CN116650451A - Application of resveratrol in preparing tumor cell iron death inducer - Google Patents
Application of resveratrol in preparing tumor cell iron death inducer Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/05—Phenols
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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Abstract
The invention discloses application of resveratrol in preparation of an iron death inducer for tumor cells. Experiments show that resveratrol can inhibit the growth of tumor cells by direct apoptosis, and can improve the anti-tumor immunotherapy effect by inducing the death function of iron. Therefore, the resveratrol is utilized to prepare the anti-tumor medicament, and the application prospect is expected to be better.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of resveratrol in preparation of a tumor cell iron death inducer.
Background
Resveratrol is an important plant secondary metabolite and has better anti-tumor activity. In recent years, many studies have found that resveratrol has an obvious therapeutic effect on various malignant tumors. Therefore, resveratrol has great significance for the research of anti-tumor mechanism and the drug development thereof.
Among the various death pathways of cells, iron death (ferroptosis) has received increasing attention in recent years as an emerging means of death. Iron death is a cell death process driven by iron-dependent phospholipid peroxidation and is regulated by a variety of cellular metabolic pathways including redox homeostasis, iron metabolism, mitochondrial activity and amino acid, lipid, sugar metabolism, and various disease-related signaling pathways. It was found that iron death can be induced in a number of ways: first, iron death is induced by depletion of intracellular glutathione; second, targeting and inactivating GPX4 protein induces iron death; third, iron death is induced by depletion of intracellular GPX4 and CoQ10 proteins; fourth, lipid peroxidation is induced by the addition of long chain fatty acids or iron oxides.
Resveratrol is a plant secondary metabolite widely applied to treating tumors, however, induction regulation of iron death of tumor cells by resveratrol has not been reported yet. Iron death is an important apoptosis mode, and is closely related to the occurrence and development of tumor cells. Therefore, research on the regulatory mechanism of resveratrol on iron death is important for deep understanding of resveratrol as an antitumor drug.
Disclosure of Invention
The invention aims to provide application of resveratrol in preparing an iron death inducer for tumor cells.
In order to explore the mechanism of Resveratrol (RES) for inhibiting breast cancer, the invention utilizes Resveratrol with different concentrations to treat tumor cells SUM159, detects the induction of apoptosis and inhibition of proliferation of tumor cells, and then detects the markers related to iron death. In the present invention, treatment of breast cancer cell line SUM159 with natural compound RES was found to significantly inhibit proliferation and viability of tumor cells (fig. 1 and 2), and induce apoptosis of tumor cells in a concentration-dependent manner (fig. 3). The experiment found that SUM159 reduced cell activity to 20% at 150. Mu.M concentration, whereas cells passing through the apoptotic pathway survived only 40%, and approximately 20% of cells by other cell death patterns, with similar results at other concentrations. In subsequent experiments, SUM159 was further treated with iron death inhibitors and inducers in combination with inhibitors of various cell death pathways (FIG. 4), and the results showed that RES alone significantly induced cell death relative to the treatment groups with iron death inducers alone (RSL 3 and FIN 56), the combination of iron death inducers with RES further reduced cell activity, while this cell activity was restored to the cell levels of the CTRL group by iron death inhibitors (Fer-1 and Lip-1) and the combination of RES and inhibitors of other pathways was only partially restored. Through the above experiments, the inventors determined that RES was able to significantly inhibit SUM159 cell activity as an iron death inducer. Subsequently, markers of iron death were further examined (fig. 5), and the results showed that RES could significantly increase the metabolite MDA of iron death while GSH was significantly down-regulated, and that changes in the levels of