New metabolic markers for preparing drugs for treating liver cancer and application thereof

Abstract

The application discloses a novel metabolic marker for preparing a medicine for treating liver cancer and application thereof. By constructing an ALDH6A1 overexpression liver cancer cell line, it is found through detection that the level of methylcitrate in the cell is inversely proportional to the proliferation and migration rate of liver cancer cells. In Aldh6a1 knockout mice, an AKT / NRAS liver cancer model is constructed by high-pressure tail vein injection, and it is found that the content of methylcitrate in the serum is inversely proportional to the levels of ALT and AST in the serum of the mice and inversely proportional to the liver cancer load of the mice. The metabolite methylcitrate 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 metabolite can effectively inhibit the formation of tumors, can be used as a metabolic marker for detecting liver cancer, and can more accurately and efficiently judge the severity of liver cancer; and can be used as a novel metabolite for treating liver cancer, and can improve the treatment effect of liver cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of function and application of metabolites, and particularly relates to a new metabolic marker for preparing a drug for treating liver cancer and application thereof. BACKGROUND

[0002] During the development of cancer, cancer cells provide fuel for continuous growth and proliferation through metabolic reprogramming, which has become a new marker of cancer. Metabolic reprogramming includes aerobic glycolysis "Warburg effect", glutamine catabolism, macromolecular synthesis and redox homeostasis. In liver cancer metabolism, metabolic reprogramming mainly includes glucose metabolism, energy metabolism and lipid metabolism reprogramming.

[0003] Methylcitrate is synthesized by citrate synthase from propionyl-CoA and oxaloacetate. Methylcitrate can be used as a biomarker for propionic acid metabolism innate error. Methylcitrate can induce brain ammonium accumulation and apoptosis, and promote brain damage caused by methylmalonic aciduria. In recent years, with the in-depth research, metabolic reprogramming has also become one of the new markers of liver cancer, and methylcitrate may play an important role in the occurrence and regulation of liver cancer.

[0004] As a first-line drug for treating advanced hepatocellular carcinoma, sorafenib provides a drug selection for improving the survival rate of hepatocellular carcinoma patients, but sorafenib only prolongs the survival period of patients for 3 months, and its therapeutic effect is limited. The therapeutic effect of sorafenib is heterogeneous, and is also accompanied by serious side effects including diarrhea, hypertension, anorexia, etc.

[0005] In the process of cancer occurrence, due to the existence of tumor heterogeneity, it is very important to understand the metabolic reprogramming of liver cancer, to excavate metabolites for early diagnosis and targeted treatment, and to improve the cure rate and prognosis of cancer. With the advent of the era of precision medicine, personalized precision treatment for diseases and specific patients can maximize the therapeutic effect in the clinical diagnosis and treatment of cancer. Multi-omics technology and large-scale sequencing provide strong support. However, the metabolic markers related to the precise diagnosis and treatment of cancer still need to be excavated and researched. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a new metabolic marker for preparing a drug for treating liver cancer and application thereof. The metabolic marker methylcitrate for liver cancer detection in the present application can predict the severity of liver cancer and improve the efficiency and accuracy of early diagnosis of liver cancer. By detecting these metabolic markers in liver cancer tissue samples, liver cancer patients can be treated individually and accurately according to the level of metabolites. The present application finds through experimental research that the metabolite level of methylcitrate in liver cancer is inversely proportional to the severity of liver cancer.

[0007] The application provides a metabolite for preparing a liver cancer marker and application thereof, namely, functions and application of methylcitrate in liver cancer detection and liver cancer treatment, namely, application as a novel drug in liver cancer treatment.

[0008] To achieve the above object, the application provides the following technical scheme.

[0009] In a first aspect, the application provides a novel metabolic marker for preparing a drug for treating liver cancer, characterized in that the metabolite is methylcitrate.

[0010] In a second aspect, application of a novel metabolic marker for preparing a drug for treating liver cancer, characterized in that methylcitrate is used as a liver cancer metabolic marker and is applied in liver cancer treatment.

[0011] As one of the preferred schemes, the methylcitrate used as a liver cancer metabolic marker has a metabolite level that is inversely proportional to the severity of liver cancer.

[0012] As the second preferred scheme, the methylcitrate can significantly inhibit liver cancer cell proliferation and subcutaneous tumor formation, and can significantly enhance the inhibitory effect of sorafenib on liver cancer cell proliferation.

