CN111973746A

CN111973746A - Application of iron death inducer in preparation of radiotherapy sensitizer

Info

Publication number: CN111973746A
Application number: CN202010989682.9A
Authority: CN
Inventors: 丁德刚; 王晓博; 高永举; 冯晗; 李晓飞; 王鹏; 杨阳; 陈战磊
Original assignee: Henan Provincial Peoples Hospital
Current assignee: Henan Provincial Peoples Hospital
Priority date: 2020-09-18
Filing date: 2020-09-18
Publication date: 2020-11-24

Abstract

The invention belongs to the technical field of medicines, and particularly provides application of an iron death inducer in preparation of a radiotherapy sensitizer. The invention discloses the sensitization and molecular mechanism of the iron death inducer in radiotherapy (or external irradiation therapy) and radionuclide therapy (or internal irradiation therapy) for the first time, and provides a new idea for the research and development of radiotherapy sensitizer medicines taking iron death as a target.

Description

Application of iron death inducer in preparation of radiotherapy sensitizer

Technical Field

The invention relates to the technical field of medicines, in particular to application of an iron death inducer in preparing a radiotherapy sensitizer, and more particularly provides application of the iron death inducer in sensitizing radiotherapy (or external irradiation therapy) and radionuclide therapy (or internal irradiation therapy).

Background

Radiotherapy is a therapeutic method for treating tumors by using radioactive rays, including alpha, beta and gamma rays generated by radioactive isotopes, and x rays, electron beams, proton beams and other particle beams generated by various x-ray therapeutic machines or accelerators. In the action mechanism, two main ways of generating biological effects by radiotherapy are that the radiation directly damages biomolecules such as DNA and lipid, and the radiation generates a large amount of free radicals mainly containing hydroxyl through ionized water molecules to further damage biomolecules such as DNA. Radiotherapy is the second largest therapeutic approach to malignant tumor after surgery, but the treatment fails because normal tissues are limited in tolerance dose and cannot give enough radiation dose to the tumor, so how to improve the sensitivity of the tumor to radiation is a prominent problem in clinical tumor radiotherapy. The radiosensitizer is used as a medicine for enhancing the sensitivity of tumor radiotherapy and improving the curative effect of the radiotherapy, and achieves the purpose of radiosensitization by increasing the radiation-induced oxygen free radicals and DNA damage and regulating and controlling the key molecular target of the radiotherapy. Currently, the types of radiosensitizers are mainly classified into DNA precursor base analogs, electrophilic radiosensitizers (including nitroimidazoles, nitroaromatics and nitroheterocycles), hypoxic cell radiosensitizers, bioreductive compounds, radiation damage repair inhibitors, sulfydryl inhibitors, oxygen utilization inhibitors, oxygen-like compounds, cytotoxic radiosensitizers, targeted radiosensitizers, gene-related tumor radiosensitizers, traditional Chinese medicines and the like. Despite decades of development of radiosensitizers, the clinical needs of radiotherapy are still not met by relevant research. Particularly, the clinical application of the existing radiosensitizer is not completely confirmed, and the action target and mechanism of the existing radiosensitizer need to be clarified to further optimize the radiosensitizer, so that the radiosensitizer becomes a more effective clinical auxiliary means. Therefore, the development of the radiosensitizer with definite target and action mechanism is one of the important directions for the development of the next-generation radiosensitizer.

In 2012, professor Stockwell, a famous scholars at the university of columbia, discovered a completely new iron-dependent cell death pattern and named it as iron death (Ferroptosis). The iron death is essentially the metabolic disorder of lipid peroxides in cells, namely, due to the depletion of Glutathione (GSH), the activity of membrane lipid repair enzyme Glutathione Peroxidase (Glutathione Peroxidase 4, GPX 4) is reduced, lipid peroxides can not be metabolized through GSH reduction reaction catalyzed by GPX4, and then abnormal metabolism is catalyzed by iron ions, so that the accumulation of lipid peroxides is caused, and the cell death is triggered. For example, Erastin and SAS can trigger iron death under oxidative stress by inhibiting the cystine/glutamate transporter (System XC-), causing a decrease in GPX4 activity. A large number of oxygen free radicals can be generated in the radiation treatment process, and whether radiotherapy sensitization can be realized or not is achieved by regulating an iron death signal channel, so that no literature report exists at present. Therefore, the research and development of the radiosensitizer drug by taking iron death as a target point is a problem which needs to be solved urgently by researchers in the field and is also a core innovation point of the invention.

Disclosure of Invention

In view of the above situation, the present invention aims to overcome the defects of the prior art and provide an application of an iron death inducer in the preparation of a radiation sensitizer.

The solution is the application of the iron death inducer in the preparation of the radiotherapy sensitizer.

Preferably, the iron death inducer achieves the radiosensitizing effect by regulating an iron death signaling pathway.

Preferably, the iron death inducer is preferably selected from Erastin, RSL3, FIN56, glutamine, cisplatin, sulfasalazine, sorafenib, artesunate, dihydroartemisinin, artemether and vitamin E.

