CN114807289A - Novel metabolic marker for preparing medicine for treating liver cancer and application thereof - Google Patents

Abstract

The invention discloses a novel metabolic marker for preparing a drug for treating liver cancer and its application. Through the construction of ALDH6A1 overexpressing liver cancer cell line, it was found that the level of methyl citrate in the cells was inversely proportional to the proliferation and migration rate of liver cancer cells. The AKT/NRAS liver cancer model was established by high pressure tail vein injection in Aldh6a1 knockout mice. It was found that the content of methyl citrate in serum was inversely proportional to the serum ALT and AST levels of mice, and was inversely proportional to the liver cancer burden in mice. The metabolite methyl citric acid of the present invention can not only effectively inhibit the proliferation of liver cancer cells alone, but also can enhance the inhibitory effect of sorafenib on the proliferation of liver cancer cells. The metabolites of the invention can effectively inhibit the formation of tumors, can be used as metabolic markers for liver cancer detection, and can more accurately and efficiently judge the severity of liver cancer; and can also be used as new metabolites for liver cancer treatment to improve the therapeutic effect of liver cancer.

Description

Novel metabolic markers for the preparation of drugs for the treatment of liver cancer and their applications

technical field

The present invention relates to the function and application fields of metabolites, in particular to a novel metabolic marker for preparing a drug for treating liver cancer and its application.

Background technique

During cancer development, cancer cells fuel continued growth and proliferation through metabolic reprogramming, which has become an emerging hallmark of cancer. Metabolic reprogramming includes the "Warburg effect" of aerobic glycolysis, glutamine catabolism, macromolecule synthesis, and redox homeostasis. In liver cancer metabolism, metabolic reprogramming mainly includes glucose metabolism, energy production metabolism and lipid metabolism reprogramming.

Methyl citrate is a citrate synthase that catalyzes the synthesis of propionyl-CoA and oxaloacetate. Methyl citrate may serve as a biomarker of inborn errors of propionic acid metabolism. Methylcitric acid induces brain ammonium accumulation and apoptosis, promoting brain damage caused by methylmalonic aciduria. In recent years, with the continuous in-depth research, metabolic reprogramming has also become one of the new markers of liver cancer. Methyl citrate may play an important role in the occurrence and regulation of liver cancer.

As a first-line drug for the treatment of advanced hepatocellular carcinoma, sorafenib provides a drug choice for improving the survival rate of patients with hepatocellular carcinoma, but sorafenib only prolongs the survival period of patients by 3 months, and its therapeutic effect is relatively limited. . There is heterogeneity in the therapeutic effect of sorafenib, and it is also accompanied by serious side effects including diarrhea, hypertension, and anorexia.

During the development of cancer, due to the existence of tumor heterogeneity, understanding the metabolic reprogramming of liver cancer, mining metabolites for early diagnosis and targeted therapy is very important to improve the cure rate and prognosis of cancer. With the advent of the era of precision medicine, in the clinical diagnosis and treatment of cancer, individualized and precise treatment of the disease and specific patients can maximize the efficacy. Multi-omics technology and large-scale sequencing provide strong support for this. However, the metabolic markers related to the precise diagnosis and treatment of cancer remain to be discovered and studied.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the purpose of the present invention is to provide a novel metabolic marker for preparing a drug for treating liver cancer and its application. The metabolic marker methyl citric acid for liver cancer detection in the present invention can predict the severity of liver cancer , to improve the efficiency and accuracy of early diagnosis of liver cancer. By detecting these metabolic markers in liver cancer tissue samples, individualized and precise treatment of liver cancer patients can be carried out according to the metabolite levels. The present invention finds through experimental research that the metabolite level of methyl citric acid in liver cancer is inversely proportional to the severity of liver cancer.

The invention provides a metabolite for preparing a liver cancer marker and its application, that is, the function and application of methyl citric acid in liver cancer detection and liver cancer treatment, that is, the application of a new drug in liver cancer treatment. Through experimental research, the present invention finds that methyl citric acid plays an important role in liver cancer, and it is found that it can significantly inhibit the proliferation rate of liver cancer cells, and can also inhibit the ability of liver cancer cells to form subcutaneous tumors.

For achieving the above object, the invention provides the following technical solutions:

In a first aspect, the present invention provides a novel metabolic marker for preparing a drug for treating liver cancer, characterized in that the metabolite is methyl citric acid.

In the second aspect, an application of a novel metabolic marker for preparing a drug for treating liver cancer is characterized in that: methyl citric acid is used as a metabolic marker for liver cancer and is used in the treatment of liver cancer.

