Application of AUR in preparation of medicine for treating colon cancer
阅读说明：本技术 Aur在制备治疗结肠癌药物中的应用 (Application of AUR in preparation of medicine for treating colon cancer ) 是由 杜前明 刘超 陈艺 张栩 谢佳乐 张岩 于 2019-07-30 设计创作，主要内容包括：本发明公开了AUR在制备治疗结肠癌药物中的应用。相对于现有技术,本发明提供了AUR治疗结肠癌的新用途,尤其对于耐5-FU结肠癌具有一定的治疗效果,并且更为显著的是,AUR能够通过上调5-FU的敏感性,提高5-FU对耐5-FU结肠癌的杀伤作用。(The invention discloses application of AUR in preparation of a medicine for treating colon cancer. Compared with the prior art, the invention provides the new application of AUR in treating colon cancer, particularly has certain treatment effect on the colon cancer resistant to 5-FU, and more remarkably, AUR can improve the killing effect of 5-FU on the colon cancer resistant to 5-FU by up-regulating the sensitivity of 5-FU.)
Application of AUR in preparation of medicine for treating 5-FU-resistant colon cancer.
Use of AUR in the manufacture of a medicament for increasing the sensitivity of 5-FU.
3. Use according to claim 1 or 2, wherein the AUR is the sole active ingredient.
4. A composition for treating colon cancer, comprising AUR and 5-FU.
5. Use of a composition according to claim 4 for the manufacture of a medicament for the treatment of colon cancer.
6. The use of claim 5, wherein the medicament comprises a 5-FU colon cancer resistant medicament.
7. The use of claim 6, wherein the medicament consists of AUR as the sole active ingredient, together with pharmaceutically acceptable excipients.
8. The use according to claim 5, wherein the medicament is composed of AUR and 5-FU as active ingredients, and pharmaceutically acceptable excipients.
9. Use according to claim 7 or 8, wherein the excipient comprises one or more of diluents, binders, disintegrants, glidants, lubricants, flavouring agents, inclusion materials and adsorbing materials.
10. The use of claim 7 or 8, wherein the medicament is in the form of granules, powder, tablets, capsules, pills, oral liquid, decoction or dripping pills.
Technical Field
The invention relates to application of AUR in preparation of a medicine for treating colon cancer, belonging to the technical field of new application of medicines.
Background
5-FU is a classical antineoplastic drug that has been discovered to be an important component of solid tumor treatment regimens. Due to the wide application of the medicine, the colon cancer is easy to generate the drug resistance phenomenon aiming at 5-FU clinically. The currently determined mechanisms for regulating and controlling the colon cancer 5-FU resistance mainly include methylation of O6-methylguanine DNA methyltransferase (O6-methylguanine DNA-transferase, MGMT) and activation of a FAK/AKT/NF-kB survival signal pathway, but no effective treatment scheme exists for the regulation and control modes, and a 5-FU resistance mechanism of the colon cancer needs to be further understood and a new drug treatment scheme needs to be explored.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the technical problems, the invention provides an application of AUR in preparing a medicine for treating 5-FU colon cancer.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:
application of AUR in preparation of medicine for treating 5-FU colon cancer resistance.
AUR (Kinnofen, CAS No.:34031-32-8, chemical structure shown below) which acts on TXNRD to transfer electrons to TXN, which in turn transfers electrons to scavenge ROS. TXN is a major cellular protein disulfide reductase, which acts as an electron donor for disulfide groups in peroxidases (peroxiredoxins) which can also be used to reduce H2O2And antioxidant molecules of peroxide, and regulate ribonucleotide reductase and methionine sulfoxide reductase. The thioredoxin system is critical for reduction-oxidation (redox) homeostasis and protects DNA from oxidative stress-related DNA damage.
AUR chemical structural formula
The invention also provides application of the AUR in preparing a medicament for improving the sensitivity of 5-FU.
Preferably, the AUR is the sole active ingredient.
The invention also provides a composition for treating colon cancer, which comprises AUR and 5-FU.
The invention also provides application of the composition in preparing a medicine for treating colon cancer.
Preferably, the drug comprises a 5-FU colon cancer resistant drug.
Preferably, the medicament consists of AUR as the only active ingredient and pharmaceutically acceptable auxiliary materials.
Preferably, the medicament consists of AUR and 5-FU as active ingredients and pharmaceutically acceptable auxiliary materials.