these markers could be restored to the cell levels of the CTRL group by the iron death inhibitors. Further, mRNA and protein levels of iron-death-related genes were detected by RT-PCR and Western Blot (FIGS. 6 and 7), and RT-PCR results showed that mRNA levels of iron-death-related genes (GPX 4, SLC7A11, ACSL3, ACSL4, tfR1, FTH 1) were not varied with RES concentration, whereas only GPX4 protein was gradually decreased with increasing RES concentration among protein levels, and these experimental results showed that: RES may control the iron wire death state of SUM159, especially the stability of GPX4 protein, by modulating GPX4 protein levels. Subsequently, ROS (fig. 8 and 9) and lipid peroxidation levels (fig. 10 and 11) induced after RES treatment of SUM159 cells were examined by flow cytometry, and the structure showed that RES could induce SUM159 cells to produce ROS and lipid peroxidation in a concentration-dependent manner, and as a result, it was further shown that RES could induce SUM159 cells to produce iron death. Next, to further verify that RES was effective in regulating iron death of SUM159 cells by GPX4, the inventors constructed SUM159 cells with GPX4 knockdown (SUM 159-GPX 4) and control cells thereof (SUM 159-V), and verified knock-down levels by Western Blot (fig. 12), and the results showed that GPX4-2 and GPX4-3 knockdown effects were better. The effect of RES on cell activity and apoptosis was subsequently verified in SUM159-GPX4 and SUM159-V cells (fig. 13 and 14). CCK8 experiments show that RES (150 mu M) has little effect on the activity of SUM159-GPX4 cells, and the activity of the SUM159-GPX4 cells is significantly higher than that of SUM159-V cells treated under the same conditions. Similar results were obtained for the apoptosis experiments. Subsequently, we examined the changes in ROS (FIGS. 15 and 16) induced after RES treatment by flow cytometry in SUM159-GPX4 and SUM159-V cells, and the results showed that ROS could be significantly induced after RES (150. Mu.M) treatment in SUM159-V cells, whereas in SUM159-GPX4 cells, the level of ROS was not significantly changed after RES treatment. Similarly, changes in lipid peroxidation induced after RES treatment were detected by flow cytometry in SUM159-GPX4 and SUM159-V cells (FIGS. 17 and 18), and the results indicate that the lipid peroxidation could be significantly induced after RES (150. Mu.M) treatment in SUM159-V cells, whereas the level of lipid peroxidation was not significantly changed after RES treatment in SUM159-GPX4 cells. The above experiments show that RES controls the iron death level of SUM159 cells by regulating the expression level of GPX4 protein. Finally, the mechanism of RES regulation of GPX4 protein level was further studied, and the result of GPX4 protein stability experiments (fig. 19) mainly shows that after RES (150 μm) treatment, GPX4 protein stability was significantly reduced, thereby promoting iron death of SUML59 cells. Thus, resveratrol can be used as an iron death inducer to inhibit the occurrence and development of breast cancer.
Compared with the research of the prior art, the invention has the beneficial effects that:
(1) The invention obtains the iron death which can obviously induce breast cancer cells through the treatment of resveratrol on breast cancer.
(2) The invention constructs a GPX4 knockout stable transgenic cell line in breast cancer SUM159 cells by using a gene editing technology. In the stable transgenic cell line, the regulation and control mechanism of resveratrol on tumor iron death is further explored.
Drawings
FIG. 1 shows the activity levels of CCK8 after detection of RES-treated SUM159 cells.
FIG. 2 is a bar graph analysis of activity levels of CCK8 detected after 48h of RES treated SUM159 cells.
FIG. 3 is an illustration of detection of apoptosis levels in RES treated SUM159 cells.
FIG. 4 is a graph showing activity level assays after treatment of SUM159 cells with various death inhibitors.
FIG. 5 is a graph showing the detection of iron death-related markers after RES treatment of SUM159 cells.
FIG. 6 shows mRNA detection of iron death-related markers before and after RES treatment of SUM159 cells.
FIG. 7 shows protein level detection of iron death-related markers before and after RES treatment of SUM159 cells.
FIG. 8 is a flow cytometry detection of ROS levels before and after SUM159 cell treatment.
FIG. 9 is a bar graph analysis of ROS levels before and after SUM159 cell treatment.