As one of the preferred solutions, the methyl citric acid is used as a liver cancer metabolic marker, and its metabolite level is inversely proportional to the severity of liver cancer.

As the second preferred solution, the methyl citric acid can significantly inhibit the proliferation and subcutaneous tumor formation of liver cancer cells, and can significantly enhance the inhibitory effect of sorafenib on the proliferation of liver cancer cells.

The above-mentioned methyl citric acid plays an important role in the early diagnosis of liver cancer and the treatment of liver cancer.

The advantages and beneficial effects of the present invention are as follows:

The invention cultivates liver cancer cells in vitro, detects the proliferation and migration rates of liver cancer cells, and detects the level of intracellular metabolites, and finds that the content of methyl citric acid is inversely proportional to the proliferation and migration rates of liver cancer cells. In the present invention, a liver cancer model is constructed in mice by high-pressure tail vein injection or intraperitoneal injection, and the metabolite levels in the serum and liver cancer tissue of the mice are detected, and it is found that the content of methyl citric acid is inversely proportional to the serum ALT and AST levels of the mice. Liver cancer burden in mice was inversely proportional. The above results show that the content of methyl citric acid is negatively correlated with the severity of liver cancer, which can be used as a metabolic marker for the early diagnosis and severity of liver cancer.

In the present invention, by adding sodium methyl citrate to human liver cancer cells for culture, it is found that the proliferation of liver cancer cells can be significantly inhibited, and the effect of sorafenib on inhibiting the proliferation of liver cancer cells can be enhanced. In the present invention, HCCLM9 cells are injected subcutaneously into BABL/C nude mice to construct a tumor model, and the treatment is performed by intraperitoneal injection of sodium methyl citrate. It is found that sodium methyl citrate can significantly inhibit the formation rate of subcutaneous tumors. The above results indicate that methyl citric acid can effectively improve and treat liver cancer.

Description of drawings

In Figure 1:

A: The level of intracellular methyl citrate;

B: Statistics of cell proliferation rate;

C: Statistics of cell migration rate;

*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

In Figure 2:

A: Schematic representation of a representative liver.

B: Serum detection of methyl citrate level;

C: Statistics of the number of liver cancer nodules in mice;

D: Serum detection of ALT and AST levels;

*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

In Figure 3:

A: HCCLM9 cells were treated with sodium methyl citrate (0, 20, 100 μM), and the cell proliferation was detected by RTCA; sodium methyl citrate significantly inhibited the cell proliferation rate of HCCLM9.

B: MHCC97L cells were treated with sodium methyl citrate (0, 50, 100 μM), and the cell proliferation was detected by RTCA; sodium methyl citrate significantly inhibited the cell proliferation rate of MHCC97L.

C: Huh7 cells were treated with sodium methyl citrate (50 μM) and sorafenib (1 μM), and RTCA detected cell proliferation; sodium methyl citrate significantly inhibited the cell proliferation rate of Huh7, and the combination with sorafenib could significantly inhibit the proliferation of Huh7 cells. Enhanced inhibition of cell proliferation.

D: HCCLM9 cells were treated with sodium methylcitrate (10μM) and sorafenib (0.5μM), and RTCA detected cell proliferation; the combination of sodium methylcitrate and sorafenib could significantly enhance the inhibitory effect on cell proliferation .

In Figure 4:

A: subcutaneous tumor picture, in the blank group (Vehicle), the sodium methyl citrate group (MCA-Na) at the end of the experiment, the tumor size of the sodium methyl citrate group was significantly lower than that of the blank group.

B: Statistical graph of subcutaneous tumor volume. The volume of subcutaneous tumors in mice was measured at different time points, and the tumor volume in the sodium methyl citrate group was significantly lower than that in the blank group.

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1: Construction of liver cancer cell model in vitro

1. Hepatoma cell culture, including HCCLM9 and HEK293T human-derived hepatoma cells, were cultured in a 37°C, 5% CO ₂ incubator, and cell passages and operations were performed in a sterile intercellular biosafety cabinet. Cells were cultured in DMEM medium, 10%

Fetal bovine serum, 1% double antibody culture conditions.

2. Construction of ALDH6A1 overexpressed stable cell line:

(1) Lentiviral packaging: 293T cells were used, and the transfection plasmid ratio was pHAGE-ALDH6A1:pMD2G:pSPAX2=2:2:1. 48h after transfection, the medium supernatant was collected, filtered with a 0.45 μM filter, and stored in a -80°C refrigerator.

(2) Lentivirus infection of HCCLM9 cells: HCCLM9 cells were inoculated into a 6-well plate one day in advance, and 2 ml of virus solution and polybrene (final concentration 10 μg/ml) were added. Change to fresh complete medium after 10h.

