CN115707487A - Use of a product containing an iron death inducer and capable of causing a tumor methionine deficiency for the preparation of a medicament for the treatment of tumors - Google Patents

Abstract

The invention belongs to the technical field of medicine, and in particular relates to the application of a product containing a ferroptosis inducer and capable of causing tumor methionine deficiency in the preparation of a drug for treating tumors. The present invention found for the first time that the combined use of methionine deficiency and ferroptosis inducer can further enhance the tumor killing ability, and the effect is obviously better than that of using methionine deficiency and ferroptosis inducer alone, indicating that the two in After combined use, there is an obvious synergistic effect, which provides a new way to overcome the resistance of tumors to the treatment of metabolic defects and to treat tumors with ferroptosis as the target. The present invention also provides a nutritional complex for inhibiting tumors and tumor-suppressive media.

Description

Products containing ferroptosis inducers and can cause tumor methionine deficiency in the preparation of therapeutic application in cancer drugs

technical field

The invention belongs to the technical field of medicine, and in particular relates to the application of a product containing a ferroptosis inducer and capable of causing tumor methionine deficiency in the preparation of a drug for treating tumors.

Background technique

Ferroptosis is a form of regulated cell death driven by iron-dependent lipid peroxidation. It was first proposed by Brent R. Stockwell in 2012. Different from general programmed death, it is obviously different from other forms of regulated cell death such as apoptosis, necrosis, and autophagy at the morphological, biological, and genetic levels. The essence of ferroptosis is the metabolic disorder of intracellular lipid peroxides, and then the abnormal metabolism under the catalysis of iron ions produces a large amount of lipids, disrupts the balance of intracellular redox reactions, and attacks biological macromolecules to trigger cell death. A large number of research reports have shown that induction of ferroptosis can be used as an important mechanism of anti-tumor, and its role in tumorigenesis, development and drug resistance has also attracted much attention. Therefore, inducing ferroptosis in tumor cells is a novel antitumor therapeutic strategy. Ferroptosis inducers include class I inducers that inhibit glutamate/cystine antitransporter SLC7A11, such as the small molecule compound Erastin, and class II inducers that inhibit glutathione peroxidase 4GPX4, such as RSL3.

Methionine (L-Met) is an essential amino acid in the human body, and it participates in the methyl transfer catalytic reaction of DNA, protein and other different substances in the body. Most tumor cells have a high demand for methionine and are methionine-dependent, that is, when L-Met is restricted or deprived in the culture medium or diet, the growth of tumor cells is inhibited, while normal mammalian cells can proliferate normally . Compared with normal cells, tumor cells rely on a large amount of exogenous preformed L-Met to maintain their growth, especially highly malignant tumors have a higher dependence on L-Met, such as GBM, astrocytoma , prostate cancer, osteosarcoma, and certain lymphomas. The high demand for L-Met by tumor cells is partly related to its proliferation rate, because L-Met is an essential substance for the production of L-cysteine (L-Cys) for protein synthesis as well as glutathione (GSH) and anti-inflammatory effects. oxidation reaction. At the same time, L-Met is also used in polyamine synthesis and S-adenosylmethionine (SAM), which is the main methyl donor for DNA and protein methylation. Aberrant methylation and increased polyamine synthesis are hallmarks of many tumors and are known contributors to tumorigenesis. Therefore, limiting the growth of L-Met by limiting the content of tumor growth is an effective tumor suppressor. Dietary interventions low in or without methionine have also been shown to be effective in stopping tumor growth and prolonging survival in cancer-bearing animals. Recombinant methionine enzyme as a more efficient and rapid method to deplete serum methionine.

SGN1 (also known as VNP20009-M) uses genetic engineering to use attenuated Salmonella VNP20009 as a carrier to carry and express L-methioninase to achieve the purpose of treating tumor growth and metastasis. VNP20009 is an attenuated strain of Salmonella typhimurium with deletion of msbB and pμr I genes. It is genetically stable and sensitive to antibiotics. The msbB gene is necessary for the acylation of lipids to endotoxin, and its deletion prevents the acylation of the lipid A terminal, which reduces the toxicity; the pμr I gene is involved in purine metabolism, and its deletion makes the bacterial reproduction require exogenous adenine. VNP20009 also reduces the tumor necrosis factor (tμmor necrosis factor, TNF) produced by the body itself, and reduces the inflammatory response caused by VNP20009. Therefore, its low pathogenicity improves the safety for clinical treatment.

