derivative and a platinum anti-cancer drug, and the molar ratio of the benzisoselenazole derivative to the platinum anti-cancer drug is (1-99) to (1-99). The immunomodulator can be used for tumor therapy, such as early tumor immunomodulator and postoperative tumor recurrence immunomodulator. The BS has the coordination of apoptosis induction and immunoregulation, so that the BS can be used as a postoperative tumor recurrence control regulator which is used for a long time and benefits the organism of a patient. Further, the use of BS in combination with cisplatin amplifies this coordination and thereby benefits more patients clinically.)
1. A tumor immunomodulator, wherein the immunomodulator comprises at least a benzisoselenazole derivative.
2. The tumor immunomodulator according to claim 1, wherein the benzoisoselenazole derivative has a structure of a compound represented by formula A, and is selected from at least one of a compound represented by formula A, a precursor thereof, an active metabolite, a stereoisomer, a pharmaceutically acceptable salt, a prodrug, and a solvate thereof,
wherein R is1、R2Identical or different, independently of one another, from H or the following radicals: c1-12Alkyl radical, C3-20A cycloalkyl group;
wherein R is selected from C1-12Alkylene, phenylene, biphenylene, triphenylene, or
3. The tumor immunomodulator according to claim 2, wherein in the compound of formula A, R is1、R2Identical or different, independently of one another, from H, -CH3、-CH2CH3、-CH(CH3)2、-C(CH3)3、-CH(CH2)4or-CH (CH)2)5(ii) a R is selected from-CH2-、-C2H4-、-C4H8-, phenylene-C6H4-。
4. The tumor immunomodulator according to any one of claims 1 to 3, wherein the benzisoselenazole derivative is selected from 1, 2-bis [2- (1, 2-benzisoselenazol-3 (2H) -one) ] -Butane (BS), which has the following structure:
5. the tumor immunomodulator according to any one of claims 1 to 4, wherein the tumor immunomodulator comprises a benzisoselenazole derivative and a platinum anticancer drug;
preferably, the platinum-based anticancer drug is selected from at least one of Cisplatin (CDDP), Carboplatin (CBP), oxaliplatin (1-OHP), and nedaplatin (CDGP).
6. The tumor immunomodulator according to claim 5, wherein the molar ratio of the benzisoselenazole derivative to the platinum anticancer drug is (1-99): (1-99);
preferably, the molar ratio of the benzisoselenazole derivative to the platinum-based anticancer drug is (50-90): (1-30), (60-70): 1-10), (1-40): 1-40), (1-25): 1-25), (1-10): 1-10), (1-6): 1-6);
more preferably, the molar ratio of the benzisoselenazole derivative to the platinum-based anticancer drug is 1:1, 2:1 (in vitro combination) or 60:1, 70:1, 80:1 (in vivo combination).
7. The tumor immunomodulator according to any one of claims 1 to 6, wherein the tumor immunomodulator further comprises at least one pharmaceutically acceptable auxiliary material.
8. Use of a tumor immunomodulator according to any one of claims 1 to 7 in the treatment of tumors; preferably, the dose of the immunomodulator is 10-500 mg/kg based on the weight of the object to be applied.
9. The use according to claim 8, of said immunomodulator in early tumour immunomodulation.
10. The use according to claim 8, of an immunomodulator in the immunomodulation of postoperative tumor recurrence.
Technical Field
The invention belongs to the technical field of tumor treatment, and particularly relates to a tumor immunomodulator and application thereof.
Background
Tumor cells evade recognition and attack by the body's immune system through various mechanisms, resulting in a phenomenon of survival and proliferation in vivo, called tumor immune escape. The immune system of the body has an immune monitoring function, and when malignant cells appear in the body, the immune system can recognize and specifically eliminate the non-self cells through an immune mechanism to resist the occurrence and development of tumors. However, malignant cells can in some cases escape immune surveillance of the body by multiple mechanisms, proliferate rapidly in vivo, and form tumors. That is to say: on the one hand, the body can resist the development of tumors by natural and acquired immunity; on the other hand, tumor cells can evade recognition and attack by the body's immunity through a variety of mechanisms. Whether a tumor occurs or not and how it is classified depends on the overall effect of both aspects. The tumor immune escape mechanism mainly has the following aspects: firstly, low immunogenicity; secondly, the antigen is identified as self-antigen; thirdly, antigen modulation; fourthly, immunosuppression induced by tumor; and fifthly, inducing the tumor to generate a exempted area.
