Application of benzisoselenazole derivative and antimetabolite in preparation of tumor treatment drug
阅读说明：本技术 苯并异硒唑衍生物与抗代谢药物联合用于制备***药物中的应用 (Application of benzisoselenazole derivative and antimetabolite in preparation of tumor treatment drug ) 是由 曾慧慧 尹汉维 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,公开了苯并异硒唑衍生物与抗代谢药物联合用于制备治疗肿瘤药物中的应用。苯并异硒唑衍生物具有如式A所示化合物的结构,所述抗代谢药物选自嘧啶拮抗剂、嘌呤拮抗剂和叶酸拮抗剂中的至少一种,所述苯并异硒唑衍生物与抗代谢药物的摩尔比为(1～10):(1～10)。本发明出人意料地发现,BS与抗代谢药物(尤其是5-FU)联合应用对肿瘤细胞增殖具有优异的协同抑制效果,尤其是对于肝癌细胞增殖抑制效果明显,对于肿瘤的治疗具有重大价值。<Image he="274" wi="700" file="DDA0001756036190000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention belongs to the technical field of tumor treatment, and discloses an application of a benzisoselenazole derivative and an antimetabolite in preparing a tumor treatment drug. The benzisoselenazole derivative has a structure of a compound shown as a formula A, the antimetabolite is selected from at least one of a pyrimidine antagonist, a purine antagonist and a folic acid antagonist, and the molar ratio of the benzisoselenazole derivative to the antimetabolite is (1-10): (1-10). The invention surprisingly discovers that the combined application of the BS and the antimetabolite (especially 5-FU) has excellent synergistic inhibition effect on the proliferation of tumor cells, especially has obvious inhibition effect on the proliferation of hepatoma cells and has great significance on the treatment of tumorsHas great value.)
1. Use of benzisoselenazole derivatives in combination with antimetabolites for the preparation of a medicament for the treatment of a tumor.
2. The use according to claim 1, wherein the benzisoselenazole derivative has a structure 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, orWherein M represents Pt, Pd or Rh.
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-。
3. Use according to claim 1 or 2, wherein the benzisoselenazole derivative is selected from 1, 2-bis [2- (1, 2-benzisoselenazol-3 (2H) -one) ] -butane, the structure of which is shown below:
4. the use according to any one of claims 1 to 3, wherein the antimetabolite is selected from at least one of a pyrimidine antagonist, a purine antagonist and a folate antagonist.
5. The use according to any one of claims 1 to 4, wherein the molar ratio of the benzisoselenazole derivative to the antimetabolite is (1-10) to (1-10).
6. The use according to any one of claims 1 to 5, wherein the tumour comprises solid tumours and non-solid tumours, both benign and malignant;
preferably, the tumor includes, but is 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.
7. The use according to any one of claims 1 to 6, wherein the benzisoselenazole derivative is used in combination with an antimetabolite to inhibit tumor cell proliferation.
8. The use of claim 7, wherein the tumor 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.
9. A method for inhibiting tumor cell proliferation by combining a benzisoselenazole derivative and an antimetabolite is characterized in that the benzisoselenazole derivative and the antimetabolite act on tumor cells according to a ratio;
preferably, the benzisoselenazole derivative has the meaning defined in claim 2 or 3, and the antimetabolite has the meaning defined in claim 4; the formulation has the meaning of claim 5;
preferably, the concentration of the benzisoselenazole derivative and the antimetabolite is 1-100 mu M;
preferably, the action time is 10-120 h.
10. A pharmaceutical composition comprising a benzisoselenazole derivative and an antimetabolite;
preferably, the benzisoselenazole derivative has the meaning defined in claim 2 or 3, and the antimetabolite has the meaning defined in claim 4; the formulation has the meaning of claim 5;
preferably, the pharmaceutical composition may further optionally comprise at least one pharmaceutically acceptable excipient.
Technical Field
The invention belongs to the technical field of tumor treatment, and particularly relates to an application of a benzisoselenazole derivative and an antimetabolite in preparation of a tumor treatment drug.
Background
Primary hepatocellular carcinoma (hereinafter, liver cancer) is one of the most common malignant tumors in humans. Liver cancer incidence is reported to be the sixth of all malignant tumor incidence worldwide, while mortality is the third. About 38.3 thousands of people die from liver cancer in China every year, accounting for 51 percent of the death cases of liver cancer worldwide, and seriously threatening the life health of human beings.
Because liver cancer cells have a high proliferation rate and are difficult to diagnose in an early stage, most patients have developed liver cancer in a late stage when liver cancer is diagnosed, and the liver cancer is often transferred in or out of the liver, and the factors are main reasons for high mortality rate of liver cancer. Therefore, the search for drugs capable of effectively inhibiting the proliferation of liver cancer cells is the key to the treatment of liver cancer.