Preferably, the radiosensitizer is a pharmaceutical composition comprising an effective amount of an iron death inducing agent and optionally a pharmaceutically acceptable carrier and/or adjuvant.

Preferably, the route of administration of the radiosensitizer comprises oral, intravenous, intramuscular, subcutaneous, nasal, intraperitoneal, sublingual or transdermal administration.

Preferably, the radiosensitizer is used in radiotherapy (or external irradiation therapy) and radionuclide therapy (or internal irradiation therapy).

The invention has the beneficial effects that: the invention discloses an application of an iron death inducer in preparing a radiation sensitizer. The invention discloses the sensitization effect and the molecular mechanism of the iron death inducer in radiotherapy (external irradiation therapy) and radionuclide therapy (internal irradiation therapy) for the first time, and provides a new idea for the research and development of radiotherapy sensitizer medicaments taking iron death as a target.

Drawings

FIG. 1 is a molecular mechanical diagram of iron death inducer sensitization radiotherapy;

FIG. 2 is a graph of cell activity after (A) A549, (B) MCF-7 and (C) HepG2 cells received different doses of radiation therapy (2, 4 and 6 Gy), iron death inducer Erastin pretreatment 12 h + different doses of radiation therapy (2, 4 and 6 Gy) and iron death inhibitor Fer-1 pretreatment 12 h + different doses of radiation therapy (2, 4 and 6 Gy), respectively;

FIG. 3 is a graph of cell activity of (A) A549, (B) MCF-7, and (C) HepG2 cells after receiving saline, 6 Gy radiation therapy, apoptosis inhibitor Z-VAD pretreatment 12 h + 6 Gy radiation therapy, iron death inhibitor Fer-1 pretreatment 12 h + 6 Gy radiation therapy, respectively;

FIG. 4 is a graph showing the content change (A) of reduced Glutathione (GSH), the expression change (B) of glutathione peroxidase-4 (GPX-4) and the content change (C) of Lipid Peroxides (LPO) in cells after the cells A549, MCF-7 and HepG2 respectively receive physiological saline, 6 Gy radiotherapy, 12 h + 6 Gy radiotherapy pretreated by iron death inducer Erastin and 12 h + 6 Gy radiotherapy pretreated by iron death inhibitor Fer-1;

FIG. 5 is a schematic diagram of an experimental study of iron death inducer sensitization A549 transplantation tumor radiotherapy;

FIG. 6 is a schematic diagram of (A) isolated tumor tissues at 12d in different treatment groups of tumor-bearing nude mice; tumor volume (B) and body weight change (C) during treatment of tumor-bearing nude mice;

FIG. 7 is a graph of hematoxylin and eosin (H & E) staining of ex vivo tumor tissue at 12d in different treatment groups of tumor-bearing nude mice;

FIG. 8 is a graph (A) showing the staining pattern of isolated tumor tissue Ki-67 and its quantitative value (B) at 12d for different treatment groups of tumor-bearing nude mice;

FIG. 9 is a Western blot of GPX-4 expression in isolated tumor tissues at 12d in nude mice bearing tumors from different treatment groups.

Detailed Description

The following embodiments are intended to illustrate the present invention and are not intended to further limit the present invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1: effect of iron death in radiation therapy of A549, MCF-7 and HepG2 cells

A549, MCF-7 and HepG2 cells were seeded in 96-well plates, each set of 6 duplicate wells. Four weeks were filled with PBS buffer (200 μ L per well) to prevent peripheral effects. Incubate at 37 ℃ in a 5% CO2 incubator. After the cells were attached to the wall, the old medium was discarded, 200. mu.L of fresh medium was added to each well, and the following treatments were performed:

control: 10 μ L of physiological saline

Treatment group 1: radiotherapy treatment (2, 4 and 6 Gy)

Treatment group 2: 10 μ L Erastin 12 h + radiation therapy (2, 4 and 6 Gy)

Treatment group 2: 10 μ L Fer-112 h + radiation therapy (2, 4 and 6 Gy)

The incubator was incubated for a further 48 h. And discarding the old culture medium after 48 hours, and measuring the activity of each group of cells by using a CCK-8 kit.

control: 10 μ L of physiological saline

Treatment group 1: 6 Gy

Treatment group 2: 10 μ L Z-VAD 12 h + radiation therapy (6 Gy)

Treatment group 2: 10 μ L Fer-112 h + radiation therapy (6 Gy)

control: 10 μ L of physiological saline

Treatment group 1: 6 Gy

Treatment group 2: 10 μ L Erastin 12 h + radiation therapy (6 Gy)

Treatment group 2: 10 μ L Fer-112 h + radiation therapy (6 Gy)

The incubator was incubated for a further 48 h. Discarding the old culture medium after 48h, and measuring the content of Glutathione (GSH) in each group of cells by using a reduced GSH content measuring kit; measuring GPX-4 expression change in each group by using western blotting; the content of Lipid Peroxide (LPO) in each group of cells was determined using a LPO detection kit.