The existing tumor treatment means comprise surgery treatment, chemotherapy, radiotherapy, immunotherapy and other treatment means, wherein the surgery, the radiotherapy and the chemotherapy are three main roles which cannot be shocked by the tumor treatment, but the immunity of the organism is reduced and the immunity of the organism is inhibited in a more serious way in the processes of the surgery, the radiotherapy and the chemotherapy, so that residual tumor cells can be continuously and further transferred, and the fatal threat is brought to tumor patients. Especially, the operation is used as a mechanical means, the local treatment is thorough, but the cancer cell is dropped after the cancer is attacked due to the metastasis of the cancer cell, and the cancer cell is proliferated or metastasized to the periphery and far through local diffusion, blood vessels, lymphatic vessels and other ways. Furthermore, the patient's own immunity is in a very weak state for a long time after the operation, and the normal immunity of the body cannot be exerted, so how to improve the immunity of the body and stabilize the immune system of the body after the operation is a difficult problem of controlling the recurrence of the tumor after the operation. According to the monitoring data of the cancer condition of the disease pre-control center in China, the recurrence and metastasis rate of cancer patients in China after operation for 3 months is 50%, the recurrence and metastasis rate of cancer patients in China after operation for 6 months is as high as 69%, and the recurrence or metastasis rate of cancer patients after operation for five years is as high as more than 90%.
After the 21 st century, with the development of oncology and immunology, the treatment of tumors around the human immune system is gradually accepted by the scientific research and industrial fields, and becomes a popular field for the research and development of new drugs. Tumor immunotherapy (I/O) refers to a method for effectively treating a tumor patient by directly or indirectly using the human immune system during the course of treatment. For example, killing or inhibiting tumors is achieved by interfering with the direct antitumor effects of cell overgrowth, transformation and metastasis or activating effector cells of the immune system and factors secreted by them.
Therefore, the development of an immunomodulator for postoperative tumor recurrence, which can improve the immunity of the organism, especially the postoperative tumor organism immunity, has low toxicity and long-term use, and can benefit the organism of a patient, is a technical problem to be solved urgently.
Disclosure of Invention
The invention provides a tumor immunomodulator, which at least comprises a benzisoselenazole derivative.
According to the invention, the benzisoselenazole derivative has a structure of a compound shown as a formula A, and is selected from at least one of the compound shown as the formula A, a precursor, an active metabolite, a stereoisomer, a pharmaceutically acceptable salt, a prodrug and a solvate thereof,
wherein R is1、R2Identical or different, independently of one another, from H or the following radicals: c1-12Alkyl radical, C3-20A cycloalkyl group; preferably, it is selected from H, or the following groups: c1-6Alkyl radical, C3-10A cycloalkyl group; illustratively, R1、R2Is selected from H.
Wherein R is selected from C1-12Alkylene, phenylene, biphenylene, triphenylene, or
Preferably, in the compound shown in the formula A, R1、R2Identical or different, independently of one another, from H, -CH3、-CH2CH3、-CH(CH3)2、-C(CH3)3、-CH(CH2)4or-CH (CH)2)5(ii) a R is selected from-CH2-、-C2H4-、-C4H8-, phenylene-C6H4-。
According to an exemplary embodiment of the present invention, the benzisoselenazole derivative is selected from 1, 2-bis [2- (1, 2-benzisoselenazol-3 (2H) -one) ] -Butane (BS), the structure of which is shown below:
according to the present invention, the tumor immunomodulator comprises a benzisoselenazole derivative and a platinum anticancer drug. Specifically, the tumor immunomodulator is formed by combining a benzoisoselenazole derivative and a platinum anti-cancer drug properly, and shows an immunoregulation effect.
