CN111568891B

CN111568891B - Application of dimethyl fumarate DMF in regulating tumor metabolism and inhibiting tumor growth

Info

Publication number: CN111568891B
Application number: CN202010000918.1A
Authority: CN
Inventors: 殷雷; 雷骏; 方嘉凌; 杨怡; 李永征
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2020-01-02
Filing date: 2020-01-02
Publication date: 2021-11-26
Anticipated expiration: 2040-01-02
Also published as: CN111568891A

Abstract

The invention belongs to the field of biotechnology and medicine, and particularly relates to application of dimethyl fumarate DMF in regulation of tumor metabolism and inhibition of tumor growth. The invention discovers the application of a small molecular inhibitor DMF in the aspect of regulating and controlling tumor metabolism so as to inhibit tumor growth for the first time; DMF can inhibit the proliferation of various tumor cells in vitro and reduce the metabolism of the tumor cells, and has certain inhibiting effect on the generation and development of tumors in vivo; by inhibiting tumor metabolism, the tumor acidic microenvironment is improved, the inhibition of T cell functions is reduced, the T cell reactivity is improved, the immunotherapy efficiency is enhanced, and a new target and a new thought are provided for tumor therapy.

Description

Application of dimethyl fumarate DMF in regulating tumor metabolism and inhibiting tumor growth

Technical Field

The invention belongs to the field of biotechnology and medicine, and particularly relates to application of dimethyl fumarate DMF in regulation of tumor metabolism and inhibition of tumor growth.

Background

Metabolism is a basic feature of life activities of the body and is the most important way for cells to obtain energy. In the process of tumor development, energy metabolism is often disordered, which changes the Tumor Microenvironment (TME) and helps tumor cells to survive, transfer and escape in immunity. Studies have demonstrated that in some tumors, glycolysis is the predominant mode of energy metabolism, as the tumor is in a hypoxic microenvironment. In the last 30 th century, doctor Otto Warburg discovered in O₂Under sufficient conditions, tumor cells still rely on aerobic glycolytic energy production. This phenomenon that the tumor cell utilizes glycolytic production energy without utilizing mitochondrial oxidative phosphorylation energy even in the presence of oxygen is calledIs the Warburg effect or is known as aerobic glycolysis. Many tumor cells produce lactate and ATP by enhancing the glycolytic pathway to consume glucose, studies have shown that: in the mouse kidney cancer model, tumor metabolic activity and activation are negatively correlated with infiltration of T cells; in melanoma and non-small cell lung cancer patients, tumor metabolic activity is enhanced, resulting in poor infiltration of T cells. Furthermore, tumor aerobic glycolysis stimulates the expression of granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF), recruits myeloid-derived suppressor cells (MDSCs) into TEM, and inhibits the ability of immune cells to respond to tumors.

TME is the cellular environment in which tumors exist, including peripheral blood vessels, extracellular matrix (ECM), other non-malignant cells, and

signaling molecules

3, 4. The preparation is characterized by low oxygen, low pH, low sugar and the like. The tumor microenvironment contains a large amount of growth factors, cell chemotactic factors and immunoinflammatory response molecules generated by various proteolytic enzymes, and the characteristics of the growth factors, the cell chemotactic factors and the immunoinflammatory response molecules are favorable for the proliferation, invasion, adhesion and angiogenesis of tumors and the resistance to radiotherapy and chemotherapy, and promote the survival, proliferation and metastasis of the tumors. The tumor microenvironment is closely related to the behaviors of tumor growth, metastasis, immune escape and the like, and the microenvironment characteristics of weak acid, hypoxia, immunosuppression and the like of the tumor are also an important reason for the tolerance of clinical antitumor treatment. Research shows that tumor aerobic glycolysis has certain influence on the treatment of tumors, such as: aerobic glycolysis of tumors causes therapeutic resistance to chemotherapy and radiotherapy, among other things. Thus, TME is considered to be one of the biggest obstacles to successful treatment of tumors. Effective treatment of tumors requires overcoming the obstacles of TME.

