Pharmaceutical composition for reducing drug resistance of liver cancer cells and application of pharmaceutical composition

Abstract

The invention belongs to the technical field of liver cancer prevention and treatment, and particularly relates to a pharmaceutical composition for reducing drug resistance of liver cancer cells and application of the pharmaceutical composition. The pharmaceutical composition comprises a component A and a component B, wherein the component A is 5-caffeoylquinic acid; and the component B is an aerobic glycolysis inhibitor, and preferably, the aerobic glycolysis inhibitor is Compound 3k. The pharmaceutical composition provided by the invention effectively solves the problem that liver cancer cells are easy to generate drug resistance to 5-caffeoylquinic acid and derivatives thereof, thereby being beneficial to the application of the pharmaceutical composition in the field of liver cancer treatment.

Description

[0001] This invention is a divisional application with application number 202111179728.1, application date October 11, 2021, and invention name “A pharmaceutical composition for reducing the drug resistance of liver cancer cells and its application”. Technical Field

[0002] The invention belongs to the technical field of liver cancer prevention and treatment, and specifically relates to a pharmaceutical composition for reducing the drug resistance of liver cancer cells and an application thereof in preparing a drug for enhancing the sensitivity of liver cancer cells to 5-caffeoylquinic acid and its derivatives and delaying the drug resistance thereof. Background Art

[0003] Hepatocellular carcinoma (LIHC) is the most common primary liver cancer, and its incidence is still increasing, especially in East Asia. Currently, the mortality rate of hepatocellular carcinoma ranks third among malignant tumors worldwide. Hepatocellular carcinoma may be more likely to occur in patients with underlying liver disease. For example, infection with hepatitis B or C virus (HBV or HCV) and long-term alcohol or aflatoxin poisoning are major risk factors for hepatocellular carcinoma. Although treatment with sorafenib and regorafenib can moderately benefit the survival of patients with liver cancer, its overall therapeutic effect is limited, and the 5-year survival rate of patients with liver cancer is still only 18%. Therefore, it is urgent to screen new anti-liver cancer compounds with clear drug targets and mechanisms, high efficiency and low toxicity.

[0004] 5-Caffeoylquinic acid (Chinese system nomenclature is 3-Caffeoylquinic acid, 3-CQA; international system nomenclature is 5-Caffeoylquinic acid), also known as chlorogenic acid (Chlorogenic acid, CGA), is a carboxyphenolic acid composed of caffeic acid (CA) and quinic acid (QA). It has multiple biological activities such as anti-inflammatory, antioxidant, antiviral, hypoglycemic and lipid-lowering, and anti-tumor. It is also one of the most abundant polyphenolic compounds in people's diet. In addition, there are more than ten 5-CQA derivatives in nature, such as methyl chlorogenate (M5-CQA), dicaffeoylquinic acid (diCQAs) and tricaffeoylquinic acid (triCQAs).

[0005] The study by Yan et al. [Yan Y, Liu N, Hou N, Dong L, Li J. Chlorogenic acid inhibits hepatocellular carcinoma in vitro and in vivo. J Nutr Biochem. 2017; 46: 68-73] showed that after incubation with 5-CQA for 24 hours, 5-CQA above 500 μM induced a significant cell cycle arrest in the S phase of HepG2 liver cancer cells, and 5-CQA may inhibit the proliferation and metastasis of liver cancer cells by inhibiting the activity of the MAPK / ERK signaling pathway and the expression of matrix metalloproteinases MMP-2 and MMP-9. Liu et al. [Liu Y, Feng Y, Li Y, Hu Y, Zhang Q, Huang Y, Shi K, Ran C, Hou J, Zhou G, Wang X. Chlorogenic Acid Decreases Malignant Characteristics of Hepatocellular Carcinoma Cells by Inhibiting DNMT1 Expression. Front Pharmacol. 2020; 11: 867] showed that after incubation with 5-CQA for 96 hours, 5-CQA above 250 μM would inhibit the proliferation of liver cancer cells HepG2 in a dose-dependent manner, and 1000 μM 5-CQA would cause HepG2 cell cycle to be significantly arrested in the S phase. Further in vitro and in vivo experimental studies have shown that 5-CQA may significantly inhibit the proliferation of HepG2 cells by downregulating DNA methyltransferase 1 (DNMT1) protein expression, significantly upregulating p53 and p21 protein expression, and inhibiting ERK1 / 2 activity. The study by Jiang et al. [Jiang Y, Nan H, Shi N, Hao W, Dong J, Chen H. Chlorogenic acid inhibits proliferation in human hepatoma cells by suppressing noncanonical NF-κB signaling pathway and triggering mitochondrial apoptosis. Mol Biol Rep. 2021] showed that after incubation with 300 μM 5-CQA for 48 hours, 5-CQA significantly inhibited the proliferation of liver cancer cells Hep-G2 and Huh-7, but had no significant inhibitory effect on the activity and proliferation of hepatocytes QSG-7701.Further RNA-seq, WB and flow cytometry analysis showed that 5-CQA may inhibit the proliferation of liver cancer cells by significantly inhibiting the non-classical NF-κB signaling pathway, and activate the mitochondrial apoptosis pathway by upregulating the expression of BBC3 (BH3-only protein Bcl-2 binding component 3), a member of the BH3-only subfamily of the Bcl-2 family.