The platinum anticancer drug is at least one selected from Cisplatin (CDDP), Carboplatin (CBP), oxaliplatin (1-OHP), nedaplatin (CDGP) and the like; illustratively, the platinum-based anti-cancer drug is selected from Cisplatin (CDDP).
According to the invention, the molar ratio of the benzisoselenazole derivative to the platinum anticancer drug is (1-99) to (1-99); for example, the molar ratio is (50-90): (1-30), (60-70): 1-10), (1-40): 1-40), (1-25): 1-25), (1-10): 1-10), (1-6): 1-6); illustratively, the molar ratio is 1:1, 2:1 (in vitro combination) or 60:1, 70:1, 80:1, etc. (in vivo combination). Specifically, when the molar ratio of the benzisoselenazole derivative to the platinum-based anticancer drug is not very different, such as the molar ratio of (1-10): 1-10), (1-6): 1-6), and exemplarily, the molar ratio is 1:1 and 2:1, the benzisoselenazole derivative and the platinum-based anticancer drug are used together as an immunomodulator and preferably can be applied to in vitro tumor cells. In vivo combination, because the in vivo metabolism of the drug to reach the tumor site concentration has a great relationship with in vivo properties, the dosage of the oral benzisoselenazole derivative is far greater than that of the platinum anticancer drug (conventional injection administration), i.e. when the dosage of the benzisoselenazole derivative is far greater than that of the platinum anticancer drug, for example, the molar ratio is (50-90): 1-30), (60-70): 1-10, exemplarily, the molar ratio is 60:1, 70:1, and 80:1 (in vivo combination), the benzisoselezole derivative and the platinum anticancer drug are used as immunomodulators, and preferably, the benzisoselezole derivatives can be applied to in vivo tumor cells.
Preferably, the tumor immunomodulator may further optionally comprise at least one pharmaceutically acceptable adjuvant.
Preferably, the pharmaceutically acceptable excipients are various excipients commonly used or known in the pharmaceutical field, including but not limited to: diluents, binders, antioxidants, pH adjusters, preservatives, lubricants, disintegrants, and the like.
For example, the diluent is selected from lactose, starch, cellulose derivatives, inorganic calcium salts, sorbitol and the like. The binder is, for example: starch, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone, and the like. For example, the antioxidant is selected from vitamin E, sodium bisulfite, sodium sulfite, butylated hydroxyanisole, and the like. For example, the pH adjusting agent is selected from hydrochloric acid, sodium hydroxide, citric acid, tartaric acid, Tris, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, and the like. For example, the preservative is selected from methyl paraben, ethyl paraben, m-cresol, benzalkonium chloride, and the like. For example, the lubricant is selected from magnesium stearate, aerosil, talc, and the like. The disintegrant is, for example: starch, methyl cellulose, xanthan gum, croscarmellose sodium, and the like.
The formulation of the immunomodulator may be in the form of an oral preparation, such as tablets, capsules, pills, powders, granules, suspensions, syrups, and the like; it can also be administered by injection, such as injection solution, powder for injection, etc., by intravenous, intraperitoneal, subcutaneous or intramuscular route. All dosage forms used are well known to those of ordinary skill in the pharmaceutical arts.
Furthermore, the invention also provides the application of the immunomodulator in tumor treatment.
Preferably, the immunomodulator is used in an amount of 10-500 mg/kg based on the weight of a subject (such as a mouse); for example, 50 to 400 mg/kg; illustratively, the amount is 90, 180, 360 mg/kg.
Preferably, the immunomodulator can be selected from benzisoselenazole derivatives, and benzisoselenazole derivatives and platinum anticancer drugs for combined application. When the immunomodulator is combined with the benzisoselenazole derivative and the platinum anticancer drug, the dosage of the platinum anticancer drug is 0.1-5 mg/kg based on the weight of an administration object (such as a mouse); for example, 0.5 to 4mg/kg, 1 to 3 mg/kg; illustratively, the amount is 2 mg/kg.