Preferably, the auxiliary materials comprise one or more of diluents, binders, disintegrants, glidants, lubricants, flavoring agents, inclusion materials and adsorbing materials.
Preferably, the dosage form of the medicine comprises granules, powder, tablets, capsules, pills, oral liquid, decoction or dripping pills.
When the AUR is used for treating colon cancer, the AUR can be used alone, can be matched with other medicines for simultaneous use, or can be prepared into a compound preparation together with other medicines for use, so that the aim of treating colon cancer can be fulfilled.
The pharmaceutically acceptable auxiliary materials refer to various conventional auxiliary materials required when different dosage forms are prepared, such as diluents, adhesives, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, adsorbing materials and the like, and the pharmaceutically acceptable auxiliary materials are prepared into any one of common oral preparations by a conventional preparation method, such as granules, powder, tablets, capsules, pills, oral liquid, decoction, dropping pills and the like.
In preclinical research, the AUR can effectively treat colon cancer, particularly has a certain treatment effect on 5-FU-resistant colon cancer, and plays a role in inhibiting drug-resistant tumors by combining growth inhibition and killing effects of 5-FU-resistant colon cancer cells and animal tumor bearing tumors, so that the AUR has a better effect. Novel methods of using AUR to treat colon cancer developing chemo-drug resistant 5-FU are provided. The concrete expression is as follows:
1) AUR can increase the sensitivity of 5-FU in a 5-FU resistant colon cancer cell line, thereby increasing the inhibition effect of 5-FU on cancer cells resistant to chemotherapy.
2) AUR combined with 5-FU can effectively inhibit the growth of drug-resistant colon cancer in a 5-FU colon cancer resistant nude mouse ectopic transplantation model, thereby achieving the effect of treating chemotherapy and resisting tumors.
The technical effects are as follows: compared with the prior art, the invention provides the new application of AUR in treating colon cancer, particularly has certain treatment effect on the colon cancer resistant to 5-FU, and more remarkably improves the killing effect of 5-FU on the colon cancer resistant to 5-FU by increasing the sensitivity of 5-FU.
Drawings
In order to more clearly illustrate the technical solution in the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below.
FIG. 1 shows the proliferation inhibition effect of different concentrations of AUR in combination with 5-FU on 5-FU-resistant HCT-8 cells in colon cancer.
FIG. 2 shows the effect of AUR combined with 5-FU at different concentrations on the proliferation inhibition of 5-FU resistant SW620 cells in colon cancer.
FIG. 3 shows the change of the volume of transplanted tumor of 5-FU combined with AUR in nude mice with SW620 cells resistant to colon cancer.
FIG. 4 shows the change of tumor weight of transplanted 5-FU combined with AUR in nude mice with SW620 cells resistant to colon cancer.
FIG. 5 shows the body weight changes of nude mice transplanted with AUR combined with 5-FU and 5-FU resistant SW620 cells for colon cancer.
Detailed Description
For further illustration of the present invention, the application of AUR provided by the present invention in the preparation of a drug for treating colon cancer will be described in detail with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
Proliferation inhibition experiment of 5-FU combined with AUR on human colon cancer resistant 5-FU cells HCT-8 and SW 620.
1. Experimental Material
(1) Medicine
AUR, white powder available from Jili chemical industry, New zone of Changzhou. Preparing mother liquor with dimethyl sulfoxide (DMSO), and storing at-20 deg.C. The required concentration was prepared just before use with DMEM medium.
5-FU, available from Selleck Bio Inc., as a white powder. Preparing mother liquor with dimethyl sulfoxide (DMSO), and storing at-20 deg.C. The required concentration was prepared just before use with DMEM medium.
(2) Cell line
5-FU resistant cell lines HCT-8/5-FU and SW620/5-FU were constructed by the inventors using HCT-8 and SW620 supplied from the institute of biochemistry and cell biology, the Shanghai institute of bioscience, China academy of sciences, and cultured in DMEM medium containing 10% fetal bovine serum.
(3) Reagent
Culture solution: DMEM medium, product of GIBCO USA. Dissolving DMEM powder in 1000mL sterile distilled water, and adding NaHCO3Adjusting the pH value to 7.3-7.4, filtering and sterilizing by a cylindrical filter, and storing in a refrigerator at 4 ℃. Before use, 10% fetal calf serum, 100U/mL penicillin and 100mg/L streptomycin are added.