[0006] In the 1860s, Pasteur proposed that cells would undergo a metabolic shift in an oxygen-deficient environment, from oxidative phosphorylation to glycolysis (Pasteur effect). The Pasteur effect profoundly affects the survival and function of various cells and tissues. This mechanism is believed to optimize the efficiency of energy (ATP) generation while preventing excessive production of ROS. In the 1950s, Warburg proposed that cancer cells obtain energy mainly through the "aerobic glycolysis" pathway (Warburg effect). He believed that this metabolic shift was the cause of cell carcinogenesis. According to literature reports, increasing the level of ROS in tumor cells may promote glucose metabolism (Warburg effect). In addition, tumor cells improve their adaptation to hypoxia and mitochondrial respiratory chain damage by activating glucose metabolism, which is a key resistance mechanism that leads to their insensitivity to chemotherapy [Xu RH, Pelicano H, Zhou Y, Carew JS, Feng L, Bhalla KN, Keating MJ, Huang P. Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia. Cancer Res. 2005; 65: 613-21].

[0007] In summary, although 5-CQA and its derivatives have good anti-liver cancer activity, liver cancer cells will develop drug resistance to them during the anti-liver cancer process, affecting the anti-tumor effect. Summary of the invention

[0008] In order to solve the technical problem that liver cancer cells develop drug resistance to 5-CQA and its derivatives during the anti-liver cancer process, the present invention provides a pharmaceutical composition for reducing the drug resistance of liver cancer cells, thereby reducing the drug resistance of liver cancer cells to 5-CQA and its derivatives, thereby facilitating their clinical application.

[0009] The technical solution of the present invention is as follows:

[0010] A pharmaceutical composition for reducing drug resistance of liver cancer cells, comprising component A and component B:

[0011] Wherein component A is at least one of 5-caffeoylquinic acid, caffeoylquinic acid methyl ester and 3,5-dicaffeoylquinic acid;

[0012] Component B is at least one of a glucose analog, a hexokinase II inhibitor, and an aerobic glycolysis inhibitor.

[0013] Preferably, the glucose analogue is 2-deoxy-D-glucose.

[0014] Preferably, the molar concentration of the 2-deoxy-D-glucose is 1-2 mM.

[0015] Preferably, the hexokinase II inhibitor is 3-bromopyruvate.

[0016] Preferably, the molar concentration of the 3-bromopyruvic acid is 10 μM.

[0017] Preferably, the aerobic glycolysis inhibitor is a PKM2 kinase inhibitor.

[0018] Preferably, the PKM2 kinase inhibitor is Compound 3k.

[0019] Preferably, the molar concentration of Compound 3k is 40-100 nM.

[0020] Preferably, the molar concentration of 5-caffeoylquinic acid, caffeoylquinic acid methyl ester and 3,5-dicaffeoylquinic acid is greater than 200 and less than 400 μM.

[0021] The invention discloses an application of the pharmaceutical composition in preparing a drug for enhancing the sensitivity of liver cancer cells to caffeoylquinic acid and its derivatives and delaying their drug resistance.

[0022] The present invention has the following innovative features and technical advantages:

[0023] 1. It is the first report that 5-CQA and its derivatives significantly induce abnormal activation of the ROS-GLUT1 / 3 signaling axis, which is the key molecular mechanism that causes liver cancer cells to develop resistance to 5-CQA and its derivatives. Therefore, the present invention targets 5-CQA and its derivatives that significantly induce high expression of glycokinase II and M2-type pyruvate kinase (PKM2) that is specifically highly expressed in liver cancer tissues, and uses 3-bromopyruvic acid (Bromopyruvic acid) that is specific for glycokinase II and Compound 3k small molecule inhibitors of PKM2 kinase to form a pharmaceutical composition that can synergistically inhibit the proliferation of liver cancer cells, thereby effectively reducing the drug resistance of liver cancer cells.

[0024] 2. For the first time, relatively high-sugar culture conditions were disclosed. Sugar-free or low-sugar culture can significantly increase the inhibitory effect of the drug combination on liver cancer cell proliferation by 1.5-3 times, thus facilitating its application in the treatment of liver cancer.

[0025] Technical effects of the present invention:

[0026] 1. The pharmaceutical composition of the present invention can increase the sensitivity of liver cancer cells to 5-CQA by 1.2-1.8 times (p < 0.05), reduce drug resistance, and increase the therapeutic effect against liver cancer;

[0027] 2. Compared with 5-CQA, low-sugar culture can increase the sensitivity of liver cancer cells to MCGA and 3,5-diCQA by about 1.5 times (p < 0.05), thereby reducing drug resistance and enhancing the therapeutic effect of 5-CQA derivatives against liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is an in vitro experiment to evaluate the effect of 5-CQA on the proliferation of human hepatoma cells.

[0029] Figure 2 5-CQA stimulates glucose uptake in liver cancer cells by significantly inducing the expression of glucose transporters GLUT1 / 3 and hexokinase HKDC1.

