CN111893096B - Method for preparing genitourinary system tumor cell iron death model based on ferric ammonium citrate and application of prepared antitumor drug - Google Patents
Method for preparing genitourinary system tumor cell iron death model based on ferric ammonium citrate and application of prepared antitumor drug Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention relates to a method for preparing a genitourinary system tumor cell iron death model based on ferric ammonium citrate and application of the method in preparing an anti-tumor medicament, and belongs to the technical field of anti-tumor medicaments. The invention provides a method for preparing a genitourinary system tumor cell iron death model based on ferric ammonium citrate, which comprises the following steps: and when the density of the cultured tumor cells reaches 65-75%, adding ferric ammonium citrate, and continuously culturing for 20-30 h to obtain the iron death model. The ferric ammonium citrate is used for treating tumor cells of the genitourinary system, is a very simple and efficient preparation method of an iron death tumor cell model, and provides a theoretical basis for subsequent research and development and clinical treatment due to the antitumor activity expressed by the ferric ammonium citrate.
Description
Technical Field
The invention relates to the technical field of antitumor drugs, in particular to a method for preparing a genitourinary system tumor cell iron death model based on ferric ammonium citrate and application of the method in preparing the antitumor drugs.
Background
The urogenital system tumor seriously harms the life health of human beings, and the existing radiotherapy, chemotherapy and operation treatment effects are not ideal. The incidence of neoplasms of the genitourinary system has recently shown a rapid growth trend year by year, especially with the dominance of breast, ovarian, prostate and renal cancers. Generally, most genitourinary tumors do not have many somatic symptoms in their early stages and, once found, have entered the intermediate-to-late stage. At present, of many chemotherapeutic drugsHas large toxic and side effects, and can cause great damage to normal cells of the organism while killing tumor cells. Therefore, the development of a number of biochemical drugs against urogenital tumors that are broad-spectrum, highly effective, low-toxic, relatively safe and inexpensive is urgently needed. Iron death (Ferroptosis) is an iron-dependent, novel programmed cell death modality distinguished from apoptosis, necrosis, and autophagy. The main mechanism of iron death is that under the action of ferrous iron or ester oxygenase, unsaturated fatty acid highly expressed on cell membranes is catalyzed to generate lipidosome peroxidation, so that cell death is induced; in addition, it was also shown that the expression level of the antioxidant system (lipid peroxide) of GPX4 was reduced. The GPX4 enzyme is the only Glutathione Peroxidase (GPX) used for liposome peroxides. GPX4 can convert the peroxide bond of lipid peroxidation to hydroxyl, losing its peroxide activity. It was found that when iron ions are continuously supplied to the cells and are ensured to be present as ferrous iron, liposome peroxidation is initiated when an iron overload condition is reached. Peroxidized liposomes can significantly deplete the activity of GPX4 in vivo, inducing iron death. When a cell undergoes iron death, the cell mitochondria become smaller, membrane density increases, and cristae decreases, accompanied by increased lipid peroxidation in the cytosol and increased ROS. The research and development of the medicine causing the death of the tumor cells of the genitourinary system are very meaningful for realizing the clinical treatment of the medicine, but the current medicine contains Fe 2+The drugs have high toxicity, and the dosage of the drugs is greatly different on different cells, so that the stability of a model constructed by the drugs is not high.
Disclosure of Invention
The invention aims to provide a method for preparing a genitourinary system tumor cell iron death model based on ferric ammonium citrate and application of the method in preparing an anti-tumor medicament. The ferric ammonium citrate is used for treating tumor cells of the genitourinary system, has no toxicity and high stability, can generate an iron death effect in the same concentration in at least 4 common cell lines of the genitourinary system tumors, is a very simple and efficient preparation method of an iron death tumor cell model, and provides a theoretical basis for subsequent research and development and clinical treatment due to the antitumor activity expressed by the ferric ammonium citrate.
The invention provides a method for preparing a genitourinary system tumor cell iron death model based on ferric ammonium citrate, which comprises the following steps: and when the density of the cultured tumor cells reaches 65-75%, adding ferric ammonium citrate, and continuously culturing for 20-30 h to obtain the iron death model.