(3) Resistance screening: After adding the corresponding resistance screening for 48h, the surviving cells were verified to express the established stable ALDH6A1 overexpression cell line.

3. Detection of intracellular metabolites: Take 10 ⁷ cells, wash three times with PBS, centrifuge at 3000 rpm for 1 min, and remove the supernatant. The levels of methyl citrate metabolites were detected by liquid chromatography-mass spectrometry (HPLC-MC).

4. RTCA to detect cell proliferation: xCELLigence Cell FunctionAnalyzer (DP System) is used to detect cell proliferation. Adherent cells were trypsinized and resuspended in complete DMEM medium at a density of approximately 4.0×10 ⁴ /mL. The E-Plate View 16 was then filled with 50 μL of complete DMEM medium to perform a baseline check (cell index should be less than 0.063). After that, 100 μL of cell suspension was added to each well, and then different drug treatments were added to each well, and real-time detection was started for about 96 hours.

5. RTCA to detect cell migration: xCELLigence Cell FunctionAnalyzer (DP System) is used to detect cell migration. Adherent cells were trypsinized and resuspended in serum-free DMEM medium at a density of about 4.0×10 ⁵ /mL. Add 165 μL of complete medium to the lower chamber of the CIM-Plate, mount the upper chamber on the lower chamber, and add 30 μL of serum-free DMEM medium to the upper chamber. The assembled CIM-Plate was equilibrated for 1 h in a 37°C, 5% _CO2 incubator to perform a baseline check (the cell index should be less than 0.063). After that, 100 μL of cell suspension was added to each well, and real-time detection was started for about 96 hours.

The present invention uses HCCLM9 human-derived liver cancer cells to construct ALDH6A1 overexpression to construct a liver cancer cell model. The results show that after ALDH6A1 overexpression, cell proliferation and migration rate are inhibited, and the level of intracellular methyl citrate increases. These results suggest that methyl citrate can be used as a metabolic marker of liver cancer severity.

Example 2: Construction of liver cancer model in mice by high pressure tail vein injection

1. Experimental animals and feeding: species, gender, age and source of experimental animals: C57BL/6 (WT) mice and C57BL/6 background Aldh6a1 knockout (Aldh6a1 ^-/- ) mice, male, 8 weeks old.

2. Animal feeding and environmental conditions: All experimental mice were kept in the SPF animal room of the School of Life Sciences, Wuhan University. Alternate lighting every 12 hours, temperature 24±2°C, humidity 40-70%, mice were allowed to drink and eat freely.

3. Sleep beauty high-pressure tail vein mouse liver cancer model construction:

Select WT male mice and Aldh6a1 ^-/- male mice, 8 weeks old. PT3-myr-AKT-HA, pCMV(CAT)T7-SB100 and pT/Caggs-NRASV12 were placed in normal saline and injected under high pressure into the tail vein of mice. AKT and NRAS entering the liver can be integrated into the mouse genome and stably expressed under the action of transposase, and finally induce liver cancer. The injection needs to be done within 5-7 seconds. After injection, mice's state and body weight changes were recorded weekly. Materials were collected 6-8 weeks after injection. Whole blood was placed at 4 degrees overnight, 2500 rpm, 4 degrees, centrifuged for 3 min, and the supernatant was serum, which was quick-frozen in liquid nitrogen. The mouse liver was taken out and photographed, and the mouse liver cancer and adjacent tissues were isolated and snap-frozen in liquid nitrogen.

4. Detection of serum metabolite levels: whole blood was placed at 4 degrees overnight, 2500 rpm, 4 degrees, centrifuged for 3 minutes, the supernatant was serum, and 50 μL of liquid nitrogen was quickly frozen. The levels of methyl citrate metabolites were detected by liquid chromatography-mass spectrometry (HPLC-MC).

5. ALT detection (NJJC, C009-2-1) Add 20 μL of pre-warmed alanine aminotransferase substrate solution at 37°C to each of the assay wells and control wells, add 5 μL of the sample to be tested in the assay wells, pipette and mix well, Place in a 37°C water bath for 30 minutes. Then, 20 μL of 2,4-dinitrophenylhydrazine liquid was added to each measurement well and control well, and 5 μL of sample was added to the control well. Mix by pipetting and place in a 37°C water bath for 20 minutes. Finally, 200 μL of 400 mM NaOH was added to each well. Shake gently, let stand for 15 minutes, and measure absorbance at 510 nm.