[0030] Figure 3 Reactive oxygen species (ROS) are the upstream signaling molecules that mediate 5-CQA-stimulated glucose uptake in liver cancer cells.

[0031] Figure 4 Sugar-free culture and inhibition of aerobic glycolysis pathway can synergize with 5-CQA to inhibit the proliferation of liver cancer cells.

[0032] Figure 5 To evaluate the effects of methyl caffeoylquinate (MCGA) and 3,5-dicaffeoylquinic acid (3,5-diCQA) on the proliferation of human hepatoma cells in vitro.

[0033] Figure 6 Abnormal activation of the ROS-GLUT1 / 3 signaling pathway is the key molecular mechanism that induces liver cancer cells to develop resistance to MCGA and 3,5-diCQA.

[0034] Figure 7 Low-glucose culture and inhibition of aerobic glycolysis pathway can significantly enhance the sensitivity of liver cancer cells to MCGA and 3,5-diCQA.

[0035] Figure 8 This is the molecular structure of 5-caffeoylquinic acid.

[0036] Fig. 9 This is the molecular structure of methyl caffeoylquinate.

[0037] Fig.10 This is the molecular structure diagram of 3,5-dicaffeoylquinic acid. DETAILED DESCRIPTION

[0038] The present invention will be further described below by way of examples.

[0039] In the present invention, 5-caffeoylquinic acid, caffeoylquinic acid methyl ester and 3,5-dicaffeoylquinic acid are all purchased from Chengdu Aifa Biotechnology Co., Ltd.

[0040] The molecular formula of 5-caffeoylquinic acid is C 16 H 18 O9, CAS number is 327-97-9, structural formula is Figure 8 As shown; chlorogenic acid methyl ester, molecular formula is C 17 H 20 O9, CAS number is 123483-19-2, molecular structure is Fig. 9 As shown; 3,5-dicaffeoylquinic acid compound molecular formula C 25 H 24 O 12 , CAS number is 2450-53-5, molecular structure is Fig.10 shown.

[0041] Example 1. Glucose analogue 2-DG (1 mM), hexokinase II inhibitor Bromopyruvic acid (10 μM) or inhibitor of PKM2 kinase in aerobic glycolysis pathway Compound 3k (100 nM) and 5-caffeoylquinic acid synergistically inhibit the proliferation of liver cancer cells

[0042] 1. To investigate the half inhibitory concentration of 5-caffeoylquinic acid in inhibiting the proliferation of liver cancer cells

[0043] 1.1 Experimental method: The liver cancer cell lines MHCC97H and HCCLM3 were counted by flow cytometry and then seeded in 12-well plates at a density of 2×10 4 cells per well. After 24 hours of cell plating, the cells were uniformly replaced with DMEM (5% FBS) medium without or with gradient concentrations of 5-CQA (three replicate wells per group) and incubated in a cell culture incubator at 37°C and 5% CO2 for 96 hours. Finally, each group of cells was digested with trypsin, resuspended in an equal volume of culture medium, and viable cells were counted using a flow cytometer.

[0044] 1.2 Experimental results: Flow cytometry was used to count the number of live cells in the sample after incubation with gradient concentrations of 5-CQA for 4 days, and its effect on the proliferation of liver cancer cell lines HCCLM3 and MHCC97H was analyzed. Based on the above experimental results, the dose-response curve of 5-CQA acting on liver cancer cells was drawn and the half-inhibitory concentration (IC50 value) of 5-CQA acting on different liver cancer cell lines was calculated. The values ​​in the figure are mean±SD values ​​(n=3), *p<0.05, ***p<0.001.

[0045] like Figure 1 As shown, the cells were incubated with 5-CQA for 96 h, and the IC50 values ​​were determined by counting the number of viable cells using flow cytometry. MHCC97H and IC50 HCCLM3 The values ​​were 546.8 μM and 452 μM, respectively. Therefore, the present invention used four concentration gradients of 5-CQA at Vehicle, 150, 300 and 600 μM for subsequent research.

[0046] 2.5-Caffeoylquinic acid significantly induces glucose uptake in liver cancer cells

[0047] 2.1 Extraction of total cell RNA

[0048] 1) Aspirate the culture medium, wash the cells twice with PBS, add 1 ml of Trizol to the culture dish, blow the cells after the cells are detached from the wall, collect them in a 1.5 ml enzyme-free EP tube, let it stand at 4°C for 5 min, then add 200 μl of chloroform, shake thoroughly on a vortex shaker for 15 s, and let it stand at 4°C for 5 min.

[0049] 2) After separation, centrifuge at 4°C and 12,000 g for 10 min. Carefully pipette the colorless portion of the upper layer into a new enzyme-free EP tube, add an equal volume of isopropanol, gently invert and mix 10 times, let stand at room temperature for 10 min, and centrifuge at 12,000 g for 10 min.

[0050] 3) The white precipitate at the bottom is RNA. Discard the supernatant and try to absorb the remaining solution. Then add 1ml of 75% ethanol (prepared with DEPC water), vortex and wash, centrifuge at 7500g for 5min, discard the supernatant, and repeat the operation. Discard the supernatant, centrifuge at 7500g, and use the enzyme removal pipette to absorb the remaining solution, and let it stand at room temperature for 5-10min. When the white precipitate at the bottom of the tube begins to become transparent, add 20μl of DEPC water to dissolve it, vortex and mix it, and use a nucleic acid detector to determine the concentration. Then adjust the concentration of each sample to 500ng / μl, re-measure the concentration and use it for reverse transcription.