The invention also provides the application of the ferric ammonium citrate in preparing the antitumor drugs.
The invention also provides the application of the ferric ammonium citrate in preparing the medicine for resisting the tumor of the genitourinary system.
The invention also provides application of the ferric ammonium citrate in preparing a medicament for inhibiting proliferation of tumor cells of the genitourinary system.
The invention also provides the application of ferric ammonium citrate in preparation of medicines for improving Fe in tumor cells of the genitourinary system2+The use in the manufacture of a medicament for treating or preventing a disease.
The invention also provides application of the ferric ammonium citrate in preparing a medicament for promoting the oxidative stress injury of tumor cells of the genitourinary system.
The invention also provides an application of the ferric ammonium citrate in preparing a medicament for targeted regulation and control of the expression of the protein related to the iron death of tumor cells of the genitourinary system, wherein the protein related to the iron death comprises an iron death negative regulatory factor and an iron death related activating factor; the negative regulator of iron death comprises cell proliferation related protein Ki67, iron death inhibitor GPX4 and iron death inhibitor FTH 1; the iron death-related activators include iron death-promoting factor RPL8 and iron death-promoting factor NOX 1.
The invention also provides application of the ferric ammonium citrate in preparing a medicament for down-regulating the expression level of the genitourinary system tumor cell iron death negative regulatory factor, wherein the iron death negative regulatory factor comprises cell proliferation related protein Ki67, an iron death inhibitor GPX4 and an iron death inhibitor FTH 1.
The invention also provides application of the ferric ammonium citrate in preparing a medicament for up-regulating the expression level of the activation factors related to the iron death of tumor cells of the genitourinary system, wherein the activation factors related to the iron death comprise an iron death promoting factor RPL8 and an iron death promoting factor NOX 1.
Preferably, the genitourinary system tumor comprises breast cancer, ovarian cancer, prostate cancer, and renal cancer.
The invention provides a method for preparing a genitourinary system tumor cell iron death model based on ferric ammonium citrate. Ferric ammonium citrate can obviously induce tumor cell strains of genitourinary system to generate in-vitro proliferation inhibition, and can improve Fe in tumor cells2+Concentration, inducing the development of oxidative stress damage; molecular biological experiments show that ferric ammonium citrate can effectively increase the expression of iron death related activating factors, inhibit the expression of iron death negative regulatory factors and finally promote tumor cells of the genitourinary system to generate iron death. The ferric ammonium citrate is used for treating tumor cells of the genitourinary system, is a very simple and efficient preparation method of an iron death tumor cell model, and provides a theoretical basis for subsequent research and development and clinical treatment due to the antitumor activity expressed by the ferric ammonium citrate.
Drawings
FIG. 1 is a chemical structural formula of ammonium ferric citrate provided by the present invention;
FIG. 2 is a graph showing the results of AFC significantly inhibiting the in vitro proliferation of tumor cell lines of the genitourinary system;
FIG. 3 is a graph showing the results of AFC significantly promoting the oxidative stress damage of tumor cell lines of the genitourinary system;
FIG. 4 is a graph showing the expression result of AFC-targeted regulation of proteins associated with iron death in tumor cell strains of the genitourinary system;
FIG. 5 shows that AFC provided by the invention can increase tumor cell strain Fe of genitourinary system2+Concentration result chart of (1).