At present, SGN1 has been used in tumor treatment research, and can act on a variety of solid tumor models, including breast cancer (patent number: ZL201310062253.7), pancreatic cancer (patent number: ZL201310688936.3), prostate cancer (patent number: ZL201410183149. 8), liver cancer (patent number: 201510546063.1), lung cancer (patent number: 201710213446.6), malignant sarcoma (patent number: 201710216811.9) and other cancers.

Contents of the invention

In order to overcome the deficiencies and shortcomings of the prior art, the object of the present invention is to provide an application of a product containing a ferroptosis inducer and capable of causing tumor methionine deficiency in the preparation of a drug for treating tumors.

Another object of the present invention is to provide a tumor-suppressing nutritional complex.

Another object of the present invention is to provide a medium for suppressing tumor cells.

The purpose of the present invention is achieved through the following technical solutions:

Application of a product containing a ferroptosis inducer and capable of causing tumor methionine deficiency in the preparation of a drug for treating tumors;

The methionine deficiency includes but is not limited to dietary or medium methionine deficiency, using bacteria or viruses as vectors, carrying plasmids to express recombinant methionine enzymes, consuming methionine to cause tumor methionine Defects, or tumor methionine deficiencies caused by other means;

The diet or culture medium deficiency refers to the methionine deficiency caused by the essential nutrient complex that does not contain methionine;

The ferroptosis inducers include but are not limited to class I and class II ferroptosis inducers;

The class I inducers include but are not limited to sorafenib, sulfasalazine, small molecule compound Erastin;

The class II ferroptosis inducer but not limited to RSL3;

The tumor includes but not limited to glioma;

A tumor-suppressing nutrient complex, which comprises a basal nutrient complex and a ferroptosis inducer, wherein the basal nutrient complex is a methionine-deficient basal nutrient complex;

The ferroptosis inducer is preferably Erastin;

The concentration of the ferroptosis inducer is preferably 0.5-5 μM;

A medium for suppressing tumor cells, the medium comprises a basal medium and a ferroptosis inducer, wherein the basal medium is a methionine-deficient basal medium;

The basal medium is methionine-deficient DMEM or RPMI 1640;

The ferroptosis inducer is preferably Erastin;

The concentration of the ferroptosis inducer is preferably 0.5-5 μM;

Compared with the prior art, the present invention has the following advantages and effects:

(1) For the first time, the present invention creatively utilizes the combination of methionine deficiency and ferroptosis inducer to further enhance the tumor killing ability, and the effect is obviously better than that of using methionine deficiency and ferroptosis inducer alone, It shows that the combination of the two has a significant synergistic effect, which provides a new way to overcome the resistance of tumors to metabolic defect therapy and tumor therapy targeting ferroptosis, and is also expected to provide a new way for the anti-tumor drug independently developed by the team. The clinical use of SGN1 in combination with other drugs indicates a new direction.

(2) The present invention relates to a tumor-suppressing trophic complex, which can treat cancer, especially glioma, through the combined use of a methionine-deficient basal trophic complex and a ferroptosis inducer, for The treatment of cancer patients provides a new technical means.

(3) The present invention relates to a medium for inhibiting tumor cells, which can inhibit tumor cells, especially glioma cells, through the combined use of a methionine-deficient basal medium and a ferroptosis inducer, It provides technical means and ideas for the study of tumor-related mechanisms.

Description of drawings

Figure 1 shows the growth status of human glioma cell line Snb19 cultured under different conditions (ferroptosis inducer is Erastin), in which, A: normal group, B: Erastin group, C: methionine-deficient group , D: Methionine-deficient + Erastin group.

Fig. 2 is an analysis diagram of the difference in the number of cells of the human glioma cell line Snb19 cultured under different conditions (ferroptosis inducer is Erastin) for 72 hours.

Fig. 3 is an analysis diagram of the difference in the number of cells of the human glioma cell line U87 cultured under different conditions (ferroptosis inducer is Erastin) for 72 hours.

Figure 4 shows the growth state of the human brain glioma cell line U138 cultured under different conditions (ferroptosis inducer is Erastin), in which, A: normal group, B: Erastin group, C: methionine Deficiency group, D: methionine deficiency+Erastin group.