FIG. 10 is a flow cytometry detection of lipid peroxidation levels before and after SUM159 cell treatment.
FIG. 11 is a bar graph analysis of lipid peroxidation levels before and after SUM159 cell treatment.
FIG. 12 is a validation of GPX4 gene knockout in SUM159 cells.
FIG. 13 is a graph showing the detection of the level of cellular activity before and after RES treatment of SUM159-V and SUM159-GPX4 cells.
FIG. 14 is a graph showing the detection of apoptosis levels before and after RES treatment of SUM159-V and SUM159-GPX4 cells.
FIG. 15 is a flow cytometry detection of ROS levels in RES-treated SUM159-V cells.
FIG. 16 is a flow cytometry detection of ROS levels in RES treated SUM159-GPX4 cells.
FIG. 17 is a flow cytometry detection of lipid peroxidation levels in RES treated SUM159-V cells.
FIG. 18 is a flow cytometry detection of lipid peroxidation levels in RES treated SUM159-GPX4 cells.
FIG. 19 shows the stability test of GPX4 protein before and after RES treatment.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
1. Treatment of breast cancer cells with varying concentrations of RES
The breast cancer cell line selected in this section was SUM159, and the procedure was as follows.
1.1 cell culture and plating
(1) The complete medium used for the breast cancer cell line SUM159 was DMEM with 10% FBS at 37℃and 5% CO 2 。
(2) And (5) carrying out cell passage. When the cell density reaches 80% -90%, selecting passage, and putting the cells after passage into 37 ℃ and 5% CO again 2 The cell culture was continued in the incubator.
1.2 treatment of breast cancer cells with resveratrol at different concentrations
(1) Cells (SUM 159) with better inoculation state are pre-cultured in a 6-well plate for 12 hours;
(2) After the cell density reached 70%, resveratrol (0, 5, 10, 50, 100 and 150 μm respectively) was added to each well at the indicated concentration, and culturing was continued for 24 hours;
(3) Protein and mRNA were collected separately, and the change in expression of iron-death-related genes was detected.
2. Detection of the level of cellular Activity before and after RES-treated SUM159 cells
In this experiment, the CCK-8 reagent was used to detect the changes of the activity of cells with time before and after the treatment of breast cancer cells SUM159 with different concentrations of RES, the concentration of RES treatment was 0,5, 10, 50, 100 and 150. Mu.M, and the detection time was selected from 0h, 12h, 24h, 36h and 48h, respectively.
3. Flow cytometry detection of apoptosis levels before and after SUM159 cell treatment
Wild-type SUM159 was treated with RES at various concentrations for 48h, and then the apoptosis level of SUM159 tumor cells in each treatment group was determined using Annexin V-PE/7-AAD Apoptosis Detection Kit (Vazyme).
As shown in fig. 1 and 2, RES significantly inhibited proliferation and viability of tumor cells; as shown in fig. 3, apoptosis of tumor cells was induced in a concentration-dependent manner.
Example 2
SUM159 cells treated with inhibitors of various death pathways in combination with resveratrol
Wild-type SUM159 cells were treated with different kinds of cell death inhibitors in combination with RES, respectively, while iron death inducers RSL3 and FIN56 and iron death inhibitors Fer-1 and Lip-1 were also used in combination. Inhibitor species: apoptosis inhibitor Z-VAD-FMK, autophagy inhibitor 3-methylglucine (3-ME), and pyroapoptosis inhibitor Bay 11-7821 (BAY). The experimental set was set as follows: CTRL (non-medicated treatment group), RSL3 treatment group, FIN56 treatment group, RES-combined RSL3 treatment group, RES-combined FIN56 treatment group, RES-combined Fer-1 treatment group, RES-combined Lip-1 treatment group, RES-combined Z-VAD-FMK treatment group, RES-combined 3-ME treatment group, RES-combined BAY treatment group. The level of cellular activity under each treatment condition was measured using CCK8 reagent.