In another example, the immunomodulator is used for inhibiting the expression of PD-L1 protein on the surface of tumor cells and promoting the secretion of cytokines (such as IFN-gamma, IL-2 and TNF- α), and in another example, the immunomodulator is used for promoting lymphocyte CD8 +An increase in the percentage of subpopulation positive cells; as another example, the immunomodulator is used to promote secretion of interferons (e.g., granzyme B, Gz-B).
According to another embodiment of the present invention, the immunomodulator can also be used for immunomodulation of postoperative tumor recurrence. Illustratively, the tumor immunomodulator is used for promoting the secretion of immune active substances (such as perforin and granzyme B), and improving the killing activity of cytotoxic T lymphocytes and NK cells; as another example, the tumor immunomodulator is used to promote lymphocyte proliferation; in another example, the tumor immunomodulator is used to upregulate expression of a lymphocyte-surface NKG2D activating receptor, preferably, expression of a lymphocyte-surface NKG2D ligand RAE-1.
According to the invention, the tumors include solid tumors and non-solid tumors, and may be benign and malignant. For example, the tumors include, but are not limited to: liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, gastric cancer, skin cancer, bladder cancer, ovarian cancer, prostate cancer, bone cancer, brain cancer, rectal cancer, esophageal cancer, tongue cancer, lymph cancer, oral epithelial cancer, epithelial cervical cancer or chronic myelogenous leukemia. Specifically, tumor cells can be selected, and the tumor cells include human cancer cells and mouse cancer cells. Such human cancer cells include, but are not limited to: human liver cancer cell HepG2, human liver cancer cell Be17402, human liver cancer cell Huh7721, human colorectal cancer cell LoVo, human colorectal cancer cell RKO, human colorectal cancer cell SW480, human lung cancer cell A549, human lung cancer cell H1299, human lung cancer cell SPCA-1, human epithelial cervical cancer cell HeLa, human breast cancer cell MCF-7, human chronic myelogenous leukemia cell k562, human esophageal cancer cell KYSE150, human esophageal cancer cell KYSE450 or human esophageal cancer cell KYSE 510. The mouse cancer cells include, but are not limited to: mouse liver cancer cell H22, mouse liver cancer cell Hepa 1-6, mouse erythroleukemia cell MEL, mouse kidney cancer cell Renca, mouse lymphoma cell EL-4, mouse lung cancer cell Lewis, and mouse breast
Further, the present invention also provides a method of treating tumor or postoperative tumor recurrence by the above-mentioned tumor immunomodulator, by administering a therapeutically effective amount of the immunomodulator to an individual in need thereof.
Preferably, the subject may be a mammal, such as a human or a mouse.
Preferably, the tumor has the meaning as described above, preferably liver cancer, lung cancer, breast cancer.
The invention has the beneficial effects that:
the invention unexpectedly discovers that the BS has extremely low toxicity, can obviously improve the toxic damage of the drug to the organism, can also promote the coefficient of immune organs (such as spleen, thymus and the like) to be increased, and can promote the increase of the number of peripheral blood leukocytes, for example, can obviously inhibit the expression of PD-L1 protein on the surface of tumor cells, promote the secretion of cytokines (such as IFN-gamma, IL-2 and TNF- α), and can promote cytotoxic T lymphocyte CD (compact disc) of T lymphocytes8 +Subgroup positiveAn increase in the percentage of cells; can promote the secretion of interferon (such as granzyme B, Gz-B). The BS has the functions of enhancing immunity and weakening toxic injury to organisms, and can improve the cellular immunity level of the organisms when being used as an immunomodulator, thereby inhibiting the proliferation of tumor cells and the relapse of postoperative tumors.
The invention also unexpectedly discovers that when the BS is combined with a platinum anti-cancer drug (particularly cisplatin CDDP), the BS can synergistically promote the secretion of immune active substances (such as perforin and granzyme B) and improve the killing activity of cytotoxic T lymphocytes and NK cells; can promote lymphocyte proliferation; can up-regulate the expression of RAE-1, a lymphocyte surface NKG2D ligand. The BS and the CDDP can synergistically promote the secretion of immune active substances, and can improve the immunity of the organism and inhibit the proliferation of tumor cells and postoperative tumor recurrence as an immunomodulator.