Detailed Description
The invention provides a method for preparing a genitourinary system tumor cell iron death model based on ferric ammonium citrate, which comprises the following steps: and when the density of the cultured tumor cells reaches 65-75%, adding ferric ammonium citrate, and continuously culturing for 20-30 h to obtain the iron death model. Ferric ammonium citrate, also known as ferric ammonium citrate(Ammonium ferric citrate, AFC), iron Ammonium 2-hydroxy-1, 2, 3-propanetricarboxylate, is a double salt of iron citrate and Ammonium citrate (fig. 1). AFC is usually added into dairy products, wheat flour for bread, biscuits, milk powder and the like, is used for nutritional supplements (iron fortifiers) and is a very safe chemical substance. The AFC is a recognized safe food additive-grade iron supplement (used for treating iron-deficiency anemia), can be eaten, and has high safety factor and small toxic and side effects, and many existing chemotherapeutic drugs have very high toxicity. Generally, AFC is used as a food additive, but it has not been reported so far whether it has an anticancer effect. The invention firstly provides that ferric ammonium citrate can be used for preparing a genitourinary system tumor cell iron death model. The invention finds that the AFC has the effect of inducing the death of the tumor iron, and provides a theoretical basis for developing the AFC into a tumor treatment medicament; no report shows that the iron supplement can induce iron death, and the invention discovers the iron death inducing function of AFC for the first time. In the invention, the concentration range of the ammonium ferric citrate used in the model construction process is preferably 0.1 mg/ml. In the present invention, the solvent for dissolving the ferric ammonium citrate preferably comprises a PBS solution. In the present invention, preferably, when the tumor cell density reaches 70%, ferric ammonium citrate is added, and preferably, the culture is continued for 24h, so as to obtain an iron death model. In the invention, the tumor cells preferably comprise breast cancer cells, ovarian cancer cells, prostate cancer cells and kidney cancer cells, more preferably comprise breast cancer cells MCF7, ovarian cancer cells SK-OV-3, prostate cancer cells 22Rv1 and kidney cancer cells A-498, and particularly 20000/ml human genitourinary system tumor cell lines with good growth state, including breast cancer cells MCF7, ovarian cancer cells SK-OV-3, prostate cancer cells 22Rv1 and kidney cancer cells A-498, are respectively and independently inoculated in one hole of a 6-hole plate. In the present invention, the tumor cells are preferably cultured in McCOY's 5A (SIGMA, M4892) medium comprising NaHCO 32.2g/L and 10 percent of high-quality fetal bovine serum. In the present invention, the environment of the cell culture is preferably a gas phase: air, 95%; 5 percent of carbon dioxide. Temperature: 37 degrees celsius.
The invention also provides the application of the ferric ammonium citrate in preparing the antitumor drugs.
The invention also provides the application of the ferric ammonium citrate in preparing the medicine for resisting the tumor of the genitourinary system.
The invention also provides the application of the ferric ammonium citrate in preparing the medicine for inhibiting the proliferation of the tumor cells of the genitourinary system.
The invention also provides the application of ferric ammonium citrate in preparing medicines for improving Fe in tumor cells of genitourinary system2+The use of a concentration of a drug.
The invention also provides application of the ferric ammonium citrate in preparing a medicament for promoting the oxidative stress injury of tumor cells of the genitourinary system.
The invention also provides an application of the ferric ammonium citrate in preparing a medicament for targeted regulation and control of the expression of the protein related to the iron death of tumor cells of the genitourinary system, wherein the protein related to the iron death comprises an iron death negative regulatory factor and an iron death related activating factor; the negative regulator of iron death comprises cell proliferation related protein Ki67, iron death inhibitor GPX4 and iron death inhibitor FTH 1; the iron death-related activators include iron death-promoting factor RPL8 and iron death-promoting factor NOX 1.
The invention also provides application of the ferric ammonium citrate in preparing a medicament for down-regulating the expression level of the genitourinary system tumor cell iron death negative regulatory factor, wherein the iron death negative regulatory factor comprises cell proliferation related protein Ki67, an iron death inhibitor GPX4 and an iron death inhibitor FTH 1.
The invention also provides application of the ferric ammonium citrate in preparing a medicament for up-regulating the expression level of the activation factors related to the iron death of tumor cells of the genitourinary system, wherein the activation factors related to the iron death comprise an iron death promoting factor RPL8 and an iron death promoting factor NOX 1.
In the present invention, the genitourinary system tumor includes breast cancer, ovarian cancer, prostate cancer, and renal cancer.
The method for preparing a genitourinary system tumor cell iron death model and the application of preparing an anti-tumor drug based on ferric ammonium citrate of the present invention are further described in detail with reference to the following specific examples, and the technical solutions of the present invention include, but are not limited to, the following examples.