② fetal bovine serum: hangzhou Sijiqing bioengineering company product. Inactivating in 56 deg.C water bath for 30min, subpackaging, and storing in-20 deg.C low temperature refrigerator.
③ PBS buffer solution: weighing 8.0g of NaCl, 0.20g of KCl and Na2HPO4·H2O 1.56g、KH2PO42.0g, dissolved in 1000mL of triple distilled water, autoclaved, and stored in a refrigerator at 4 ℃.
0.25% pancreatin: weighing pancreatin 0.25g, dissolving in 100ml PBS buffer solution, filtering and sterilizing.
Fifth, DMSO solution: Sigma-Aldrich, USA (St. Louis, Mo).
Sixthly, MTT solution: 50mg of MTT powder (Bioshrp) was weighed out and placed in 10mL of PBS, and sterilized by filtration through a 0.22um filter.
2. An experimental instrument:
(1) YJ-875 type medical purification workbench: suzhou purification plant.
(2)3111 type water jacket CO2An incubator: product of Thermo electron corporation, usa.
(3) Model OlympusIX51 inverted fluorescence microscope: a product of Olympus corporation of japan.
(4) An electronic balance: product of Beijing Sidolis Instrument systems, Inc.
(5) ZW-A type micro oscillator: tan, Tan.
(6) 35800 enzyme linked immunosorbent assay device, available from BioTeK corporation of usa.
3. Experiment of drug effect
Tetramethylazoazolium (MTT) experiment is adopted to investigate the inhibitory effect and IC of AUR combined with 5-FU on different 5-FU-resistant colon cancer cells50The value is obtained.
The HCT-8/5-FU and SW620/5-FU cells in logarithmic growth phase were digested with 0.25% trypsin, centrifuged, resuspended and counted to make a cell suspension at 5X 104Adding the cell concentration into a 96-well enzyme label plate with 100 mu L of each well, arranging 3 wells, placing at 37 ℃ and 5% CO2Culturing in an incubator for about 12h until the cells are completely attached to the wall. Then dosing was performed as follows: the first group is added with 5-FU and low-concentration AUR, the final concentration of 5-FU in each hole is 0.1 μ M, 1 μ M, 10 μ M, 50 μ M, 100 μ M and 200 μ M, and the final concentration of AUR is 0.1 μ M; adding 5-FU and AUR in the second group, wherein the final concentration of 5-FU in each well is 0.1. mu.M, 1. mu.M, 10. mu.M, 50. mu.M, 100. mu.M, 200. mu.M, and the final concentration of AUR is 0.2. mu.M; the third group was supplemented with 5-FU and AUR at a final concentration of 0.1. mu.M, 1. mu.M, 10. mu.M, 50. mu.M, 100. mu.M, 200. mu.M and 0.4. mu.M in each well. Adding medicine into each hole, incubating for 24h, adding 20 mu L of 5mg/mL MTT solution, incubating for 4h at 37 ℃ in the dark, discarding all supernatants, adding 100 mu L DMSO into each hole, oscillating for 2-3 min on a micro oscillator, and determining the absorbance value A at the wavelength of 570nm by using an enzyme linked immunosorbent assay detector after crystals are completely dissolved. The larger the value of A, the larger the number of viable cells. The experiment was repeated 3 times, and the growth inhibition rate of the drug on the cells was calculated from the A value.
Growth inhibition rate of 1-AMedicine adding device/AControl group
Calculated according to the Sun's synthesis method (modified Kouzhou's method)Median inhibitory concentration IC50(i.e., the concentration of drug required to inhibit 50% of cell growth).
The experimental results are shown in FIGS. 1-2, and it can be seen that when 5-FU is used alone, the drug-resistant cells have no obvious inhibition effect, but when 5-FU and AUR are used in combination, the combination can significantly inhibit the proliferation of the drug-resistant cells (p is less than 0.05) compared with the case of simply administering 5-FU, which indicates that AUR achieves the effect of killing the drug-resistant cells by increasing the sensitivity of the drug-resistant cells to 5-FU.
Example 2
The treatment effect of 5-FU combined with AUR on 5-FU resistant nude mouse transplantation tumor of colon cancer is investigated.
1. Experimental Material
(1) Medicine
AUR, white powder available from Jili chemical industry, New zone of Changzhou. Preparing mother liquor with dimethyl sulfoxide (DMSO), and storing at-20 deg.C. The required concentration was prepared just before use with DMEM medium.