Fig. 5 is an analysis diagram of the difference in the number of cells of the human brain glioma cell line U138 cultured under different conditions (ferroptosis inducer is Erastin) for 72 hours.

Fig. 6 is an analysis diagram of the difference in the number of cells of the human acute myeloid leukemia cell line HL-60 cultured under different conditions (ferroptosis inducer is Erastin) for 72 hours.

Figure 7 shows the growth state of the human glioma cell line Snb19 cultured under different conditions (ferroptosis inducer is RSL3), wherein, A: normal group, B: RSL3 group, C: methionine-deficient group , D: Methionine-deficient+RSL3 group.

Fig. 8 is an analysis diagram of the difference in the number of cells of the human glioma cell line Snb19 cultured under different conditions (ferroptosis inducer is RSL3) for 72 hours.

Fig. 9 is an analysis diagram of the difference in the number of cells of the human glioma cell line U87 cultured under different conditions (ferroptosis inducer is RSL3) for 72 hours.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the associated drawings. The following content provides the preferred embodiment of the present invention, but the present invention can be realized by many different forms, is not limited to the embodiment described in this research, and these embodiments just facilitate the disclosure content of the present invention to be done more thoroughly and comprehensively understanding. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used in the description of the present invention are only for describing specific embodiments, and are not intended to limit the present invention.

MEM medium containing 10% fetal bovine serum, DMEM medium containing 10% fetal bovine serum, and RPMI 1640 medium containing 10% fetal bovine serum were purchased from Gibco, USA, and Erastin and RSL3 were purchased from selleck.

Snb19 cell line (human glioma cell line), U138 cell line (human brain glioma cell line), U87 cell line (human glioma cell line), HL-60 cell line (human acute myeloid leukemia cell line) cell lines) were purchased from the Shanghai Cell Institute of the Chinese Academy of Sciences.

Example 1

1. Experimental group

In this embodiment, each tumor cell was divided into four groups: normal group, ferroptosis-induced (Erastin/RSL3) group, methionine-deficient group, and methionine-deficient+Erastin/RSL group.

2. Experimental method

(1) Culture of tumor cells

①Snb19 cell line (human glioma cell line), M138 cell line (human brain glioma cell line), M87 cell line (human glioma cell line) and other glioma cell lines were resuscitated and inoculated in a 10cm In the cell culture dish, the seeding density is 1×10 ⁶ cells/dish, wherein, the culture medium of Snb19 cells and M138 cells is MEM medium containing 10% fetal bovine serum, and the medium of M87 cells is containing 10% fetal bovine serum DMEM medium; after inoculation, the cells were cultured in an incubator at 37°C and 5% CO ₂ for 8 hours to allow the cells to adhere to the wall.

② Resuscitate and inoculate HL-60 cell line (human myeloid leukemia cell line) in T25 culture flask at a seeding density of 1×10 ⁶ cells/dish, wherein the culture medium of HL-60 cells contains 10% fetal bovine Serum-based RPMI 1640 medium; after inoculation, culture in an incubator at 37°C and 5% CO ₂ for 8 hours to allow the cells to adhere to the wall.

(2) Preparation of ferroptosis inducer

① Prepare 10mM Erastin, dissolve 10mg Erastin in 1.82mL DMSO, dilute 2000 times to a final concentration of 5μM before use, and store in a -20°C refrigerator away from light.

② Prepare 10mM RSL3, dissolve 5mg RSL3 in 1.2mL DMSO, then dilute 10 times to 1mM, dilute to a final concentration of 0.5μM before use, and store in a -20°C refrigerator away from light.

(3) Preparation of methionine-deficient medium

Purchase DMEM (No L-Met, no methionine) and RPMI 1640 (No L-Met) from Gibco, USA, add L-Gl μtamine to a final concentration of 20 mM before the medium is used, and the normal group and the ferroptosis induction group need Add L-Met to a final concentration of 30 mg/L.