Antimetabolites, drugs that specifically bind to in vivo metabolites to affect or antagonize metabolic function, are generally similar in chemical structure to nucleic acid or protein metabolites in the body. The fluorouracil (5-FU) is a typical antimetabolite, is a first-line anticancer drug for various cancers, is suitable for various cancers such as liver cancer, ovarian cancer, lung cancer and the like, and can play a broad-spectrum anticancer role through various mechanisms. However, the antimetabolites generally show bone marrow toxicity in the second week after administration, often manifested as leukopenia and thrombocytopenia, and thus, when the antimetabolites are used alone as anticancer drugs, they have high toxic and side effects, resulting in poor anticancer effects.
Disclosure of Invention
The invention provides an application of a benzisoselenazole derivative and a uracil derivative in preparation of a tumor treatment drug in a combined manner.
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, the structure of which is shown below:
according to the present invention, the antimetabolite drug may be selected from at least one of a pyrimidine antagonist, a purine antagonist, and a folic acid antagonist.
For example, the pyrimidine antagonist may be selected from uracil-derived agents and/or cytosine-derived agents. Preferably, the pyrimidine antagonist may be at least one selected from Fluorouracil (5-Fluorouracil, abbreviated as 5-FU), tegafur (Ftorafur), Carmofur (Carmofur), 5-acetyleneuridine (Eniluracil), doxifluridine, cytarabine hydrochloride, and the like; for example, the pyrimidine antagonist is selected from fluorouracil, tegafur or carmofur; illustratively, the pyrimidine antagonist is selected from fluorouracil (5-FU). For example, the purine antagonist may be selected from the group consisting of derivatives of hypoxanthine and guanine; preferably, the purine antagonist may be selected from mercaptopurine. For example, the folate antagonist can be selected from methotrexate.
According to the invention, the molar ratio of the benzisoselenazole derivative to the antimetabolite is (1-10) to (1-10); for example, the molar ratio is (1-8), (1-7), (1-6), (1-5), (1-4) and (1-4); illustratively, the molar ratio is 1:1, 2:1, 1:2, 1: 3.
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. Preferably, the tumor is a drug-resistant tumor, which means resistance to an antitumor drug acting on tumor cells in a dividing and proliferating state.
Further, the invention provides the use of the benzisoselenazole derivative in combination with an antimetabolite for inhibiting tumor cell proliferation. For example, the use of a benzisoselenazole derivative in combination with an antimetabolite to inhibit the proliferation of tumor cells in vitro or in vivo; preferably, for use in inhibiting the proliferation of tumor cells in vitro.
For example, the tumor 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. According to the technical scheme, the tumor cells are selected from human liver cancer cells HepG 2.
According to an exemplary embodiment of the present invention, the benzisoselenazole derivative is used in combination with an antimetabolite to inhibit the proliferation of human hepatoma cell HepG 2. For example, for inhibiting the proliferation of human liver cancer cells HepG2 in vitro; preferably, it causes S-phase (DNA synthesis phase) retardation of HepG2 cells, and inhibits TrxR (thioredoxin oxidoreductase) activity in HepG2 cells.
Furthermore, the invention provides a method for inhibiting tumor cell proliferation by combining the benzisoselenazole derivative and an antimetabolite, wherein the benzisoselenazole derivative and the antimetabolite act on tumor cells according to a certain proportion.
Preferably, the certain mixture ratio is as follows: the molar ratio of the benzisoselenazole derivative to the antimetabolite is (1-10) to (1-10); for example, the molar ratio is (1-8), (1-7), (1-6), (1-5), (1-4) and (1-4); illustratively, the molar ratio is 1:1, 2:1, 1:2, 1: 3. The proposed method is based on the idea that to achieve the desired concentration at the tissue site in the body, the differences that may occur due to the desired metabolism in the body are additionally discounted by the metabolic characteristics of the drug.
Preferably, the concentration of the benzisoselenazole derivative and the antimetabolite is 1-100 μ M, such as 5-60 μ M and 10-50 μ M; illustratively, the concentration is 15, 20, 30, 40 μ M.
Preferably, the dosage of the benzisoselenazole derivative is 0.04-1 mg/g based on the weight of tumor cells; for example, the dosage is 0.045-0.8 mg/g, 0.0.06-0.0.1 mg/g, 0.15-0.5 mg/g; illustratively, the amounts administered are 0.18mg/g, 0.045mg/g, 0.09 mg/g.
Preferably, the dosage of the antimetabolite is 0.005-0.05 mg/g based on the weight of tumor cells; for example, the dosage is 0.008 to 0.04mg/g, 0.01 to 0.03 mg/g; illustratively, the amounts administered are 0.01mg/g, 0.013mg/g, 0.026mg/g, 0.039 mg/g.