[0051] 2.2 RNA reverse transcription

[0052] Reverse transcription was performed using the reverse transcription kit from Nanjing Novozymes.

[0053] 1) Removal of possible residual genomes

[0054]

[0055] Prepare the reaction system according to the above table and incubate at 42°C for 2 min.

[0056] 2) Reverse transcription

[0057]

[0058] PCR program: 50°C, 5 min, 85°C, 5 s.

[0059] 2.3 Fluorescence quantitative PCR

[0060] 1) Fluorescence quantitative PCR primers were designed as follows:

[0061]

[0062] 2) Fluorescence quantitative PCR system

[0063]

[0064] The template amount can be adjusted later based on the experimental results.

[0065] 3) Perform qPCR reaction according to the following conditions

[0066]

[0067] In addition, it is necessary to determine whether the reaction is specific by performing a melting curve and performing nucleic acid electrophoresis on the qPCR product.

[0068] 2.4 Western blot experimental steps:

[0069] (1) Sample treatment: The protein samples whose concentrations were determined by the BCA method were diluted with 5× SDS-PAGE Loading buffer to a uniform protein concentration of 2 μg total protein / μl, mixed, heated at 95°C for 5 min, and centrifuged at 15,000 g for 5 min.

[0070] (2) Sample loading volume: 20-60 μg total protein / lane. For the remaining lanes, add an equal volume of 1× SDS-PAGE Loading buffer. The loading volume of Rainbow Mark is 5 μl / lane.

[0071] (3) Protein electrophoresis: Electrophoresis was performed using Nanjing GenScript ExpressPlus PAGE Gel gradient precast gel and buffer at 120 V constant voltage for approximately 60 min.

[0072] (4) Transfer

[0073] 1) Preparation of transfer buffer: Dissolve 1 pack of GenScript transfer buffer powder in 900 ml of deionized water, then add 100 ml of methanol and mix thoroughly.

[0074] 2) PVDF membrane and filter paper treatment: First cut the PVDF membrane and six filter papers of the same size as the glue. Then immerse the PVDF membrane in methanol for 2-3 minutes, then soak it in deionized water for 2 minutes, and then put the membrane in 1× transfer buffer to balance for 5 minutes; the filter paper can be directly placed in 1× transfer buffer to balance.

[0075] 3) Transfer clip: black corresponds to the negative pole, white corresponds to the positive pole

[0076] From black to white, the sequence is: sponge → 3 layers of filter paper → gel → PVDF membrane → 3 layers of filter paper → sponge (slowly place from one corner of the transfer clip, pay attention to remove bubbles), close the clip (all done below the liquid surface, pay attention to remove bubbles), quickly put it into the rack of the electrophoresis tank containing 1L transfer buffer, and put an ice box in the electrophoresis tank. When transferring the membrane, the outside of the electrophoresis tank should be embedded with ice cubes.

[0077] 4) Transfer current and time: 300mA constant current. The transfer time is generally calculated based on the molecular weight of the target protein, 1kDa / min.

[0078] Note: After the transfer is completed, be sure to distinguish the front and back sides of the membrane. The side of the PVDF membrane close to the black negative electrode is the front side (the side where the protein is imprinted). It is best to mark the front side of the membrane with a ballpoint pen and make the rainbow mark darker to prevent the color from fading.

[0079] 5) Electroporation effect detection: Place the PVDF membrane in the reaction box (with the protein side facing up), add Ponceau red, and stain for 1-3 minutes. At this time, if there is protein on the membrane, you can see the band (it doesn’t matter if there is no band). Then cut out the target band according to the position of the rainbow mark and the band stained with Ponceau red.

[0080] (5) Closed

[0081] Preparation of 10×TBS: NaCl 80g / L, KCl 2g / L, Tris-base 30g / L, HCl to adjust pH to 7.4, make up to 1L, and store at 4℃.

[0082] Preparation of 1×TBST: 30ml 10×TBS, 1.5ml Tween-20, dilute to 300ml and store at room temperature.

[0083] Western Blot blocking solution: Weigh 2.5 g skim milk powder and dissolve it in 50 ml 1× TBST solution.

[0084] The target band cut out after Ponceau staining was taken out, washed with TBST until the Ponceau color faded, and then blocked with Western Blot blocking solution at room temperature for 1.5 hours with gentle shaking.

[0085] Take out the PVDF membrane blocked with Western Blot blocking solution, wash the PVDF membrane with distilled water (2 times, 5 min each time), and shake it slowly on a shaker at room temperature to remove excess blocking solution;

[0086] (6) Incubation with primary antibody

[0087] 1) Dilute the primary antibody with TBST or Beyotime Western Blot Primary Antibody Diluent. For details on the dilution ratio, please refer to the antibody manual.

[0088] 2) Add the PVDF membrane washed with distilled water and incubate overnight on a shaker at 4°C. Note that in order for the primary antibody to fully bind to the target protein, the volume of primary antibody used must completely cover the membrane.