As shown in FIGS. 2 and 3, in 549, MCF-7 and HepG2 cells, cell death was induced by administration of different doses of radiation. Cell death was significantly increased after the addition of the iron death inducer Erastin. However, in the treatment group to which the iron death inhibitor Fer-1 was added, cell death was suppressed; as a control, the apoptosis inhibitor Z-VAD was added in response to the control, and the inhibition of cell death was relatively weak. These findings indicate that iron death occurs during radiotherapy and that iron death inducers can sensitize radiotherapy.

As shown in FIG. 4, GSH content in various cells was significantly reduced, GPX-4 expression was increased, and LPO content was increased 48h after radiotherapy treatment. In the treatment group given with the iron death inducer Erastin, the GSH content is further reduced, the GPX-4 expression is reduced, and the LPO content is obviously increased; in the treatment group given the iron death inhibitor, Fer-1, both GSH and GPX-4 levels increased and LPO levels decreased. These results indicate that, in radiotherapy, although iron death occurs, it also induces an increase in GPX-4 expression, resistant to radiotherapy. Upon administration of an iron death inducing agent, GPX-4 expression was significantly down-regulated, further inducing the occurrence of iron death. Thus, iron death induction and radiation therapy produce a synergistic effect. The iron death inducer can be used as a radiotherapy sensitizer and has sensitization effect in radiotherapy.

Example 2: experimental study on iron death inducer sensitization A549 transplantation tumor radiotherapy

Selecting BALB/c nude mice with the week age of 4-5 weeks, subcutaneously inoculating about 107A 549 cells, establishing a lung cancer transplantation tumor model, randomly dividing into 4 groups when the tumor volume reaches 100-200 mm3, and respectively performing the following treatment (figure 5): a, injecting normal saline into the abdominal cavity every day; b10 Gy radiation therapy; after C10 Gy radiotherapy, Erastin (15 mg/kg) was given to the peritoneal cavity daily; after D10 Gy radiation therapy, daily intraperitoneal injections of Fer-1 (10 mg/kg) were given. The weight and the tumor volume of the tumor-bearing nude mice are monitored every 2 days for 12 days. Nude mice were sacrificed at 12d, tumor tissue was taken, stained with hematoxylin and eosin (H & E) and Ki-67; and detecting the change of the content of GPX-4 by Western blot.

As shown in fig. 6, tumor volume decreased after a549 lung cancer tumor was treated with 10 Gy radiation for 12 days; the tumor volume was significantly reduced by further administration of the iron death inducer group; in contrast, when the group of iron death inhibitors was further administered, the tumor volume became large. During the course of treatment, the tumor-bearing nude mice in each group slightly lost weight. H & E and Ki-67 staining indicated that tumor cell necrosis was evident and proliferation index was significantly reduced in the radiation therapy in combination with iron death inducer group (fig. 7 and 8). Western blot detection GPX-4 expression experiments show that after radiotherapy, GPX-4 expression is up-regulated, and GPX-4 expression can be remarkably down-regulated by further administration of an iron death inducer (figure 9). These results indicate that iron death induction and radiation therapy produce a synergistic effect. The iron death inducer can be used as a radiotherapy sensitizer and has sensitization effect in radiotherapy.

Finally, it should be noted that the above-mentioned embodiments are only preferred examples of the present invention, and are not intended to limit the present invention, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of them. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. The invention has been described in detail with respect to a general description and specific embodiments thereof, but it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Therefore, any modification or improvement made without departing from the spirit and substance of the present invention shall fall within the scope of the claimed invention.

Claims

1. Application of iron death inducer in preparing radiotherapy sensitizer.

2. The use of an iron death inducer according to claim 1 in the manufacture of a therapeutic radiosensitizing agent, wherein the iron death inducer achieves the radiosensitizing effect by modulating the iron death signaling pathway.

3. Use of the iron death inducer according to claim 1 or 2, in the preparation of a radiosensitizer for treatment, wherein said iron death inducer is preferably selected from the group consisting of Erastin, RSL3, FIN56, glutamine, cisplatin, sulfasalazine, sorafenib, artesunate, dihydroartemisinin, artemether and vitamin E.

4. Use of an iron death inducer according to claim 3 in the preparation of a therapeutic radiosensitizer, wherein said radiosensitizer is a pharmaceutical composition comprising an effective amount of iron death inducer, optionally together with pharmaceutically acceptable carriers and/or adjuvants.

5. Use of an iron death inducing agent according to claim 4 in the preparation of a therapeutic radiosensitizer, wherein the route of administration of the radiosensitizer comprises oral, intravenous, intramuscular, subcutaneous, nasal, intraperitoneal, sublingual or transdermal administration.

6. Use of an iron death inducer according to claim 5 in the preparation of a therapeutic radiosensitizer, wherein said radiosensitizer is used in radiotherapy (or external irradiation therapy) and radionuclide therapy (or internal irradiation therapy).