(4) plank administration

A. For Erastin:

①Digest the four kinds of cells cultured in step (1) with trypsin, collect by centrifugation and pipette to make cell suspension (concentration is 2×10 ⁶ cells/ml), and then press 1×10 ⁵ cells/well The density is evenly seeded in a six-well plate, and then 2ml of medium is added to each well according to the cell grouping and the following ratio:

Normal group (Met+): DMEM (10% FBS) + 1 μl DMSO

Ferroptosis induction group: DMEM (10% FBS) + 1μl Erastin (10mM)

Deficient group (Met-): DMEM (No L-Met, 10% FBS) + 1 μl DMSO

Combined group: DMEM (No L-Met, 10% FBS) + 1μl Erastin (10mM)

② After administration, the four groups continued to observe the cell state for three days, and collected the cells after 72 hours and counted the difference in the number of cell growth.

B. For RSL3:

The four kinds of cells cultured in step (1) were digested with trypsin respectively, collected by centrifugation and blown to make cell suspension (concentration is 2×10 ⁶ cells/ml), and then press the density of 1×10 ⁵ cells/well Inoculate evenly in a six-well plate, and then add 2ml of medium to each well according to the cell grouping and the following ratio:

Normal group (Met+): DMEM (10% FBS) + 1 μl DMSO

Ferroptosis induction group: DMEM (10% FBS) + 1 μl RSL3 (1 mM)

Deficient group (Met-): DMEM (No L-Met, 10% FBS) + 1 μl DMSO

Combined group: DMEM (No L-Met, 10% FBS) + 1μl RSL3 (1mM)

Among them, the combination group needs to add 1μl 1mM RSL3 after two days of deficient culture in DMEM (No L-Met, 10% FBS);

② After administration, the normal group, the ferroptosis induction group and the deficiency group were observed for three consecutive days, and the cells were collected after 72 hours and counted to count the difference in the number of cells; the combination group was co-cultured for 24 hours after adding 0.5 μM RSL3 Collect cell statistics.

(5) Experimental results

3 days after administration, the growth of the cells was significantly different. The state of the cells was photographed under a microscope at a magnification of 10×, and then the cells were collected after trypsinization and centrifugation, resuspended in 500 μL of PBS and counted using a cell counting plate. In the two glioma cell lines of Snb19 and U87, the number of cells in the combination group was significantly less than that of the other three groups, and there was a statistical difference, indicating that the combination of methionine deficiency and Erastin had a significant effect on the killing of glioma cells. Significant enhancement (Figure 1 ~ Figure 9).

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (10)

1. Application of a product containing a ferroptosis inducer and capable of causing tumor methionine deficiency in the preparation of a drug for treating tumors. 2. The application of the product containing a ferroptosis inducer according to claim 1 and which can cause tumor methionine deficiency in the preparation of a drug for treating tumors, characterized in that: The methionine deficiency includes but is not limited to dietary or medium methionine deficiency, using bacteria or viruses as vectors, carrying plasmids to express recombinant methionine enzymes, consuming methionine to cause tumor methionine Defects, or tumor methionine deficiencies caused by other means. 3. The application of the product containing a ferroptosis inducer according to claim 2 and which can cause tumor methionine deficiency in the preparation of a drug for treating tumors, characterized in that: The said diet or culture medium deficiency refers to the methionine deficiency caused by the essential nutrient complex that does not contain methionine. 4. The application of the product containing a ferroptosis inducer according to claim 1 and which can cause tumor methionine deficiency in the preparation of a drug for treating tumors, characterized in that: The ferroptosis inducers include but are not limited to class I and class II ferroptosis inducers. 5. The application of the product containing a ferroptosis inducer according to claim 4 and which can cause tumor methionine deficiency in the preparation of a drug for treating tumors, characterized in that: The class I inducers include but are not limited to sorafenib, sulfasalazine, small molecule compound Erastin; The class II ferroptosis inducer is but not limited to RSL3. 6. The application of the product containing a ferroptosis inducer according to claim 1 and which can cause tumor methionine deficiency in the preparation of a drug for treating tumors, characterized in that: Said tumor includes but not limited to glioma. 7. A tumor-suppressing nutrient complex, characterized in that the nutrient complex comprises a basal nutrient complex and a ferroptosis inducer, wherein the basal nutrient complex is a methionine-deficient basal nutrient complex. 8. The nutritional complex for suppressing tumors according to claim 7, characterized in that: The ferroptosis inducer is Erastin. 9. A medium for suppressing tumor cells, characterized in that the medium comprises a basal medium and a ferroptosis inducing agent, wherein the basal medium is a methionine-deficient basal medium. 10. the medium for suppressing tumor cells according to claim 9, characterized in that: The ferroptosis inducer is Erastin.

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