[0089] 3) Recover the primary antibody (can be reused), then wash the PVDF membrane with TBST buffer by shaking for a total of 3 times, each wash for about 10 minutes.

[0090] (7) Incubation with secondary antibody

[0091] 1) Dilute the HRP-conjugated secondary antibody with TBST diluent and incubate it with the PVDF membrane bound to the primary antibody at room temperature for 1 hour.

[0092] 2) Remove the secondary antibody and wash the membrane three times with TBST, each time for about 10 minutes.

[0093] (8) Exposure and Development

[0094] Gently absorb the residual liquid on the PVDF membrane strip with absorbent paper, place the membrane face up on the plastic film, evenly cover the prepared ECL Luminol / Enhancer solution (A solution: B solution = 1:1) on the PVDF membrane, protect from light, incubate at room temperature for 1 minute, absorb the ECL working solution, and fix the plastic film in an X-ray photography dark folder; in the dark room, press the strip with X-ray exposure film, and wash the film with an automatic film developer. Finally, mark the position of the corresponding standard protein marker, the name of the target protein, the sample name, the date and other information on the washed X-ray film.

[0095] 2.5 Glucose uptake experiment

[0096] The culture medium of liver cancer cells incubated with or without gradient concentrations of 5-CQA for 96 h was sucked out by a vacuum pump, and then DMEM (5% FBS) sugar-free culture medium containing 100 μM 2-NBDG was added and incubated in a 37°C incubator for 30 min. The cells were then digested with trypsin and washed 3 times with PBS. Finally, the green fluorescence intensity emitted by intracellular 2-NBDG was detected using a flow cytometer.

[0097] 2.6 Experimental results: Figure 2 AB) Real-time fluorescence quantitative PCR detection of the response effect of glucose transporters GLUT1 (A) and GLUT3 (B) in liver cancer cells to concentration gradient 5-CQA at the gene level; ( Figure 2 C) Real-time fluorescence quantitative PCR analysis of the effect of 5-CQA on the expression level of HKDC1 gene; ( Figure 2 D) Western blotting was used to verify the effect of gradient concentrations of 5-CQA on the protein expression levels of glucose transporters GLUT1, GLUT2 and GLUT3; ( Figure 2 E) Flow cytometry quantitative analysis of the uptake capacity of glucose analogs (2-NBDG fluorescent probe) by liver cancer cells after incubation with gradient concentrations of 5-CQA for 96 h.

[0098] like Figure 2 As shown in Figure 2, qRT-PCR assay showed that 5-CQA significantly upregulated the mRNA expression of glucose transporters Glut1 / 3 and Hkdc1 (p < 0.05) in a concentration-dependent manner ( Figure 2 AC). WB detection also showed that 5-CQA significantly upregulated the expression of GLUT1 and GLUT3 proteins in a concentration-dependent manner ( Figure 2 D). Further studies have found that 5-CQA significantly promotes sugar absorption in liver cancer cells (p < 0.001), and there is also a concentration-dependent effect ( Figure 2E). Therefore, the above research results suggest that 5-CQA promoting sugar absorption is the key molecular mechanism leading to drug resistance in liver cancer cells.

[0099] 3. The significant accumulation of ROS is the upstream signaling molecule that mediates 5-caffeoylquinic acid-induced glucose uptake in liver cancer cells

[0100] 3.1 Detection of total intracellular reactive oxygen species (ROS)

[0101] 1) Prepare Carboxy-H2DCFDA fluorescent probe stock solution with DMSO to a final concentration of 20mM (25mg Carboxy-H2DCFDA dissolved in 2.353ml DMSO) and mix well. Dilute the stock solution with PBS to a final concentration of 50μM working solution before use. Keep the operation away from light;

[0102] 2) The liver cancer cells incubated with or without gradient concentrations of 5-CQA (three replicates for each concentration) for 96 h were digested with trypsin, and the cells were collected after centrifugation at 4°C, 1000 rpm for 4 min, the supernatant was discarded, and the cell concentration was uniformly adjusted to 5×10 5 cells / tube, centrifuged at 1000 rpm for 4 min, and discarded the supernatant;

[0103] 3) Add 1 ml of Carboxy-H2DCFDA working solution to each tube of cell pellet, wrap it with tin foil, and incubate it in a 37°C, 5% CO2 incubator for 20 min. Mix it several times every 5 min to allow the probe to fully enter the cells. Cells not incubated with the probe serve as negative controls.

[0104] 4) Centrifuge at 4°C, 1000 rpm for 4 min, remove the supernatant, and then wash the cells three times with PBS to remove the Carboxy-H2DCFDA fluorescent probe that has not entered the cells;

[0105] 5) Finally, add 0.5 ml of ice-cold PBS to resuspend the cells and use flow cytometry to detect the average green fluorescence intensity of 20,000 cells. Keep the operation away from light throughout the process.