[0013] The above methylcitrate plays an important role in early diagnosis of liver cancer and treatment of liver cancer.

[0014] The application has the following advantages and beneficial effects.

[0015] The application in vitro culture of liver cancer cells, detection of liver cancer cell proliferation, migration rate and detection of intracellular metabolite level, found that the content of methylcitrate is inversely proportional to the proliferation and migration rate of liver cancer. The application constructs a liver cancer model in mice by high-pressure tail vein injection or intraperitoneal injection, detects the metabolite level in the serum and liver cancer tissue of the mice, and finds that the content of methylcitrate is inversely proportional to the serum ALT and AST levels of the mice and inversely proportional to the liver cancer load of the mice. The above results show that the content of methylcitrate is negatively correlated with the severity of liver cancer, and can be used as a metabolic marker for early diagnosis and severity of liver cancer.

[0016] The application adds sodium methylcitrate to human liver cancer cells in culture and finds that it can significantly inhibit liver cancer cell proliferation and enhance the effect of sorafenib on inhibiting liver cancer cell proliferation. The application also injects HCCLM9 cells into the subcutaneous tumor model of BABL / C nude mice, and treats by intraperitoneal injection of sodium methylcitrate, and finds that sodium methylcitrate can significantly inhibit the formation speed of subcutaneous tumor. The above results show that methylcitrate can effectively improve and treat liver cancer. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 In:

[0018] A: The level of intracellular methylcitrate;

[0019] B: Cell proliferation rate statistics;

[0020] C: Cell migration rate statistics;

[0021] *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0022] Figure 2 In:

[0023] A: Representative liver schematic diagram.

[0024] B: Serum methylcitrate level detection;

[0025] C: Mouse liver cancer nodule number statistics;

[0026] D: Serum ALT, AST level detection;

[0027] *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0028] Figure 3 In:

[0029] A: HCCLM9 cells were treated with sodium methylcitrate (0, 20, 100 μΜ), and cell proliferation was detected by RTCA. Sodium methylcitrate significantly inhibited the cell proliferation rate of HCCLM9.

[0030] B: MHCC97L cells were treated with sodium methylcitrate (0, 50, 100 μΜ), and cell proliferation was detected by RTCA. Sodium methylcitrate significantly inhibited the cell proliferation rate of MHCC97L.

[0031] C: Huh7 cells were treated with sodium methylcitrate (50 μΜ) and sorafenib (1 μΜ), and cell proliferation was detected by RTCA. Sodium methylcitrate significantly inhibited the cell proliferation rate of Huh7, and the combination of sodium methylcitrate and sorafenib significantly enhanced the inhibition of cell proliferation.

[0032] D: HCCLM9 cells were treated with sodium methylcitrate (10 μΜ) and sorafenib (0.5 μΜ), and cell proliferation was detected by RTCA. The combination of sodium methylcitrate and sorafenib significantly enhanced the inhibition of cell proliferation.

[0033] Figure 4 In:

[0034] A: Subcutaneous tumor pictures, at the end of the experiment, the tumor size of the methyl citrate sodium group was significantly lower than that of the blank group.

[0035] B: Subcutaneous tumor volume statistics chart. The volume of subcutaneous tumors in mice was measured at different time points, and the tumor volume of the methyl citrate sodium group was significantly lower than that of the blank group. DETAILED DESCRIPTION

[0036] The application will be further described in detail below in conjunction with the examples and drawings, but the embodiments of the application are not limited thereto.

[0037] Example 1: In vitro construction of hepatocellular carcinoma cell model

[0038] 1. Hepatocellular carcinoma cell culture, including HCCLM9, HEK293T human hepatocellular carcinoma cells, cultured at 37°C in a 5% CO2 incubator, and cell passage and operation were carried out in a sterile intercellular biological safety cabinet. The cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double-antibiotic culture conditions.

[0039] fetal bovine serum, 1% double-antibiotic culture conditions.

[0040] 2. Construction of ALDH6A1 overexpression stable cell line:

[0041] (1) Lentivirus packaging: 293T cells were used, and the transfection plasmid ratio was pHAGE-ALDH6A1: pMD2G: pSPAX2 = 2:2:1. After 48h of transfection, the culture medium supernatant was collected, filtered with a 0.45μM filter, and stored in a-80℃ refrigerator.