The immune regulation function of the BS and the combined use of the BS and the cisplatin plays an important role in controlling postoperative tumor recurrence. At present, the postoperative recurrence control is realized by directly killing tumors with treatment and/or chemotherapy drugs clinically, so as to control the tumor recurrence. However, the chemical killing drugs used in clinical practice at present may damage the immune function of the body to a certain extent and cause immunosuppressive reactions, for example, adverse reactions (for example, renal toxicity: repeated administration at multiple times and in a short period of time, irreversible renal dysfunction, bone marrow suppression: reduction of leukocytes and/or platelets) generated by the body of a patient after platinum drugs are used may gradually lead to the final body being unable to bear the adverse reactions.
The BS and the cisplatin jointly show that the activity of the BS on NK is improved, the function of the additive pharmacological action is generated on the basis of the apoptosis action of the BS on the inhibition of the tumor growth, the BS medicament has the coordination action of apoptosis induction and immunoregulation and extremely low toxicity of the BS, the toxic injury brought to an organism by long-term administration can be obviously improved, and the BS can be used as a postoperative tumor recurrence control regulator which is used for a long time and benefits the organism of a patient. Further, the use of BS in combination with cisplatin amplifies this coordination and thereby benefits more patients clinically.
Definition and description of terms
Unless otherwise indicated, the definitions of groups and terms described in the specification and claims of the present application, including definitions thereof as examples, exemplary definitions, preferred definitions, definitions described in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. The definitions of the groups and the structures of the compounds in such combinations and after the combination are within the scope of the present specification.
The term "C1-12Alkyl is understood to preferably mean a straight-chain or branched, saturated monovalent hydrocarbon radical having from 1 to 12 carbon atoms, preferably C1-6An alkyl group. "C1-6Alkyl "is understood to preferably mean a straight-chain or branched, saturated monovalent hydrocarbon radical having 1,2, 3, 4, 5, 6 carbon atoms. The alkyl group is, for example, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, an isopropyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an isopentyl group, a 2-methylbutyl group, a 1-ethylpropyl group, a 1, 2-dimethylpropyl group, a neopentyl group, a 1, 1-dimethylpropyl group, a 4-methylpentyl group, a 3-methylpentyl group, a 2-ethylbutyl group, a 1-ethylbutyl group, a 3, 3-dimethylbutyl group, a 2, 2-dimethylbutyl group, a 1, 1-dimethylbutyl group, a 2, 3-dimethylbutyl group, a 1, 3-dimethylbutyl group or a 1, 2-dimethylbutyl group. In particular, the radicals have 1,2, 3, 4, 5, 6 carbon atoms ("C)1-6Alkyl groups) such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, tert-butyl, more particularly groups having 1,2, 3 or 4 carbon atoms ("C)1-4Alkyl groups) such as methyl, ethyl, n-propyl, isopropyl or butyl.
The term "C3-20Cycloalkyl is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 20 carbon atoms, preferably "C3-10Cycloalkyl groups ". The term "C3-10Cycloalkyl radicals "shouldIs understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Said C is3-10Cycloalkyl groups may be monocyclic hydrocarbon groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or bicyclic hydrocarbon groups such as decalin rings.
The term "C1-12Alkylene is understood to mean preferably a straight-chain or branched, saturated monovalent hydrocarbon radical having from 1 to 12 carbon atoms, preferably C, with the loss of two hydrogen atoms1-4An alkylene group. "C1-4Alkylene "is understood to preferably mean a straight-chain or branched, saturated monovalent hydrocarbon radical having 1,2, 3, 4 carbon atoms which has lost two hydrogen atoms. The alkylene group is, for example, methylene, ethylene, propylene, butylene.