Preferably, the action time is 10-120 h, such as 15-105 h and 24-96 h; the action times are, for example, 24h, 48h, 72 h.
Further, the present invention also provides a pharmaceutical composition comprising the benzisoselenazole derivative and an antimetabolite. Preferably, the benzisoselenazole derivative and the antimetabolite have the meaning and concentration ratio as described above.
Preferably, the pharmaceutical composition may further optionally comprise at least one pharmaceutically acceptable excipient.
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 dosage form of the pharmaceutical composition may be in the form of oral preparations 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.
Further, the present invention also provides a method for treating tumors by using the above pharmaceutical composition, and a therapeutically effective amount of the pharmaceutical composition is administered to an individual in need thereof.
Preferably, the subject may be a mammal, such as a human.
Preferably, the tumor has the meaning as described above, preferably liver cancer.
The invention has the beneficial effects that:
the invention unexpectedly discovers that the combined application of the BS and the antimetabolite (especially 5-FU) has excellent synergistic inhibition effect on the proliferation of tumor cells, especially has obvious inhibition effect on the proliferation of liver cancer cells, and has great value for treating tumors. The combination of the two can cause S phase (DNA synthesis phase) retardation of HepG2 cells and inhibit TrxR (thioredoxin oxidoreductase) activity in HepG2 cells.
Specifically, the use of BS effectively makes up the defects that 5-FU has no obvious growth inhibition effect on liver cancer cells and is easy to generate drug resistance when being taken at an early stage, and the BS also well exerts the anti-tumor activity and the relatively thorough cell killing power. The two are used together, compared with the single 5-FU medicament, the dosage of the 5-FU with larger toxicity is obviously reduced, and the safety of anticancer medicament is improved.
The mechanism of tumor inhibition by the combination of BS and 5-FU:
5-FU, a typical antimetabolite, is first converted in vivo into fluorouracil (5-FUR) and fluorouracil deoxynucleoside (5-FUdR), which are further converted into the corresponding nucleoside phosphate and deoxynucleoside (5-FUdRP). 5-FUdRP can inhibit the activity of Thymidylate Synthase (TS), thereby blocking the process of dUMP methylation to form dTMP, and stopping cell proliferation in S phase (DNA synthesis phase) to die, but has obvious toxic and side effects. The BS has the inhibition effect on thioredoxin reductase and can promote the death of tumor cells through apoptosis induction, so that the BS and the 5-FU can play different action mechanisms when being combined for application, a synergistic effect is generated, and the anti-tumor effect is excellent.
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 "is 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 as preferably meaning missingA straight or branched chain saturated monovalent hydrocarbon radical of two hydrogen atoms having 1,2, 3, 4 carbon 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 evaluation of the interaction of BS and 5-FU administered in combination with HepG2 cells in example 3;
wherein (A) is a curve showing the change of CI with the cell proliferation inhibition rate after 24 hours, 48 hours and 72 hours of HepG2 cells treated in example 3; (B) the profile of inhibition of cell proliferation as a function of DRI for HepG2 cells treated for 48 hours for examples 1-4; (C) for example 3 and 5-FU single drug treatment of HepG2 cells for 48 hours, 5-FU dosage was plotted as a function of cell proliferation inhibition rate. Data values are mean ± sd, n is 3.
FIG. 2 is a graph of cell cycle profiles of 15. mu.M 5-FU, 15. mu.M BS and a combination of both (15. mu.M 5-Fu: 15. mu.M BS ═ 1:2) on HepG2 cells for 48 h.
Wherein, A: a control group; b: 15 μ M5-FU; c: 15 μ M BS; d: 15 μ M5-Fu: 15 μ M BS ═ 1: 2.
FIG. 3 is a graph of the effect of 15. mu.M 5-FU, 15. mu.M BS and a combination of both (15. mu.M 5-Fu: 15. mu.M BS ═ 1:2) on HepG2 cell cycle for 48 h. The data values are mean values. + -. standard deviations, n.gtoreq.3.
FIG. 4 is a graph of protein changes after 48h of 15. mu.M 5-FU, 15. mu.M BS and a combination of both (15. mu.M 5-Fu: 15. mu.M BS ═ 1:2) on HepG2 cells;
wherein, A: protein expression levels of TrxR, Trx in cells; b: relative protein expression of TrxR; c: relative protein expression of Trx. The data are mean values. + -. standard deviation, n is more than or equal to 3.*P<0.05 indicates a significant difference compared to the control;#P<0.05 indicated a significant difference compared to the 5-FU group;※P<0.05 represents a significant difference compared with the single drug group.