In addition to tumor cells, tumors also have a variety of non-cancerous cells, including fibroblasts, vascular endothelial cells, and immune cells, including T cells, macrophages, and neutrophils, which function differently. Initial T cell dependent oxidative phosphorylation maintains energy requirements; when naive T cells are stimulated with antigen, they proliferate and differentiate to form effector T cells. The rapid replication of T cells and the secretion of cytokines increase the biological energy and anabolismAnd (4) demand. To address these needs, activated T cells have increased glucose and amino acid uptake and utilization by enhancing pathways such as glycolysis, glutamine breakdown, and branched chain amino acid catabolism. Glucose is a key substrate for T cells and M1-type macrophages, and its role in tumor development relies on aerobic glycolysis to maintain activation and effector function. Nutrition and O in the tumor microenvironment₂The balance of (a) controls the function of the immune cells. Also, in the tumor microenvironment, tumor cells and immune cells are in a metabolic competitive relationship. Tumor cells and proliferation-activated T cells share similar metabolic pathways as well as key substrates. However, tumor cells rapidly consume glucose and amino acids, depriving immune cells of nutrients to maintain effector function. Poorly perfused tumor regions elicit hypoxic responses in tumor cells and T cells. Under hypoxic or other mechanisms, HIF-1 α activity is increased, promoting glycolysis, inhibiting increased concentrations of metabolites and leading to acidification of the local environment. Lactic acid is further secreted outside the tumor cells to trigger the acidic microenvironment of the tumor, further damaging the function of the T cells. The Warburg effect is a metabolic mode specific to tumors, and rapidly dividing tumors exhibit high levels of aerobic glycolytic capacity, rapidly depleting the surrounding environment of glucose. Thus, this abnormal metabolic pattern of tumor cells deprives T cells of nutrients, inhibits T cell immune metabolism, impairs T cell glycolytic metabolism, decreases T cell secreted cytokines, and ultimately leads to the conversion of effector T cells into null cells.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide the application of dimethyl fumarate DMF in the aspects of regulating tumor metabolism and inhibiting tumor growth.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

application of dimethyl fumarate DMF in preparation of medicines for regulating and controlling tumor metabolism is provided.

Application of dimethyl fumarate DMF in preparation of medicines for inhibiting tumor growth is provided.

Use of dimethyl fumarate DMF in the preparation of a medicament for increasing T cell reactivity.

Application of dimethyl fumarate DMF in preparation of medicines for enhancing tumor immunotherapy efficiency.

Application of dimethyl fumarate DMF in preparation of medicines for reducing side effects of IL-2 in treating tumors.

Application of dimethyl fumarate DMF and IL-2 pharmaceutical composition in preparation of drugs for inhibiting tumor growth.

The invention has the beneficial effects that: the invention discovers the application of a small molecular inhibitor DMF in regulating and controlling tumor metabolism so as to inhibit the growth of tumors for the first time; DMF can inhibit the proliferation of various tumor cells in vitro and reduce the metabolism of the tumor cells, and has certain inhibiting effect on the generation and development of tumors in vivo; by inhibiting tumor metabolism, the tumor acidic microenvironment is improved, the inhibition of T cell functions is reduced, the T cell reactivity is improved, the immunotherapy efficiency is enhanced, and a new target and a new thought are provided for tumor therapy.

Drawings

FIG. 1 shows the inhibition of the proliferation of various tumor cells by DMF in example 1.

FIG. 2 is a graph showing the use of DMF to regulate the metabolic processes of various tumor cells in example 2.

FIG. 3 is the inhibition of tumor growth in tumor-bearing mice by DMF as in example 3.

FIG. 4 is a graph of example 4 showing the use of bioinformatics to analyze GAPDH expression differences in tumor tissue specimens and negative correlation with patient prognosis.

FIG. 5 is a graph showing the improvement of proliferation and normal function of immune-related cells after improving tumor microenvironment using DMF in example 5.

FIG. 6 is a structural mechanism of inhibition of GAPDH enzyme activity by DMF in example 6, which is analyzed by structural biology techniques.

FIG. 7 is a graph showing that DMF can be used to reduce side effects of IL-2 treatment on tumors in example 7.

FIG. 8 is a graph of the inhibition of tumor growth in tumor-bearing mice using DMF in combination with IL-2 therapy in example 8.

Detailed Description

In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.