The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound of the present invention sufficient to effect the intended use, including but not limited to the treatment of a disease as defined below. The therapeutically effective amount may vary depending on the following factors: the intended application (in vitro or in vivo), or the subject and disease condition being treated, such as the weight and age of the subject, the severity of the disease condition and the mode of administration, etc., can be readily determined by one of ordinary skill in the art. The specific dosage will vary depending on the following factors: the particular compound selected, the dosage regimen to be followed, whether to administer it in combination with other compounds, the timing of administration, the tissue to be administered and the physical delivery system carried.
Drawings
FIG. 1 is a graph showing the effect of BS on mouse body weight and tumor volume in example 1;
wherein, A: change in body weight of the mouse; b: mice relative body weight changes. Data are presented as mean ± standard deviation (n ═ 6).
FIG. 2 is a graph showing the tumor growth time and tumor growth rate of each group of mice in example 1.
FIG. 3 is a graph showing tumor growth in the groups of mice in example 1;
wherein (a): photographs of each group of tumors at the end of the experiment; (b) the method comprises the following steps Tumor volume changes in mice of each group. .
FIG. 4 is a graph showing the effect of BS on the inhibition of the expression of PD-L1 in tumor cells in example 1.
FIG. 5 shows lymphocyte CD4 in example 1+、CD8+Graph of percentage of positive cells of subpopulations;
wherein, A: control group (control); b: BSL; c: BSM; d: BSH.
FIG. 6 is a graph showing the change in body weight of mice in example 1;
wherein (a): the weight change of the mice in the administration process; (b) the method comprises the following steps Weight gain during administration in each group of mice.
FIG. 7 shows the change in the body weight of mice in example 2;
wherein (a): the weight change of the mice in the administration process; (b) the method comprises the following steps Weight gain during administration in each group of mice.
FIG. 8 shows the change in the body weight of mice in example 2;
wherein (a): the weight change of the mice in the administration process; (b) the method comprises the following steps Weight gain during administration in each group of mice.
FIG. 9 shows RAE-1 protein expression levels in mouse tumor tissues in example 3;
wherein (a): mouse tumor tissue RAE-1 expression level; (b) the method comprises the following steps Relative expression level of RAE-1. Data are presented as mean ± standard deviation, n ═ 3. P <0.05, indicating a statistical difference compared to the control group.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. The following examples are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of the invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
The experimental method comprises the following steps:
1. test drugs, cells and animals:
BS: the chemical name of the product is 1, 2-bis [2- (1, 2-benzisoselenazol-3 (2H) -ketone) ] -butane, which is referred to Chinese patent No. 02158917.8.
Cisplatin (CDDP): purchased from Hai Wei, China (Beijing) Gene science and technology, Inc.
Mouse hepatoma cell line H22: presented by a Chinese toon researcher in the institute of tumor prevention and treatment of Beijing university.
Mouse lung cancer cell line Lewis: purchased from basic medical college of cooperative medical university.
Mouse breast cancer cell line 4T 1: purchased from basic medical college of cooperative medical university.
Kunming inbred mice (KM mice): male, 4 weeks, with a weight of 18-22g, purchased from the experimental animals center of the department of medicine of Beijing university, the license number of experimental animals: SYXK (Kd) 2012 and 0036. The breeding environment is clean, the breeding temperature is 25 +/-2 ℃, the illumination is 12 hours and the darkness is 12 hours after 12 hours, and the food and water supply is sufficient.
Balb/c mice: the experimental animal is purchased from the center of experimental animals of department of medicine of Beijing university, and the production license number SYXK (Jing) 2012-0036 of the experimental animals, wherein the experimental animal is 4 weeks old, the weight of the experimental animal is 16-18 g, and male (lung cancer and postoperative recurrence of liver cancer) and female (postoperative recurrence of breast cancer). The breeding environment is clean, the breeding temperature is 25 +/-2 ℃, the illumination is 12h for 12h, the breeding environment is dark, and the food and water supply is sufficient.