At least 3 replicates were established for each experiment. Data are presented as mean ± the Standard Error (SE). Statistical analysis of differences between groups was performed using Student's t-tests, with a P value <0.05 as there was a significant statistical difference.
Example 1
1. And (3) cell culture:
the human genitourinary system tumor cell strain comprises breast cancer cells MCF7, ovarian cancer cells SK-OV-3, prostate cancer cells 22Rv1 and kidney cancer cells A-498 which are all purchased from the cell resource center of Shanghai Life science research institute of Chinese academy of sciences. The above cells were cultured in McCOY's 5A (SIGMA, M4892) medium containing NaHCO32.2g/L, 10 percent of high-quality fetal bovine serum. The culture environment is gas phase: air, 95%; 5 percent of carbon dioxide. Temperature: 37 ℃ is carried out.
MTT assay:
ferric ammonium citrate was purchased from SIGMA. The storage concentration was set to 100mg/ml with sterile PBS and sterilized by suction filtration using a 0.22 μm pore size filter. Take 2X 103The well-grown tumor cells were seeded in 96-well cell culture plates per ml. The next day, AFC was added to concentrations of 0.001, 0.01, 0.1, 1.0mg/ml, 3 duplicate control wells per group were set, and equal volumes of PBS were used as negative controls. Culturing for 24h under the same environment. According to the MTT test kit instructions (Beyotime, Shanghai, China), 10. mu.l of MTT solution (5mg/ml) was added per well and incubation was continued for 4h in a cell incubator. Add 100. mu.l of Formazan lysis solution to each well, mix well, and incubate further in the cell culture box. Until formazan was completely dissolved as observed under a normal light microscope. Absorbance was measured at 570 nm.
The results show that AFC significantly inhibits the in vitro proliferation of tumor cell strains of the genitourinary system:
four human genitourinary tumor cells were treated with different concentrations of AFC (0.001mg/ml, 0.01mg/ml, 0.1mg/ml, 1.0mg/ml) for 24h, and an equal volume of PBS was used as a control group. The results of the MTT assay indicated that, in addition to 0.001mg/ml AFC, all three concentrations significantly inhibited the in vitro proliferation of the cell lines (fig. 2, AFC significantly inhibited the in vitro proliferation of the genitourinary tumor cell line, where a is the MTT assay result of MCF 7. the results of the MTT assay indicated that, in addition to 0.001mg/ml AFC, all 3 concentrations significantly inhibited the in vitro proliferation of the cell line. B is the MTT assay result of SK-OV-3. the results of the MTT assay indicate that, in addition to 0.001mg/ml AFC, all 3 concentrations significantly inhibited the in vitro proliferation of the cell line. C is the MTT assay result of 22Rv 1. the results of the MTT assay indicate that, in addition to 0.001mg/ml AFC, all 3 concentrations significantly inhibited the in vitro proliferation of the cell line. D is the results of a-498. the results of the MTT assay result indicates that, in addition to 0.001mg/ml AFC, the other 3 concentrations all significantly inhibited the in vitro proliferation of this cell line. (. p <0.01vs PBS control group;. p <0.05vs PBS control group; n.4 (4 replicates of the same group of samples);. ttest)).
SOD assay:
briefly, 1X 10 was taken out according to the SOD activity detection kit instructions (Beyotime, Shanghai, China)6Mu.l of sample lysate was added to each ml of cells, the mixture was thoroughly aspirated, centrifuged at 12000g at 4 ℃ for 5min, and the supernatant was collected. And (3) fully and uniformly mixing 151 mu l of SOD detection buffer solution, 8 mu l of WST-8 and 1 mu l of enzyme solution to obtain the WST-8 enzyme working solution. And simultaneously setting up a concentration gradient SOD standard reference substance, diluting the SOD standard substance to 100U/ml, 50U/ml, 20U/ml, 10U/ml, 5U/ml, 2U/ml and 1U/ml, and detecting with the sample at the same time. Mu.l of cell lysis supernatant and standard solution were added to 160. mu.l of freshly prepared WST-8 enzyme working solution and 20. mu.l of reaction starting solution, respectively, mixed well and incubated at 37 ℃ for 30 min. Absorbance was measured at 450 nm.