6. For the detection of AST (NJJC, C010-2-1), add 20 μL of aspartate aminotransferase substrate solution preheated at 37°C to each of the assay wells and the control wells, and add 5 μL of the sample to be tested for measurement. Mix with a pipette. Homogenize and place in a 37°C water bath for 30 minutes. Then, 20 μL of 2,4-dinitrophenylhydrazine liquid was added to each measurement well and control well, and 5 μL of sample was added to the control well. Mix by pipetting and place in a 37°C water bath for 20 minutes. Finally, 200 μL of 400 mM NaOH was added to each well. Shake gently, let stand for 15 minutes, and measure absorbance at 510 nm.

In the present invention, the AKT/NRAS liver cancer model was constructed in WT and Aldh6a1 ^-/- male mice with Sleep beauty high pressure tail vein, and the results found that after ALDH6A1 knockout, the liver cancer load was enhanced, the number of tumor nodules, blood ALT levels, blood AST levels increased, the level of methyl citrate in the serum of the mice was significantly reduced, which was inversely proportional to the liver cancer burden. These results suggest that methyl citrate can be used as a metabolic marker of liver cancer severity.

Example 3: Culture test of adding sodium methyl citrate and sorafenib to human hepatoma cells

1. Hepatoma cell culture, including HCCLM9, MHCC97L, Huh7 and other human-derived hepatoma cells, were cultured in a 37°C, 5% _CO2 incubator, and cell passages and operations were carried out in a sterile intercellular biosafety cabinet. Cells were cultured in DMEM medium, 10% fetal bovine serum, and 1% double antibody.

2. RTCA to detect cell proliferation: xCELLigence Cell FunctionAnalyzer (DP System) is used to detect cell proliferation. Adherent cells were trypsinized and resuspended in complete DMEM medium at a density of approximately 4.0×10 ⁴ /mL. The E-Plate View 16 was then filled with 50 μL of complete DMEM medium to perform a baseline check (cell index should be less than 0.063). After that, 100 μL of cell suspension was added to each well, and then different drug treatments were added to each well, and real-time detection was started for about 96 hours.

In the present invention, human liver cancer cells such as HCCLM9, MHCC97L, Huh7 are added to the cell culture by adding sodium methyl citrate (MCA-Na), sorafenib (Sorafenib), sodium methyl citrate and sorafenib to the cell culture In this study, a liver cancer cell model was constructed, and it was found that sodium methyl citrate could significantly inhibit the cell proliferation rate, and the combination with sorafenib could significantly enhance the inhibitory effect of sorafenib on the proliferation of liver cancer cells. The above results indicate that sodium methyl citrate can effectively improve and treat liver cancer.

Example 4: Construction of mouse subcutaneous tumor model by intraperitoneal injection of sodium methylcitrate

1. Subcutaneous tumor formation of BABLC-nude nude mice: 4-week-old BALB/c-nude nude mice were raised in SPF animal room. Nude mice were randomly divided into four groups, HCCLM9 cells in logarithmic growth phase were taken, and the cells were resuspended in PBS to 7×10 ⁷ /ml. Add 1:1 volume of Matrigel thawed at 4°C to the cell suspension and mix well. In an ultra-clean bench, 200ul of the cell suspension was inoculated subcutaneously in nude mice, and the tumor size at the inoculation site was measured regularly. Seven days after subcutaneous injection, saline-blank group (Vehicle) and sodium methyl citrate (MCA-Na) were injected intraperitoneally, once every other day. About 14 days later, when the tumor in the Vehicle group grew to about 1000 mm ³ , the nude mice were killed by neck dislocation, and the tumor mass was taken out and photographed.

In the present invention, HCCLM9 cells are subcutaneously injected into BABL/C nude mice, and a mouse model is constructed by intraperitoneal injection of sodium methyl citrate. It is found that sodium methyl citrate can significantly inhibit the formation of subcutaneous tumors. The above results indicate that sodium methyl citrate can effectively improve and treat liver cancer.

Claims (4)

1. A novel metabolic marker for preparing a medicament for treating liver cancer is characterized in that: the metabolite is methyl citric acid.

2. The application of a novel metabolic marker for preparing a medicine for treating liver cancer is characterized in that: the methyl citric acid is used as a liver cancer metabolic marker and is applied to liver cancer treatment.

3. Use of the novel metabolic markers for the preparation of a medicament for the treatment of liver cancer according to claim 2, characterized in that: the methyl citric acid is used as a liver cancer metabolic marker, and the metabolite level of the methyl citric acid is inversely proportional to the severity of liver cancer.

4. Use of the novel metabolic markers for the preparation of a medicament for the treatment of liver cancer according to claim 2, characterized in that: the methyl citric acid can obviously inhibit liver cancer cell proliferation and subcutaneous tumor formation, and can enhance the inhibition effect of sorafenib on liver cancer cell proliferation.

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