2. The test method comprises the following steps:
mouse H22 liver cancer/Lewis lung cancer/
Respectively taking out H22 (liver cancer) cryopreserved cells/Lewis (lung cancer) cryopreserved cells/4T 1 (breast cancer) cryopreserved cells, taking out after the cells become liquid at 37 ℃ for 1-2 min; and (4) centrifuging after resuspension, discarding the supernatant after centrifuging at 1200rpm/min for 2min, resuspending in 1mL serum-free medium, and injecting cells into the abdominal cavity.
Approximately one week later, when the abdomen of the mouse was touched to have a swelling sensation, ascites was extracted. The abdominal cavity outer skin of the mouse is slightly reduced with a small opening by scissors and tweezers, then the two sides of the hemostatic forceps are slightly pulled open to expose the abdomen, and the ascites is extracted by a syringe.
Preparing ascites of Balb/c mouse into single cell suspension, counting the number of living cells by trypan blue dye exclusion method, and ensuring high cell survival rateAt a rate of 95%; injecting the tumor cell suspension into the right axilla of male Balb/c mice with the weight of 16g-18g, 10g each6And (4) cells.
2.1 mouse H22 model for excision of liver cancer transplantable tumor
Balb/c mice subcutaneously transplanted with mouse hepatoma cells H22 were subjected to local tumor resection as follows: when the right axillary tumor of the mouse reaches about 500mm3In this case, 5% chloral hydrate solution (0.08mL/10g) was intraperitoneally injected for anesthesia and hair preparation. After the mice enter the general anesthesia state, a 1-2cm incision is cut near the tumor, the tumor tissue is blunt stripped and cut off 90%, and the residual is about 50mm3Finally, the tumor tissue is sutured by using sterile medical silk threads, and the wound is coated with iodophor for disinfection. The above operations are all completed in a super clean bench. Mice were placed in a clean, warm place and given sterile aqueous glucose for their consumption after awakening. The day of surgery is considered to be 0 days after surgery. After the mice were fully awakened, the mice were randomly divided into 4 groups of 6 mice each, and the drug treatment protocol was as follows:
a blank control group (5% CMC-Na, i.g., q.d.);
BS Low dose group (90 mg. kg)-1,i.g.,q.d.)；
BS Medium dose group (180 mg. kg)-1,i.g.,q.d.)；
BS high dose group (360 mg. kg)-1,i.g.,q.d.)。
Mice body weight, tumor volume and activity were recorded at intervals during the experiment. The mice were sacrificed eight days after administration, and the eyeballs were removed to obtain blood for the detection of TrxR activity in serum. Tumors, liver, kidney, thymus and spleen were rapidly isolated, observed and weighed. Tumor tissues were kept in liquid nitrogen for subsequent experiments.
2.2 mouse Lewis Lung cancer transplantation tumor excision model
As above, Balb/c mice subcutaneously transplanted with murine lung carcinoma cells Lewis were subjected to local tumor resection as follows: when the right axillary tumor of the mouse reaches about 500mm3In this case, 5% chloral hydrate solution (0.08mL/10g) was intraperitoneally injected for anesthesia and hair preparation. After the mice enter the general anesthesia state, a 1-2cm incision is cut near the tumor, and the tumor group is divided into two groupsThe blunt tissue is stripped off and cut 90% with a residual of approximately 50mm3Finally, the tumor tissue is sutured by using sterile medical silk threads, and the wound is coated with iodophor for disinfection. The above operations are all completed in a super clean bench. Mice were placed in a clean, warm place and given sterile aqueous glucose for their consumption after awakening. The day of surgery is considered to be 0 days after surgery. After the mice were fully awakened, the mice were randomly divided into 4 groups of 6 mice each, and the drug treatment protocol was as follows:
a blank control group (5% CMC-Na, i.g., q.d.);
BS Low dose group (90 mg. kg)-1,i.g.,q.d.)；
BS Medium dose group (180 mg. kg)-1,i.g.,q.d.)；
BS high dose group (360 mg. kg)-1,i.g.,q.d.)。
Mice body weight, tumor volume and activity were recorded at intervals during the experiment. The mice were sacrificed eight days after administration, and the eyeballs were removed to obtain blood for the detection of TrxR activity in serum. Tumors, liver, kidney, thymus and spleen were rapidly isolated, observed and weighed. Tumor tissues were kept in liquid nitrogen for subsequent experiments.