As shown in fig. 4, RES alone significantly induced cell death compared to the iron death inducer alone (RSL 3 and FIN 56) treated groups, the combination of iron death inducer and RES further reduced cell activity, while this cell activity was restored to the cell level of CTRL groups by iron death inhibitors (Fer-1 and Lip-1) and RES in combination with inhibitors of other pathways was only partially restored.
Example 3
Detection of iron death products before and after RES treatment
Wild SUM159 cells were selected and treated with RES and iron death inhibitors Fer-1 and Lip-1, and the experimental groups were as follows: CTRL (non-medicated treatment group), RES treatment group, RES combined with Fer-1 treatment group, RES combined with Lip-1 treatment group. Levels of iron death-related products MDA and GSH in each experimental group were detected using MDA and GSH detection kits.
As shown in fig. 5, RES was able to significantly increase the metabolite MDA of iron death while GSH was significantly down-regulated, and the level changes of these markers were able to be restored to the cellular levels of the CTRL group by the iron death inhibitors.
Example 4
1. mRNA detection of iron death-related markers before and after RES treatment
Wild-type SUM159 was treated with RES at various concentrations of 0,5, 10, 50, 100 and 150. Mu.M, respectively, and cells were collected 24 hours after treatment. Total RNA was prepared from RNA-easy Isolation Reagent and reverse transcribed into cDNA by HiScript II Q RT SuperMix (Vazyme) for use in qPCR (AceQ qPCR SYBR Green Master Mix) (Vazyme). The genes detected mainly include: GPX4, SLC7a11, ACSL4, ACSL3, tfR1 and FTH1.
2. Protein detection of iron death-related markers before and after RES treatment
Wild-type SUM159 was treated with RES at various concentrations of 0,5, 10, 50, 100 and 150. Mu.M, respectively, and cells were collected after 48h of treatment. Cells were collected and protein was extracted with a mixture of RIPA buffer and protease inhibitor (mixing ratio 100:1). The proteins detected mainly include: GPX4, SLC7a11, ACSL4, ACSL3, tfR1 and FTH1.
As shown in FIGS. 6 and 7, the RT-PCR results showed that mRNA levels of iron-death-related genes (GPX 4, SLC7A11, ACSL3, ACSL4, tfR1, FTH 1) did not vary with RES concentration, whereas in protein levels, only GPX4 protein was gradually decreased with increasing RES concentration,
example 5
1. Flow cytometry detection of ROS level detection before and after SUM159 cell treatment
Wild-type SUM159 was treated with RES at various concentrations of 0,5, 10, 50, 100 and 150. Mu.M, respectively, and cells were collected 24 hours after treatment. The level of ROS in each group of cells was detected using C11-BODIPY.
2. Detection of lipid peroxidation level before and after SUM159 cell treatment by flow cytometry
Wild-type SUM159 was treated with RES at various concentrations of 0,5, 10, 50, 100 and 150. Mu.M, respectively, and cells were collected 24 hours after treatment. The level of lipid peroxidation was then detected in each group of cells using DCFH-DA.
As shown in fig. 8, 9, 10 and 11, RES was able to induce SUM159 cells to produce ROS and lipid peroxidation in a concentration-dependent manner, and the results further indicate that RES may induce SUM159 cells to produce iron death.
Example 6 verification of GPX4 Gene knockout in SUM159 cells
Three sgrnas (sgRNA 1, sgRNA2, and sgRNA 3) were designed for gene GPX 4. GPX4 is knocked out from a breast cancer cell SUM159 by using a CRSPR Cas9 gene editing technology, a stable transfer cell line SUM159-GPX4 is constructed, and a control cell line SUM159-V is constructed. And collecting cells, and verifying the knocking-out effect of GPX4 by using western blot.
As shown in FIG. 12, GPX4-2 and GPX4-3 knockdown effects were better.