EXAMPLE 1 inhibition of the proliferation Process of various tumor cells Using DMF

MTT experiment is used for determining the inhibition effect of DMF with different concentrations on the proliferation of 4T1 breast cancer cells and CT26 colon cancer cells, and the MTT experiment comprises the following steps:

(1) taking cells in logarithmic growth phase, digesting and counting by using a blood counting chamber;

(2) add 1X 10 to 96-well plates⁴Cells, 100 μ Ι _ per well; adding DMF (dimethyl formamide) with different concentrations for treating for 24 hours or 48 hours after the wall adhesion is carried out for 2-4 hours;

(3) adding 10 mu L of CCK8 reagent into each hole, and detecting the absorbance of each hole at 450nm by using a microplate reader after 24 or 48 hours;

(4) a standard curve is drawn and the inhibition of tumor cell proliferation by DMF at different concentrations is calculated.

The inhibition of tumor cell proliferation by DMF obtained as described above is shown in FIG. 1. The results in FIG. 1 show that: i treated 4T1 breast cancer cells and CT26 colon cancer cells with DMF in vitro and found that the proliferation of tumor cells could be inhibited.

EXAMPLE 2 use of DMF to regulate the metabolic Processes of various tumor cells

The extracellular lactate release levels as well as intracellular ATP and ROS levels were measured by treating 4T1 breast cancer cells and CT26 colon cancer cells with DMF at various concentrations.

And (3) lactic acid release detection:

will be 5X 10⁵The tumor cells of (2) were seeded in each well of a 6-well plate and treated with DMF for 48 hours. Collecting the cell culture medium, and detecting the content of lactic acid by using a lactic acid detection kit.

And (3) detecting the ATP level in the cell:

will be 5X 10⁵The tumor cells of (2) were seeded in each well of a 6-well plate and treated with DMF for 48 hours. Collecting the cell culture medium, and detecting the ATP content by using an ATP detection kit.

And (3) detecting the intracellular ROS level:

(1) plating on the day before detection, planting cells with certain density in a 6-hole plate, and treating with DMF for 48 hours;

(2) discarding the old culture medium, and cleaning twice by using a serum-free culture medium;

(3) before loading the probe, diluting DCFH-DA with serum-free culture solution according to a ratio of 1:1000 to make the final concentration of the DCFH-DA 10 mu M;

(4) adding DCFH-DA working solution diluted by a proper volume, and incubating for 30min in a cell culture box at 37 ℃ in a dark place;

(5) washing the cells for 1-2 times by using a serum-free culture solution to fully remove DCFH-DA which does not enter the cells;

(6) digesting cells by pancreatin, stopping digestion by completely culturing the medium at 1,000rpm for 3min, removing the culture medium and pancreatin, and detecting by a flow-type computer;

the experimental results obtained according to the above method are shown in fig. 2, and we treated 4T1 breast cancer cells and CT26 colon cancer cells with DMF in vitro, analyzed the energy metabolism of the tumor cells, and examined lactate secretion, ATP production and reactive oxygen species levels. And (4) prompting by a result: after tumor cells were treated with DMF, extracellular lactate secretion decreased, i.e., glycolytic capacity decreased (fig. 2A), but ATP level increased, i.e., oxidative phosphorylation was recovered, and active oxygen level increased (fig. 2B).

Example 3 inhibition of tumor growth in tumor-bearing mice Using DMF

Establishing a corresponding mouse tumor model until the size of the tumor is about 100-200 mm³Mice were randomly divided into control and experimental groups after time.

A. Control group: intravenous PBS, daily for 7 weeks;

dmf targeted therapy group: carrying out intravenous injection on the DMF-loaded self-assembled nano-polypeptide, wherein the injection lasts for 7 weeks every day;

tumor volume was measured every two days, the formula was calculated: v1/2 × L × W²L is the longest diameter of the tumor, and W is the shortest diameter of the tumor. And detecting the weight change of the mice.

The experimental results obtained according to the above-described method are shown in FIG. 3. Establishing a mouse tumor model, and starting administration after the tumor grows to a certain size. And (4) prompting by a result: DMF has certain inhibitory effect on 4T1 breast cancer and CT26 colon cancer (figure 3A) and (figure 3C).