[0106] 3.2 Detection of reactive oxygen species (ROS) in mitochondria

[0107] 1) Dissolve 50 μg MitoSOX mitochondrial superoxide fluorescent probe in 13 μl DMSO to prepare 5 mM MitoSOX fluorescent probe stock solution. Keep it away from light;

[0108] 2) Prepare 5 μM MitoSOX fluorescent probe working solution using HBSS / Ca / Mg buffer;

[0109] 3) Resuspend the cells with 1 ml of 5 μM MitoSOX fluorescent probe working solution to a concentration of about 5×10 5 Incubate cells / tube in a 37°C incubator in the dark for 10 min, invert and mix several times every 3 min.

[0110] 4) Wash the cells three times with ice-cold PBS to remove the MitoSOX fluorescent probe that has not entered the cells;

[0111] 5) Finally, add 0.5 ml of ice-cold PBS to resuspend the cells, and use flow cytometry to detect the average red fluorescence intensity of 20,000 cells.

[0112] 3.3 Experimental results:

[0113] like Figure 3 As shown, ( Figure 3 AB) Flow cytometry analysis of total intracellular ROS (A) and mitochondrial ROS (B) levels indicated by Carboxy-H2DCFDA and MitoSOX fluorescent probes; ( Figure 3 C) Flow cytometry analysis of the effect of antioxidant NAC (2 mM) on the toxicity of 5-CQA to liver cancer cells; ( Figure 3 D) Western blotting was used to analyze the effect of NAC on the expression of glucose transporter GLUT1 / 3 induced by 5-CQA; ( Figure 3 E) Real-time fluorescence quantitative PCR was used to detect the effect of NAC on the expression of HKDC1 gene induced by 5-CQA; ( Figure 3 F) Flow cytometry analysis of the effect of NAC on 5-CQA-stimulated glucose uptake in liver cancer cells; ( Figure 3 G) Bar graph of quantitative statistical analysis of the effect of NAC on the glucose uptake ability of 5-CQA-stimulated liver cancer cells. The data in the figure are mean±SD values ​​(n=3), *p<0.05, **p<0.01, ***p<0.001.

[0114] After incubation with 5-CQA for 96 h, 5-CQA induced total ROS in liver cancer cells ( Figure 3 A) and mitochondrial ROS ( Figure 3 B) increased significantly, and both were concentration- and time-dependent. Figure 3AB also showed that mitochondrial ROS is the main source of intracellular ROS. Although 600μΜ 5-CQA significantly inhibited the proliferation of liver cancer cells, the antioxidant NAC (2mM) was able to effectively relieve or alleviate the inhibitory effect of 5-CQA on the proliferation of liver cancer cells (p < 0.001). Interestingly, WB and qRT-PCR tests showed that NAC was also able to effectively relieve the induction effect of 5-CQA on the expression of glucose transporters GLUT1 / 3 and HKDC1. Further flow cytometry analysis also showed that NAC would relieve the promoting effect of 5-CQA on the glucose uptake ability of liver cancer cells. Therefore, the above experimental results suggest that the significant accumulation of ROS is a "double-edged sword" signal molecule that mediates the toxicity of 5-CQA liver cancer cells and causes liver cancer cell resistance.

[0115] 4. A pharmaceutical composition consisting of a small molecule inhibitor that can limit glucose absorption or downregulate aerobic glycolysis pathway and 5-caffeoylquinic acid will synergistically inhibit the proliferation of liver cancer cells, thereby promoting its clinical application by reducing the resistance of liver cancer cells to 5-caffeoylquinic acid

[0116] 4.1 Sugar-free culture significantly enhanced the inhibitory effect of 5-caffeoylquinic acid on the proliferation of hepatoma cells

[0117] Hepatoma cell lines MHCC97H and HCCLM3 were seeded in 96-well plates at a seeding density of 5 × 10 3 / well, incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours, then replaced with DMEM medium containing 10% FBS or 5% FBS but without glucose or containing high concentration of glucose, and added 5-CQA (400 μM) at a specified concentration in the experimental group, with 3 parallel wells set in each group, incubated in a cell culture incubator at 37°C and 5% CO2 for 48 hours. Finally, the MTT cell activity assay was used to analyze the cell viability of each group.

[0118] 4.2 Evaluation of the anti-liver cancer effect of a pharmaceutical composition for reducing drug resistance of liver cancer cells

[0119] The cell seeding density of MHCC97 and HCCLM3 hepatoma cell lines was 4 × 10 3 / well, incubated in a cell culture incubator at 37°C and 5% CO2 for 24h, and then replaced with DMEM (10% FBS) high-glucose medium containing a small molecule inhibitor (glucose analog 2-DG (1mM), hexokinase II inhibitor Bromopyruvic acid (10μM) or inhibitor Compound 3k (100nM) of the key protein PKM2 kinase in the aerobic glycolysis pathway), 5-CQA (400μM), the small molecule inhibitor (a pharmaceutical combination of glucose analog 2-DG (1mM) and 5-CQA (400μM), a pharmaceutical combination of hexokinase II inhibitor Bromopyruvic acid (10μM) and 5-CQA (400μM), a pharmaceutical combination of PKM2 kinase, a key protein in the aerobic glycolysis pathway, Compound 3k (100nM)) and 5-CQA (400μM), three parallel wells were set for each group, and incubated at 37°C and 5% CO2 for 24h. The cells were incubated in a CO2 concentration cell culture incubator for 96 h, and the medium was changed every 48 h. Finally, the cell viability was detected by MTT reagent to evaluate the inhibitory effect of each group of drugs on the proliferation of liver cancer cells, where the cell viability of the blank control group was defined as 100%, and the relative cell viability of each drug group was calculated.