ATP assay:
briefly, 1X 10 was taken according to the instructions of the enhanced ATP detection kit (Beyotime, Shanghai, China)6Mu.l of sample lysate was added to each ml of cells, the mixture was thoroughly aspirated, centrifuged at 12000g at 4 ℃ for 5min, and the supernatant was collected. Setting ATP standard reference substance at the same time, adjusting ATP standard substance to several concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μ M, andthe samples were tested simultaneously. And preparing a fresh detection working solution according to the Kit requirements. Adding 100 μ l ATP detection working solution into detection well and the sample well, and standing at room temperature for 5 min. Subsequently, 20. mu.l of the sample or the standard was added to the wells, mixed well rapidly, left at room temperature for 5 seconds, and then the RLU value was measured using a Luminometer.
MDA experiment:
according to the specification of enhanced MDA detection kit (Nanjing institute of Biotechnology), 1 × 10 is selected6To each ml of cells, 200. mu.l of the sample lysate was added, sufficiently aspirated, centrifuged at 12000g at 4 ℃ for 5min, and the supernatant was collected. And simultaneously setting ATP standard reference substances, adjusting the ATP standard reference substances to several concentrations of 1, 3, 6, 12, 25, 50 and 100 mu Mol/mg, and detecting the ATP standard reference substances and the samples at the same time. And preparing a fresh detection working solution according to the Kit requirements. Adding 100 μ l MDA detection working solution into detection well and the target product well, mixing well, and incubating at 37 deg.C for 30 min. Absorbance was measured at 450 nm.
The results of steps 3, 4 and 5 show that AFC significantly promotes the oxidative stress damage of the tumor cell lines of the genitourinary system (FIG. 3, a result graph of AFC significantly promotes the oxidative stress damage of the tumor cell lines of the genitourinary system, wherein A is the content of Malondialdehyde (MDA) of each cell, after four tumor cells of the human genitourinary system are respectively treated by AFC with the concentration of 1.0mg/ml for 24h, the detection result shows that the level of the AFC in the cell lines of the treated group is significantly higher than that of the cells of the PBS control group, B is the content of superoxide dismutase (SOD) of each cell, after four tumor cells of the human genitourinary system are respectively treated by AFC with the concentration of 1.0mg/ml for 24h, the detection result shows that the level of the SOD in the cell lines of the AFC treated group is significantly lower than that of the cells of the PBS control group, C is the content of Adenosine Triphosphate (ATP) of each cell, after four tumor cells of the human genitourinary system are respectively treated by AFC with the concentration of 1.0mg/ml for 24h, the detection result shows that the ATP level in the AFC treatment cell strain is obviously lower than that of the PBS control cell. (. p <0.01vs PBS control; n-4; ttest)):
Oxidative stress damage in cells is one of the necessary conditions that lead to their iron death. After four human genitourinary system tumor cells are respectively treated by AFC with the concentration of 1.0mg/ml for 24 hours, the detection result shows that the MDA level in the AFC treatment group cell strain is obviously higher than that in the PBS control group cell (A in figure 3). The levels of SOD and ATP in the AFC-treated cell lines were significantly lower than those of PBS control cells (B and C in fig. 3).
Westernblot experiment:
the total proteins of the cells of each group were subjected to 12% SDS-PAGE denaturing gel electrophoresis, and after completion, they were transferred to a PVDF membrane (Millipore). After sealing and membrane washing, the primary antibody (Anti-Ki67 antibody [ EPR3610] (ab92742), Anti-GPX4 antibody [ EPNCIR144] (ab125066), Anti-Ferritin Heavy Chain antibody (ab65080), Anti-RPL8 antibody [ EPR11630] (ab169538), Anti-NOX1 antibody (ab121009), Anti-GAPDH antibody [ EPR16891] -Loading Control (ab181602)) was incubated at 37 ℃ for 45 min. After washing the membrane sufficiently, the secondary antibody (Goat Anti-Rabbit IgG H & L (HRP) (ab97051)) was incubated at 37 ℃ for 45 min. The membrane was washed 4 times with TBST at room temperature for 14min each time. Then, the resultant was developed by ECL enhanced chemiluminescence (ECL kit, Pierce Biotechnology) exposure.