2.3 mouse 4T1 Breast cancer graft tumor excision model
Meanwhile, Balb/c mice subcutaneously transplanted with mouse mammary cancer cells 4T1 were subjected to local tumor resection as follows: when the right axillary tumor of the mouse reaches about 500mm3In this case, 5% chloral hydrate solution (0.08mL/10g) was intraperitoneally injected for anesthesia and hair preparation. After the mice enter the general anesthesia state, a 1-2cm incision is cut near the tumor, the tumor tissue is blunt stripped and cut off 90%, and the residual is about 50mm3Finally, the tumor tissue is sutured by using sterile medical silk threads, and the wound is coated with iodophor for disinfection. The above operations are all completed in a super clean bench. Mice were placed in a clean, warm place and given sterile aqueous glucose for their consumption after awakening. The day of surgery is considered to be 0 days after surgery. After the mice were fully awakened, the mice were randomly divided into 4 groups of 6 mice each, and the drug treatment protocol was as follows:
a blank control group (5% CMC-Na, i.g., q.d.);
BS inhibitorDosage group (90mg kg)-1,i.g.,q.d.)；
BS Medium dose group (180 mg. kg)-1,i.g.,q.d.)；
BS high dose group (360 mg. kg)-1,i.g.,q.d.)。
Mice body weight, tumor volume and activity were recorded at intervals during the experiment. The mice were sacrificed eight days after administration, and the eyeballs were removed to obtain blood for the detection of TrxR activity in serum. Tumors, liver, kidney, thymus and spleen were rapidly isolated, observed and weighed. Tumor tissues were kept in liquid nitrogen for subsequent experiments.
2.4 Western Blot method:
extracting total protein of tissue, determining protein concentration (30-50 μ g), adding 5 × loading buffer solution, adding RIPA lysate to make up volume, mixing, and performing denaturation treatment at 95 deg.C for 10 min. After fully and uniformly mixing, adding a clean 1.5mm rubber plate to a proper position, and sealing with n-butanol. Adding 1mL of 1 XSEPARATION GEL buffer solution, and standing at room temperature overnight; inserting a 1.5mm sample adding comb, and standing at room temperature for 2h to wait for gelation.
Pulling out the sample adding comb, and loading the sample by a conventional method; and (4) performing constant voltage electrophoresis at 80V until the front edge of the bromophenol blue enters the separation gel, increasing the voltage to 160V, continuing the constant voltage electrophoresis, and stopping the electrophoresis until the bromophenol blue migrates to the end of the separation gel.
Accurately shearing 6 pieces of filter paper with the same size as the separation gel and a 0.2 mu m PVDF membrane, soaking the PVDF membrane in methanol for 10s, soaking in deionized water for 3min, and soaking in an electrotransformation buffer solution for 3 min;
the electricity changes sandwich and lays by the order of positive pole to negative pole in proper order: one sponge, three pieces of filter paper, a PVDF membrane, separation glue, three pieces of filter paper and one sponge (all soaked in an electric transfer buffer solution in advance), air bubbles are expelled from the layers by a glass rod, and an electric transfer clamp and an ice box are clamped and put into an electric transfer tank;
and (5) placing the electric rotating tank in an ice-water bath, and rotating for 2 hours at a constant current of 250 mA.
After the electrotransfer is finished, taking out the PVDF membrane, and placing the PVDF membrane in a 5% closed state; washing the membrane with TBST for 3 times, sealing the PVDF membrane and a diluted secondary antibody solution with a proper proportion in a hybridization bag, incubating for 1h at room temperature, washing the membrane with TBST for 3 times, and developing;
and opening a gel imaging system, uniformly mixing the solution A and the solution B of the ECL luminous liquid in equal volume, uniformly distributing the mixture on the PVDF membrane, and carrying out exposure analysis.
2.5 ELISA method: detection was performed according to the ELISA commercial kit protocol.