[0120] 4.3 Experimental results: (4A) MTT cell activity assay to analyze the effects of serum concentration and glucose content in DMEM medium on the toxic effect of 5-CQA on liver cancer cells. The cell seeding density was 5×10 3 / well, 96-well plate, after culturing for 24h, replaced with drug-added DMEM medium (5% or 10% FBS), incubated in a 37℃, 5% CO2 incubator for 48h; (B) MTT cell activity assay to analyze the effects of glucose analog 2-DG (1mM), hexokinase II inhibitor Bromopyruvic acid (10μM) and Compound 3k (100nM), an inhibitor of PKM2 kinase, a key protein in the glycolytic pathway, on the toxicity of 5-CQA to liver cancer cells. The cell seeding density was 4×10 3 / well, 96-well plate, after 24h of culture, replaced with drug-added DMEM medium (10% FBS), and replaced every 48h for a total of 96h. The values ​​in the figure are mean±SD values ​​(n=3), *p<0.05, **p<0.01, ***p<0.001.

[0121] like Figure 4As shown in A, although changing the serum concentration from 10% to 5% did not significantly affect the proliferation of liver cancer cells, it significantly enhanced the sensitivity of liver cancer cells to 5-CQA; the sugar-free culture experiment showed that regardless of whether the serum concentration was 10% or 5%, 400 μM 5-CQA significantly inhibited the proliferation of liver cancer cells in the sugar-free DMEM medium for 48 hours, with an inhibition rate of 20-60% ( Figure 4 A). However, after incubation in DMEM (10% FBS) high glucose (4.5 g / L glucose) medium for 48 h, 400 μM 5-CQA had no significant effect on the proliferation of liver cancer cells ( Figure 4 A). In particular, the pharmaceutical composition consisting of glucose analogue 2-DG (1 mM), hexokinase II inhibitor Bromopyruvic acid (10 μM) or PKM2 kinase inhibitor Compound 3k (100 nM) respectively with 5-CQA has a significant synergistic inhibitory effect on the proliferation of liver cancer cells ( Figure 4 B).

[0122] Example 2. A drug combination for reducing the drug resistance of liver cancer cells to 5-CQA derivatives and its application

[0123] 1. Compared with 5-caffeoylquinic acid, chlorogenic acid methyl ester and 3,5-dicaffeoylquinic acid have greater application potential in the treatment of liver cancer

[0124] The initial cell seeding density of hepatoma cell lines MHCC97H and HCCLM3 was 4×10 3 Cells / well (96-well plate), 24 hours after cell plating, uniformly replaced with DMEM (10% FBS) medium without drug or with gradient concentrations (0, 50, 100, 200, 400 and 800 μM) of MCGA or 3,5-diCQA, 3 parallel wells were set up in each group, incubated in a cell culture incubator at 37°C and 5% CO2 for 96 hours, and the medium was changed every 48 hours. Finally, MTT reagent was used to detect cell viability and evaluate the effect of MCGA and 3,5-diCQA on the proliferation of liver cancer cells.

[0125] 2.5-Caffeoylquinic acid derivatives also induce drug resistance in liver cancer cells by activating the ROS-GLUT1 / 3 signaling axis

[0126] 2.1. The initial seeding density of cells in the hepatoma cell lines MHCC97H and HCCLM3 and the hepatoma cell line L-02 was 4×10 5Cells / 10 cm dish were plated and replaced with DMEM (10% FBS) medium with or without drug (350 μM) after 24 hours. The medium was changed every 48 hours and incubated for 96 hours. Then, cells of each group were collected by trypsin digestion. Finally, the induction effect of 5-CQA derivatives on the expression of glucose transporter GLUT1 / 3 was analyzed by WB method.

[0127] 2.2. The initial seeding density of the hepatoma cell lines MHCC97H and HCCLM3 was 2×10 3 Cells / well, 96-well plates, 24 hours after plating, uniformly replaced with DMEM (5% FBS) medium with or without drug (350 μM), 2 mM NAC, and NAC combined with 5-CQA derivatives, and the medium was changed once every 48 hours for 96 hours. Finally, MTT reagent was used to detect cell viability in each group.

[0128] 3. Low-concentration glucose culture conditions will significantly enhance the potential of 5-caffeoylquinic acid derivatives in the treatment of liver cancer

[0129] The initial seeding density of the hepatoma cell lines MHCC97H and HCCLM3 was 4×10 3 Cells / well (96-well plate), after 24 hours of plating, replaced with DMEM low-glucose (1g / L glucose) medium with or without drug (350μM), changed the medium every 48 hours, and incubated for 96 hours. Finally, MTT reagent was used to detect the cell viability of each group. The cell viability of each experimental group was divided by the cell viability of the control group, where the cell viability ratio of the 5-CQA group was defined as 1, and the cell viability ratio of the 5-CQA derivative group divided by the cell viability ratio of the 5-CQA group was defined as the relative cell viability ratio.