[0042] (2) Lentivirus infection of HCCLM9 cells: HCCLM9 cells were inoculated in a 6-well plate one day in advance, and 2ml of virus liquid and polybrene (final concentration 10μg / ml) were added. 10h later, replace with fresh complete culture medium.

[0043] (3) Resistance screening: add corresponding resistance screening for 48h, and the surviving cells are verified for expression to obtain the constructed ALDH6A1 overexpression stable cell line.

[0044] 3. Intracellular metabolite detection: take 10 7 cells, wash with PBS three times, centrifuge at 3000rpm for 1min, and remove the supernatant. The level of methyl citrate metabolites was detected by liquid chromatography-mass spectrometry (HPLC-MC).

[0045] 4. RTCA detection of cell proliferation: xCELLigence Cell Function Analyzer (DP System) is used to detect cell proliferation. After trypsin digestion, adherent cells are resuspended in complete DMEM medium at a density of about 4.0 x 10 4 / mL. Then fill the E-Plate View 16 with 50 μL of complete DMEM medium to perform baseline check (cell index should be less than 0.063). Then add 100 μL of cell suspension to each well, and then add different drug treatments to each well, and start real-time detection for about 96 hours.

[0046] 5. RTCA detection of cell migration: xCELLigence Cell Function Analyzer (DP System) is used to detect cell migration. After trypsin digestion, adherent cells are resuspended in serum-free DMEM medium at a density of about 4.0 x 10 5 / mL. Add 165 μL of complete medium to the lower chamber of the CIM-Plate, install the upper chamber on the lower chamber, and add 30 μL of serum-free DMEM medium to the upper chamber. After the assembled CIM-Plate is equilibrated at 37°C, 5% CO2 incubator for 1 h, perform baseline check (cell index should be less than 0.063). Then add 100 μL of cell suspension to each well, and start real-time detection for about 96 hours.

[0047] The present application constructs an ALDH6A1 overexpression construct liver cancer cell model with HCCLM9 human liver cancer cells. The results show that after overexpression of ALDH6A1, cell proliferation and migration rate are inhibited, and the level of methylcitrate in the cell is increased. The above results show that methylcitrate can be used as a metabolic marker for the severity of liver cancer.

[0048] Example 2: Construction of liver cancer model in mice by high-pressure tail vein injection

[0049] 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.

[0050] 2. Animal feeding and environmental conditions: All experimental mice were fed in the SPF animal room of the School of Life Sciences, Wuhan University. The light was turned on every 12 hours, the temperature was 24 ± 2°C, the humidity was 40-70%, and the mice were free to drink water and eat.

[0051] 3. Sleep beauty high-pressure tail vein mouse liver cancer model construction:

[0052] 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 into the tail vein of the mice under high pressure. AKT and NRAS entering the liver can be integrated into the mouse genome under the action of transposase and stably expressed, ultimately inducing liver cancer. The injection needs to be completed within 5-7 seconds. After injection, the state and weight changes of the mice were recorded every week. The samples were taken 6-8 weeks after injection. Whole blood was placed at 4°C overnight, centrifuged at 2500 rpm for 3 min at 4°C, and the supernatant was serum, which was frozen in liquid nitrogen. The mouse liver was taken out and photographed, and the mouse liver cancer and paracancerous tissue were separated and frozen in liquid nitrogen.

[0053] 4. Serum metabolite level detection: whole blood was placed at 4°C overnight, centrifuged at 2500 rpm for 3 min at 4°C, and the supernatant was serum, 50 μL of which was frozen in liquid nitrogen. The level of methylcitrate metabolites was detected by high performance liquid chromatography-mass spectrometry (HPLC-MC).

[0054] 5. Detection of ALT (NJJC, C009-2-1): 20 μL of preheated alanine aminotransferase substrate solution at 37°C was added to each of the determination hole and the control hole, 5 μL of the sample to be tested was added to the determination hole, and the mixture was mixed by pipetting and placed in a 37°C water bath for 30 minutes. Then 20 μL of 2,4-dinitrophenylhydrazine liquid was added to each of the measurement hole and the control hole, and 5 μL of the sample was added to the control hole. The mixture was mixed by pipetting and placed in a 37°C water bath for 20 minutes. Finally, 200 μL of 400 mM NaOH was added to each hole. Gently shake, stand for 15 minutes, and measure the absorbance at 510 nm.