FIG. 5 is a line of BS and 5-FU inhibition of HepG2 cell proliferation; wherein, in the figure (A), 5-FU acts on HepG2 cells; panel (B) shows BS action on HepG2 cells.
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 and cells:
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.
5-FU: purchased from third Hospital, Beijing university.
HepG2 cells: purchased from cell center of basic medical college of the university of medical science.
2. The test method comprises the following steps:
2.1 assay of inhibition of cell proliferation (SRB method):
(1) the cells cultured by adherence are digested into single cell suspension, the cell concentration is adjusted to be 2 multiplied by 104/mL, and the single cell suspension is evenly inoculated to a 96-well plate (Corning), wherein each well is 180 mu L;
(2) placing the 96-well plate in an incubator for 24h, and adding 20 mu L of compound per well according to the specified concentration after the cells adhere to the wall;
(3) at the end of the drug action, 100 μ L of 10% (v/v) trichloroacetic acid precooled at 4 ℃ is added into each well, and a 96-well plate is fixed for 1h at 4 ℃;
(4) carefully discarding the fixative, slowly washing with deionized water from one side of the 96-well plate for 4 times, blow-drying with electric heating blower or naturally drying at room temperature (the dried 96-well plate can be stored at room temperature for a long time);
(5) adding 100 μ L of 0.057% (w/v) SRB staining solution into each well, and staining for 30min at room temperature;
(6) carefully discarding the dye solution, washing each well with 150 μ L of 1% (v/v) acetic acid for 4 times, drying by blowing with electric heat or naturally drying at room temperature (the dried 96-well plate can be stored at room temperature for a long time);
(7) accurately adding 200 mu L of 10mM Tris destaining solution into each hole, standing at room temperature to dissolve the Tris destaining solution naturally or placing the Tris destaining solution on a shaking table to dissolve the Tris destaining solution;
(8) the absorbance of the sample at 492nm was measured using a microplate reader (Thermo Fisher Scientific). The cell proliferation inhibition rate was calculated according to the following formula:
wherein, the control well is inoculated with 180 μ L of cell suspension, and the compound is replaced by medium (20 μ L) of the same volume; for the inoculation of the blank wells, 180. mu.L of medium was added, and for the administration, 20. mu.L of medium was added. Three duplicate wells were set for each drug concentration.
2.2 Western Blot:
extracting total tissue protein, determining protein concentration (30-50 mu g), adding 5 Xloading buffer solution, supplementing volume with RIPA lysate, mixing uniformly, and performing denaturation treatment at 95 ℃ 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.
2.3 DTNB reduction:
1. adding 50 μ g protein sample into 96-well plate, adding 0.1M sodium phosphate buffer to make up volume to 80 μ L, and incubating at 37 deg.C for 30 min;
2. add 20. mu.L of 5mM NADPH solution to each sample well, add an equal volume of 0.1M sodium phosphate buffer to the control well;
3. after 100. mu.L of 10mM DTNB solution was added to each well by a line gun, the absorbance at 405nm was measured immediately with a microplate reader, and the wells were shaken for 10 seconds before the first reading, and measured every 15 seconds for 30 times. The maximum reaction rate was taken as an indicator of enzyme activity. The experiment was independently repeated three times.
2.4 flow cytometry assay:
1) cells from logarithmic growth phase were seeded in six-well plates (Corning),
2) after 24h of drug action, the recovery medium is centrifuged at 1200rpm for 2min in a 5mL centrifuge tube, and the supernatant is discarded. Rinsing adherent cells in the culture dish once by using PBS precooled at 4 ℃, adding 300 mu L of pancreatin, and placing in an incubator for digestion for 3 min;
3) and (3) blowing the cells into single cells by using 1mL of culture medium, re-suspending the precipitated cells in a centrifuge tube by using 1mL of PBS, combining the precipitated cells with the adherent cells in a culture dish of the corresponding group, centrifuging at 400rpm for 2min, and discarding the supernatant. Resuspend the cells with 1mL PBS, screen through 300 mesh cell screen;
4) resuspending the precipitated cells with 190. mu.L of binding solution, adding 10. mu.l of Annexin V-FITC, incubating at room temperature in the dark for 10min, centrifuging at 4000rpm for 2min, and discarding the supernatant;
5) the cells were resuspended in 195. mu.L of binding solution, 5. mu.L of PI solution was added and immediately detected on a flow cytometer (BDFACSCalibur).
2.5 preparation of the liquid medicine
BS medicine mother liquor: BS was weighed and dissolved in DMSO to prepare a 10mM stock solution, which was stored at-20 ℃ until use.
CDDP mother liquor: CDDP was weighed and dissolved in PBS to prepare a 10mM stock solution, which was stored at room temperature for further use.
The stock solution was diluted to the desired concentration (15, 20, 30, 40 μ M) in serum-free medium as required before use.