5-FU, available from Selleck Bio Inc., as a white powder. Preparing mother liquor with dimethyl sulfoxide (DMSO), and storing at-20 deg.C. The required concentration was prepared just before use with DMEM medium.
(2) Cell line
The 5-FU resistant cell line SW620/5-FU was constructed by the inventors using SW620 supplied from the institute of biochemistry and cell biology, cell bank of Shanghai institute of bioscience, China academy of sciences, and cultured in DMEM medium containing 10% fetal bovine serum.
(3) Reagent
Culture solution: DMEM medium, product of GIBCO USA. Dissolving DMEM powder in 1000mL sterile distilled water, and adding NaHCO3Adjusting the pH value to 7.3-7.4, filtering and sterilizing by a cylindrical filter, and storing in a refrigerator at 4 ℃. Before use, 10% fetal calf serum, 100U/mL penicillin and 100mg/L streptomycin are added.
② fetal bovine serum: hangzhou Sijiqing bioengineering company product. Inactivating in 56 deg.C water bath for 30min, subpackaging, and storing in-20 deg.C low temperature refrigerator.
③ PBS buffer solution: weighing 8.0g of NaCl, 0.20g of KCl and Na2HPO4·H2O 1.56g、KH2PO42.0g, dissolved in 1000mL of triple distilled water, autoclaved, and stored in a refrigerator at 4 ℃.
0.25% pancreatin: weighing pancreatin 0.25g, dissolving in 100ml PBS buffer solution, filtering and sterilizing.
(4) Laboratory animal
Source, strain: BALB/c-nu nude mice, provided by the university of Yangzhou, center of comparative medicine (license number: SCXK (Su) 2010-0001). The week age is as follows: 6 weeks, body weight: 16-20g, sex: and (4) male.
2. The experimental method comprises the following steps:
(1) grouping of laboratory animals
Negative control group (blank control group): 6, only one of the raw materials is used;
6 of 5-FU groups (5-FU, 50mg/kg, i.p., 5-FU once every four days);
6 AUR groups (AUR, 6mg/kg, i.g., AUR daily);
6 combined 5-FU and AUR groups (5-FU, 50mg/kg, once in four days 5-FU; AUR, 6mg/kg, i.g., AUR daily);
(2) molding method
Taking colon cancer drug-resistant cell strain SW620 in logarithmic growth phase, and preparing into 1 × 10 cell strain under aseptic condition7The cell suspension was inoculated subcutaneously in the right axilla of nude mice at 0.2 mL. Measuring the diameter of the transplanted tumor of the nude mouse by using a vernier caliper until the tumor grows to 100mm3Animals were then randomized into groups. The antitumor effect of the test object is dynamically observed by using a method for measuring the tumor size. Tumor diameter was measured 1 time every 2-3 days. The administration volume was 0.2mL/20 g. After 24 days, the mice were sacrificed and the tumor mass was surgically removed and weighed.
The formula for Tumor Volume (TV) is: TV 1/2 × a × b2Wherein a and b represent length and width, respectively.
As a result:
the experimental treatment results of the 5-FU combined with AUR on the human colon cancer drug-resistant cell SW620 nude mouse transplantation tumor are shown in the figures 3-5. The experimental results are as follows, as shown in fig. 3, after 24 days of continuous administration, compared with the blank group, 5-FU group and AUR group, the tumor volume of the 5-FU combined AUR group was different from that of the other groups at 4 days, the volume was significantly smaller than that of the other groups, and the tumor volume of the 5-FU combined AUR group at each time point thereafter was significantly smaller than that of the other groups and was statistically different. At the same time, tumor volume also decreased to some extent with AUR alone relative to the blank group. As shown in FIG. 4, the tumor weight of the combination group was significantly smaller and statistically different from that of the other groups, and the tumor weight of the AUR group was also decreased to some extent. As shown in fig. 5, there was no significant difference between the animal body weights of the groups.
4. And (4) conclusion: the 5-FU combined AUR has obvious growth inhibition effect on the heterogeneously inhibited tumors of human colon cancer resistant 5-FU cell SW620 nude mice.
According to the embodiment, the AUR provided by the invention has a certain treatment effect on the 5-FU-resistant colon cancer, and more remarkably, the AUR can improve the killing effect of 5-FU on the 5-FU-resistant colon cancer by up-regulating the sensitivity of 5-FU. The present invention provides a novel method of using AUR to treat colon cancer developing chemo-therapeutic 5-FU resistance
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.