The result shows that the AFC target regulates and controls the expression of proteins related to iron death of tumor cell strains of the genitourinary system:
after four human genitourinary system tumor cells are respectively treated by AFC with the concentration of 1.0mg/ml for 24 hours, Western blot detection results show that the expression levels of cell proliferation related protein (Ki67) and iron death inhibition factors (GPX4 and FTH1) are obviously reduced in an AFC treatment group compared with a PBS control group; the expression of iron death promoting factors (RPL8 and NOX1) is remarkably increased (FIG. 4, the expression result of AFC target-regulated and controlled protein related to the iron death of tumor cell strains of the genitourinary system, wherein A is the result of protein immunoblotting experiment (Western blot) of SK-OV-3 and 22Rv1 cells in an AFC treatment group and SK-OV-3 and 22Rv1 cells in a PBS treatment group, the result of Western blot AFC detection shows that the expression levels of cell proliferation related protein (Ki67) and iron death inhibiting factors (GPX4 and FTH1) are remarkably reduced in the AFC treatment group compared with the PBS control group, the expression of iron death promoting factors (RPL8 and NOX1) is remarkably increased, B is the result of protein immunoblotting experiment (Western blot) of MCF7 and A-498 cells in the AFC treatment group and MCF7 and A-498 cells in the AFC treatment group compared with the PBS control group, and the result of Western blot AFC treatment group shows that the protein immunoblotting experiment (Western blot) and PBS control group, the expression levels of cell proliferation related protein (Ki67) and iron death inhibitory factor (GPX4, FTH1) are reduced remarkably; while the expression of iron death promoting factors (RPL8, NOX1) was significantly increased.
7. Detection of Fe in cells by flow cytometry2+Content experiment:
according to the specifications of the Fluorescent imaging probe for the detection of iron (II) (Dye) Kit (GORYO Chemical, Inc., Japan). Take 1X 106Perml of suspended single cells, 200. mu.l of PBS buffer and then 5. mu.l of BioTracker 575 Red Fe2+The probe was stained at 4 ℃ for 30min in the dark. Subsequently, 800. mu.l of PBS buffer was added, the cells were mixed well, and each group of cells was subjected to flow cytometry (Cytomics FC 500, BECKMAN) using FL2 channel, and 20000 cells were counted for each sample.
The results show that AFC increases the Fe content in tumor cell lines of genitourinary system2+The concentration of (a):
one of the essential conditions for the cell to undergo iron death is an increase in the ferrous content. BioTracker 575 Red Probe staining was used to identify groups of intracellular Fe2+The content of (a). The results of the BioTracker 575 Red probe staining combined flow cytometry detection show that after four human genitourinary system tumor cells are respectively treated by AFC with the concentration of 1.0mg/ml for 24 hours, the proportion of the cells with positive staining is obviously higher than that of PBS control group cells (FIG. 5, AFC increases genitourinary system tumor cell strain Fe2+Wherein A is Fe in each group of cells detected by a flow cytometer after the tumor cell line of the genitourinary system is processed by AFC 2 +The probe BioTracker 575 Red marks the proportion of cells positive. B is that after the tumor cell line of the genitourinary system is treated by PBS, Fe in each group of cells is detected by a flow cytometer2+The probe BioTracker 575 Red marks the proportion of cells positive. Experimental results show that after AFC treatment, the proportion of positive cells marked by the probe is obviously higher than that of cells in a PBS control group. (. about. p)<0.01 vs PBS control; n is 4; t test)).
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.
Claims (1)
1. The method for preparing the genitourinary system tumor cell iron death model based on ferric ammonium citrate comprises the following steps: when the density of the cultured genitourinary system tumor cells reaches 65-75%, adding ferric ammonium citrate, and continuing culturing for 20-30 h to obtain an iron death model; the mass concentration of the ferric ammonium citrate is 0.1mg/mL or 1.0 mg/mL; the tumor of the genitourinary system is breast cancer, ovarian cancer, prostatic cancer or renal cancer.
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