Example 4 analysis of GAPDH expression differences in tumor tissue specimens and negative correlation with patient prognosis using bioinformatics

We used the Kaplan-Meier Plotter, cancer genome project (TCGA), on-line database to perform intensive analysis of GAPDH transcripts in different tumor tissues and tissues adjacent to the cancer, and further analyzed the correlation of GAPDH transcripts with breast cancer patient prognosis by Kaplan-Meier log rank method.

The expression difference and negative correlation between prognosis obtained according to the above-described method are shown in FIG. 4. As shown in fig. 4A, GAPDH has differences in expression of transcripts in different types of tumors. The expression level of GAPDH in the tumor tissue of the patient is obviously higher than that in the paracancerous tissue. GAPDH has expression differences between the two in breast and colon cancer patient samples (fig. 4B). The survival of 1402 breast cancer patients was analyzed by Kaplan-Meier log rank survival analysis, the results are shown in FIG. 4C: GAPDH^LowAnd GAPDH^HighHas significant difference in post-operative survival (P ═ 0.027) of breast cancer patients. The above results suggest that the abnormally high expression of GAPDH may play a certain role in the development of breast cancer.

Example 5

The improvement effect of DMF with different concentrations on the tumor microenvironment is simulated by using the lactic acid concentrations with different concentrations in the environment. The simulated analysis of the proliferation capacity of the related immune cells and the secretion capacity of the related cytokines in the improved tumor microenvironment provides a new idea for combining DMF with tumor immunotherapy.

1. Effect of lactic acid on T cell proliferation

(1) Preparing 1640 culture medium with the concentration of 0mM, 10mM and 20mM lactic acid;

(2) isolation of primary culture

CD 8T cells;

(3) CFSE staining;

(4) the CD3 and CD28 antibodies stimulate T cells to proliferate for 72 h;

(5) and (5) detecting on a flow type computer.

2. Effect of lactic acid on cytokine secretion by T cells

(2) isolation of primary culture

CD 8T cells;

(3) the CD3 and CD28 antibodies stimulate T cells to proliferate for 72 h;

(4) under the condition of different concentrations of lactic acid, PMA and ionomycin are added to non-specifically activate T cells;

(5) and (5) detecting on a flow type computer.

3. Functional analysis of tumor infiltrating lymphocytes:

(1) establishing a mouse tumor model, wherein when the tumor size is about 100-200 mm³At the time, experimental animals were grouped. Control group: gavage vehicle (0.8% methylcellulose); drug treatment group: performing intragastric administration treatment with DMF 30 mg/kg;

(2) taking out tumor tissue under aseptic condition, sufficiently shearing, and placing in collagenase IV solution containing 0.5 mu g/mL;

(3) digesting for 1h at 37 ℃;

(4) separating immune cells from 40-70% of Percoll separation liquid;

(5) setting the acceleration and deceleration of a centrifuge to be 1, centrifuging for 30min at 25 ℃ and 800g, and then, obtaining an obvious white layer, namely a lymphocyte layer after centrifugation, and carefully absorbing lymphocytes in the lymphocyte layer;

(6) washing the isolated tumor infiltrating lymphocytes twice with PBS;

(7) cell surface staining and cytokine staining are carried out according to experimental conditions;

(8) resuspending the fixed cells with 0.5mL PBS, testing on the machine and analyzing IFN-. gamma.⁺CD8⁺T cells and TNF-alpha⁺CD8⁺Change in T cells.

The experimental results obtained by the above method are shown in fig. 5, and the lactic acid environments with different concentrations simulate the improvement effect of DMF with different concentrations on the tumor microenvironment. Lactate can inhibit the proliferation of CD 8T cells and the secretion of related cytokines (fig. 5(a), fig. 5 (b)). DMF may reduce the concentration of lactate in the tumor microenvironment, thereby improving proliferation and cytokine secretion of CD 8T cells. DMF can be used in combination with tumor immunotherapy to treat tumor. After the DMF is injected into the tumor-bearing mice, the tumor microenvironment in the mice can be effectively improved, and the tumor cell metabolism is inhibited, so that the effect function of tumor infiltrating lymphocytes is improved. The results show that: after reducing tumor metabolism, tumor-infiltrating T lymphocytes have a stronger ability to secrete cytokines (fig. 5 (c)).