[0055] 6. Detection of AST (NJJC, C010-2-1): 20 μL of preheated aspartate aminotransferase substrate solution at 37°C was added to each of the determination hole and the control hole, 5 μL of the sample to be tested was added to the determination hole, and the mixture was mixed by pipetting and placed in a 37°C water bath for 30 minutes. Then 20 μL of 2,4-dinitrophenylhydrazine liquid was added to each of the measurement hole and the control hole, and 5 μL of the sample was added to the control hole. The mixture was mixed by pipetting and placed in a 37°C water bath for 20 minutes. Finally, 200 μL of 400 mM NaOH was added to each hole. Gently shake, stand for 15 minutes, and measure the absorbance at 510 nm.

[0056] The present application uses Sleep beauty high-pressure tail vein, and the transposase is injected into the tail vein of WT and Aldh6a1 - / -The results of constructing AKT / NRAS liver cancer model in male mice show that after ALDH6A1 knockout, the liver cancer load is enhanced, the number of tumor nodules, the blood ALT level, and the blood AST level rise, and the level of methylcitrate in the serum of the mice is significantly reduced, which is inversely proportional to the liver cancer load. The above results show that methylcitrate can be used as a metabolic marker of the severity of liver cancer.

[0057] Example 3: Sodium methylcitrate and sorafenib are added to human liver cancer cell culture test

[0058] 1. Liver cancer cells, including HCCLM9, MHCC97L, Huh7, and other human liver cancer cells, are cultured in a 37°C, 5% CO2 incubator, and cell passage and operation are carried out in a sterile intercell biological safety cabinet. The cells are cultured in DMEM medium, 10% fetal bovine serum, and 1% double-antibiotic culture conditions.

[0059] 2. RTCA detects cell proliferation: xCELLigence Cell Function Analyzer (DP System) is used to detect cell proliferation. After trypsin digestion, adherent cells are resuspended in complete DMEM medium at a density of about 4.0 x 10 4 / mL. Then 50 μL of complete DMEM medium is used to fill the E-Plate View 16 to perform baseline inspection (cell index should be less than 0.063). Then, 100 μL of cell suspension is added to each well, and different drug treatments are added to each well, and real-time detection is started for about 96 hours.

[0060] The present application adds sodium methylcitrate (MCA-Na), sorafenib, and a combination of sodium methylcitrate and sorafenib to the cell culture medium of HCCLM9, MHCC97L, Huh7, and other human liver cancer cells to construct a liver cancer cell model. The results show that sodium methylcitrate can significantly inhibit cell proliferation rate, and the combination of sodium methylcitrate and sorafenib can significantly enhance the inhibitory effect of sorafenib on liver cancer cell proliferation. The above results show that sodium methylcitrate can effectively improve and treat liver cancer.

[0061] Example 4: Construction of mouse subcutaneous tumor model by intraperitoneal injection of sodium methylcitrate

[0062] 1. Subcutaneous tumor formation in BABLC-nude mice: 4-week-old BALB / c-nude mice are raised in an SPF-level animal room. The nude mice are randomly divided into four groups, and log-phase HCCLM9 cells are resuspended in PBS to 7 x 10 7 / ml. The 4℃ thawed Matrigel was added to the cell suspension at a volume ratio of 1:1 and mixed well. The cell suspension was inoculated subcutaneously in nude mice in a super-clean bench, and the tumor size at the inoculation site was measured regularly. After 7 days of subcutaneous injection, the saline-vehicle group (Vehicle) and methyl sodium citrate (MCA-Na) were injected intraperitoneally, once every other day. After about 14 days, when the tumors in the Vehicle group grew to about 1000 mm 3 , the nude mice were sacrificed by cervical dislocation, and the tumor mass was removed and photographed.

[0063] The present application subcutaneously injects HCCLM9 cells into BABL / C nude mice to construct a mouse model by intraperitoneal injection of methyl sodium citrate. The results show that methyl sodium citrate can significantly inhibit the formation of subcutaneous tumors. The above results show that methyl sodium citrate can effectively improve and treat liver cancer.

Claims

1. Use of sodium methyl citrate for the preparation of a medicament, characterized in that: The medicine is used for treating liver cancer or enhancing the inhibitory effect of sorafenib on the proliferation of liver cancer cells.

2. Use according to claim 1, characterized in that: The medicine can significantly inhibit the proliferation of liver cancer cells and subcutaneous tumorigenesis.

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