Example 7
Detection of the level of Activity of RES treated GPX4 deleted SUM159 cells before and after cell
The stable transgenic cell lines SUM159-GPX4 and SUM159-V constructed as described above were selected, were treated with RES (concentration was selected to be 0 and 150. Mu.M), and the changes in the cell activities of SUM159-GPX4 and SUM159-V with time were detected using CCK-8 reagent, and the detection times were selected to be 0d, 1d, 2d, 3d, 4d, 5d, respectively.
As shown in FIGS. 13 and 14, RES (150. Mu.M) had little effect on the activity of SUM159-GPX4 cells, which was significantly higher than that of SUM159-V cells treated under the same conditions, and similar results were obtained in apoptosis experiments.
Example 8
1. Flow cytometry detection of ROS levels in RES-treated GPX 4-deleted SUM159 cells
The stable transgenic cell lines SUM159-GPX4 and SUM159-V constructed as described above were selected, and were respectively subjected to RES treatment (concentration was selected to be 0 and 150. Mu.M), and cells were collected after 24 hours of treatment. The level of ROS in each group of cells was detected using C11-BODIPY.
2. Detection of lipid peroxidation levels in RES-treated GPX 4-deleted SUM159 cells by flow cytometry
The stable transgenic cell lines SUM159-GPX4 and SUM159-V constructed as described above were selected, and were respectively subjected to RES treatment (concentration was selected to be 0 and 150. Mu.M), and cells were collected after 24 hours of treatment. The level of lipid peroxidation was then detected in each group of cells using DCFH-DA.
As shown in FIGS. 15 and 16, ROS can be significantly induced in SUM159-V cells after RES (150. Mu.M) treatment, whereas ROS levels did not change much in SUM159-GPX4 cells after RES treatment. As shown in fig. 17 and 18, the change in lipid peroxidation caused after RES treatment was detected by flow cytometry in SUM159-GPX4 and SUM159-V cells, and the results showed that the lipid peroxidation could be significantly induced after RES (150 μm) treatment in SUM159-V cells, whereas the level of lipid peroxidation was not greatly changed after RES treatment in SUM159-GPX4 cells.
Example 9
Stability detection of GPX4 protein before and after RES treatment
Selecting wild breast cancer cells SUM159, treating with concentration of 0 and 150 μm respectively, and then treating SUM159 with CHX and MG132 respectively for 0h,2h,4h,8h, wherein the corresponding experimental group is RES 0 μm (CHX 0h, MG1320 h); RES 0. Mu.M (CHX 2h, MG132 h); RES 0. Mu.M (CHX 4h, MG132 h); RES 0. Mu.M (CHX 8h, MG1320 h); RES 0. Mu.M (CHX 2h, MG132 2 h); RES 0. Mu.M (CHX 4h, MG1324 h); RES 0 μm (CHX 8h, mg132 8 h); RES 150. Mu.M (CHX 0h, MG1320 h); RES 150. Mu.M (CHX 2h, MG132 h); RES 150. Mu.M (CHX 4h, MG132 h); RES 150. Mu.M (CHX 8h, MG1320h); RES 150. Mu.M (CHX 2h, MG132 2 h); RES 150. Mu.M (CHX 4h, MG1324 h); RES 150. Mu.M (CHX 8h, MG132 8 h). The cells of each group were collected, protein was extracted, and the protein level of GPX4 was detected by using a western blot.
As shown in fig. 19, the stability of GPX4 protein was significantly reduced after RES (150 μm) treatment, thereby promoting iron death of SUML59 cells.
Claims (2)
1. Application of resveratrol in preparing tumor cell iron death inducer is provided.
2. The use according to claim 1, characterized in that: the tumor cells are breast cancer cells.
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| CN112704673A (en) * | 2020-12-24 | 2021-04-27 | 中国人民解放军军事科学院军事医学研究院 | Application of resveratrol in preparing medicine for inhibiting trophoblastic iron death and treating preeclampsia |
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| Title |
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| 倪青等编: "《中国中西医专科专病临床大系 内分泌病诊疗全书》", vol. 1, 31 October 2016, 中国中医药出版社, pages: 853 * |
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