Example 6

The structural mechanism of DMF for inhibiting GAPDH enzyme activity is analyzed by a structural biology technology:

the culture of protein crystals is usually carried out mainly by a Vapor Diffusion Method (Vapor Diffusion Method). The method mainly comprises two technologies: hanging drop vapor diffusion and sitting drop vapor diffusion. The subject selects the condition of primary screening protein crystal by sitting drop method (sitting drop), mixes the protein solution with equal volume and crystallization reagent, then closes in the high concentration solvent environment, utilizes the principle of gas phase equilibrium to make the volatile component reach equilibrium between small drop and large sample pool, the concentration of protein solution is relatively low, the water in the system is diffused into the high concentration solvent, the precipitant and protein concentration in the protein drop are gradually increased, crystal nucleus is produced in supersaturation state, finally crystal is separated out.

Preparation of protein samples

(1) Expressing and purifying GAPDH by using an escherichia coli expression system;

(2) concentrating the protein to 10mg/mL, centrifuging at 12,000rpm at 4 deg.C, and removing precipitate;

preliminary screening of crystallization conditions

(3) And (3) initially screening 1000 crystallization conditions by using a crystal screening robot, wherein the contents of protein and pool liquid in a crystallization hole are 0.1 mu L: 0.1 μ L, and observing the crystallization under a microscope;

optimization of crystallization conditions

(4) Because the volume of the mixed solution of the protein solution and the pool liquid required by the robot primary screening is small, the crystallization condition of the primary screening is repeated, and the content of the protein solution and the pool liquid is enlarged in an equal ratio. And (3) respectively carrying out two-dimensional gradient refinement on the temperature, the pH gradient, the salt ion concentration, the precipitant concentration and the like near the primary screening crystallization condition, and finally determining the optimal crystallization condition.

X-ray diffraction data collection and processing

(5) In this experiment, X-ray diffraction was performed on a Shanghai synchrotron radiation light source (SSRF) BL 17U. As for data processing, currently mainstream software is: HKL2000/3000, iMosflm, XDS. this study will use HKL2000/3000 and iMosflm processing data.

Structural analysis and correction

(6) This experiment will resolve the structure using a molecular replacement method. After the diffraction data is processed by using HKL2000/3000, the subsequent structure analysis can be directly carried out, and after the diffraction data is processed by using iMosflm, the CCP4i is required to carry out further data processing. And (3) introducing the processed data into a Phaser for molecular replacement, filling related parameters, operating software, manually building a structure model by using Coot on the basis, and correcting the structure by using Phemix.

We subject the obtained protein crystals to X-ray diffraction by using Shanghai synchrotron radiation light source (SSRF) BL 17U. As for data processing, currently mainstream software is: HKL2000/3000, iMosflm, XDS. We processed the data using HKL2000/3000 and iMosflm. We used the method of molecular replacement to resolve the structure. After diffraction data are processed by using HKL2000/3000, subsequent structure analysis can be directly carried out, and after diffraction data are processed by using iMosflm, CCP4i is required to carry out further data processing. And then, importing the processed data into Phaser for molecular replacement, filling related parameters, operating software, manually building a structure model by using Coot on the basis, and correcting the structure by using Phenix.

We resolved the crystal structure of DMF and holoenzyme, and identified the binding region of DMF and GAPDH as shown in fig. 6A, B. As can be seen by the human eye,the tetrameric enzyme protein GAPDH has three chains, each subunit is combined with DMF, and DMF is positioned in substrate NAD of GAPDH⁺Nearby. DMF occupies the position where the substrate and GAPDH bind.

We analyzed regions where DMF (red) and substrate G3P (green) bound GAPDH. The results are shown in FIG. 6C: DMF, G3P, almost completely agreed with the binding region of GAPDH, as did the residues that interact with GAPDH. Thus, we consider: DMF may occupy the binding region of G3P and GAPDH, which results in ineffective binding of G3P and GAPDH, thus reducing the enzyme activity of GAPDH, further reducing the aerobic glycolysis of tumor cells, resulting in insufficient reaction substrates and energy of synthetic biomacromolecules required by rapid division of the tumor cells, and finally inhibiting the growth of tumors.

Example 7

The side reaction generated in the process of treating the tumor by reducing IL-2 (human interleukin 2) by using DMF:

injection of IL-2 is an FDA approved therapy for metastatic melanoma and renal cancer, and is effective in activating T cells and promoting their secretion of relevant cytokines. However, IL-2 has serious toxic and side effects on treating tumors, such as pulmonary edema and the like, and DMF can reduce the side effects of treating the tumors to a certain extent.

Detection of IL-2 side effect remission:

1. normal mice were randomly divided into 4 groups: control group, DMF group, IL-2+ DMF group;

A. control group: intraperitoneal injection of PBS is carried out, and injection is carried out every day for six days; gavage 0.8% methylcellulose on

days

2, 4, and 6.

Group B.DMF: the DMF was gavaged on

days

2, 4 and 6 at 30mg/kg per mouse.

IL-2 group: intraperitoneal injection of IL-2, daily injection, for six days, 10 per mouse⁵U。

IL-2+ DMF set: intraperitoneal injection of IL-2, daily injection, six times, 10 per mouse⁵U;

day

2, 4, 6 gastric DMF 30mg/kg per mouse.

2. Mice were sacrificed on day 7, lung changes were observed and weighed;

3. drying the lung for 3 days at 65 ℃, re-weighing, and obtaining the difference value of the weight change of the lung before and after the lung is the weight of the pulmonary edema.

The results of the experiments performed as described above are shown in FIG. 7, where FIG. 7A shows the time of intragastric DMF injection and intraperitoneal injection of IL-2 in mice, and the mice were sacrificed on day 7 to observe pulmonary edema. We can see that high concentrations of IL-2 can cause severe pulmonary edema, while DMF can significantly alleviate its pulmonary edema condition (fig. 7B). The lung tumors before and after drying were weighed to find that DMF treated mice had reduced pulmonary edema (fig. 7C), effectively reducing the toxic side effects of IL-2 on the mice.

Example 8

DMF combined with IL-2 for inhibiting tumor growth in tumor-bearing mice

1. Establishing a corresponding mouse tumor model until the size of the tumor is about 100-200 mm³Mice were randomized into 4 groups after time: control, DMF, IL-2+ DMF;

A. control group: injecting PBS into the abdominal cavity on the 21 st to 26 th days after modeling, and injecting every day; gavage 0.8% methylcellulose every day on 7-20 days and 22,24 and 26 days;

group B.DMF: performing intragastric administration of DMF (dimethyl formamide) every day for 7-20 days and 22,24 and 26 days, wherein the concentration of DMF is 30mg/kg for each mouse;

IL-2 group: injecting IL-2 into abdominal cavity on 21-26 days after modeling, and injecting 10 doses of IL-2 into each mouse⁵U；

IL-2+ DMF set: injecting IL-2 into abdominal cavity on 21-26 days after modeling, and injecting 10 doses of IL-2 into each mouse⁵U; daily gavage of DMF at 30mg/kg per mouse on days 7-20 and 22,24, 26;

2. measuring the tumor volume every 2 days after successfully establishing a related tumor model, and calculating a formula: v1/2 × L × W²L is the longest diameter of the tumor, and W is the shortest diameter of the tumor.

The results of the experiments according to the above method are shown in FIG. 8, and FIG. 8A shows the time of intragastric DMF injection and intraperitoneal injection of IL-2 in mice, and the tumor size of the mice is recorded every 2 days from day 7. The results show that: DMF in combination with IL-2 treatment significantly inhibited tumor growth in mice compared to controls and had better efficacy compared to IL-2 treatment alone.

It is apparent that the above embodiments are only examples for clearly illustrating and do not limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are therefore intended to be included within the scope of the invention as claimed.

Claims

1. Use of dimethyl fumarate DMF in the preparation of a medicament for reducing the side effects of pulmonary edema caused by IL-2 drugs.

2. Application of dimethyl fumarate DMF and IL-2 medicaments in preparation of medicaments for inhibiting tumor growth and reducing side effect of pulmonary edema caused by IL-2 medicaments is disclosed.