[0130] 4. The pharmaceutical composition composed of glucose analogs, hexokinase II inhibitors or aerobic glycolysis inhibitors and 5-CQA derivatives can synergistically inhibit the proliferation of liver cancer cells and effectively reduce the drug resistance of liver cancer cells

[0131] The cell seeding density of MHCC97H and HCCLM3 hepatoma cell lines was 5×10 3 / well, 96-well plate, incubated in a cell culture incubator at 37°C and 5% CO2 for 24 h, and then replaced with medium containing a small molecule inhibitor (2-DG (1 or 2 mM), Bromopyruvic acid (10 μM) or Compound 3k (40 nM)), MCGA (200 μM), 3,5-diCQA (200 μM), 2-DG (2 mM) and MCGA (200 μM), Bromopyruvic acid (10 μM) and MCGA (200 μM), Compound 3k (40 nM)) and MCGA (200 μM), 2-DG (1 mM) and 3,5-diCQA (200 μM), Bromopyruvic acid (10 μM) and MCGA (200 μM), Compound The drug combination of 3k (40nM) and 3,5-diCQA (200μM) was cultured in high-glucose DMEM (10% FBS), with 3 parallel wells set in each group, incubated in a cell culture incubator at 37°C and 5% CO2 for 96h, and the medium was changed every 48h. Finally, the MTT reagent was used to detect cell viability to evaluate the inhibitory effect of each drug group on the proliferation of liver cancer cells, in which the cell viability of the blank control group was defined as 100%, and the relative cell viability of each drug group was calculated.

[0132] 5. Statistical analysis: All statistical analyses in the present invention were performed using GraphPad Prism 5 software, and t-test was used for comparison between the two groups. Data statistics are expressed as mean ± standard deviation (mean ± SD) (n = 3), and p < 0.05 indicates that the difference between the two groups is statistically significant.

[0133] 6. Experimental results: Figure 5 As shown in the figure, the dose-response curve analysis showed that the IC50 values ​​of MCGA and 3,5-diCQA on liver cancer cells were significantly lower than those of 5-CQA, and the IC50 values ​​of MCGA on liver cancer cell proliferation were IC50 MHCC97H =343μM, IC50 HCCLM3 =334μM, the IC50 values ​​of 3,5-diCQA for inhibiting the proliferation of liver cancer cells were IC50 MHCC97H =349μM, IC50 HCCLM3 =337μM. Further WB analysis showed that Figure 6 Consistent with 5-CQA, abnormal activation of the ROS-GLUT1 / 3 signaling pathway is the key molecular mechanism that induces resistance of liver cancer cells to MCGA and 3,5-diCQA. Figure 6 A) Western blotting was used to analyze the effects of gradient concentrations of MCGA and 3,5-diCQA on the expression of GLUT1 / 3 proteins in liver cancer cells; ( Figure 6 BC) MTT analysis of the effect of antioxidant NAC on the toxicity of MCGA (B) and 3,5-diCQA (C) to liver cancer cells. MCGA and 3,5-diCQA also significantly upregulated the protein expression of glucose transporter GLUT1 / 3, but had no induction effect on the expression of GLUT1 / 3 in liver cells ( Figure 6 A). Further analysis showed that the antioxidant NAC significantly neutralized the toxicity of MCGA and 3,5-diCQA on liver cancer cells ( Figure 6 BC). In particular, the effects of low-sugar and high-sugar DMEM culture media on the toxicity of 5-CQA and its derivatives to liver cancer cells were compared and analyzed. The experimental results showed that low-sugar culture significantly reduced the resistance of liver cancer cells to 5-CQA and its derivatives, and the cytotoxicity was as follows: 5-CQA<MCGA<3,5-diCQA( Figure 7 A. Figure 7 B), Figure 7 Low-glucose culture and inhibition of aerobic glycolysis significantly enhanced the sensitivity of liver cancer cells to MCGA and 3,5-diCQA. (AB) MTT analysis of the effect of low-glucose culture on the effects of 5-CQA, MCGA and 3,5-diCQA on liver cancer cells relative to DMEM (10% FBS) high-glucose culture medium. The initial cell seeding density was 4×10 3 Cells / well (96-well plate), 24 hours after cell plating, the medium was replaced with DMEM (10% FBS) medium with or without drug, and the medium was changed every 48 hours for 96 hours. (CD) MTT cell activity assay analyzed the effects of glucose analog 2-DG (1-2mM), Bromopyruvic acid (10μM), an inhibitor of hexokinase II, and Compound 3k (40nM), an inhibitor of PKM2 kinase, a key protein in the glycolytic pathway, on the toxicity of 5-CQA to liver cancer cells. The data in the figure are mean±SD values ​​(n=3), *p<0.05, **p<0.01, ***p<0.001. Consistent with 5-CQA, under high-glucose DMEM medium conditions, a pharmaceutical composition composed of a glucose analog 2-DG, a hexokinase II inhibitor Bromopyruvic acid or an aerobic glycolysis inhibitor Compound 3k and a 5-CQA derivative can also synergistically inhibit the proliferation of liver cancer cells and effectively reduce the drug resistance of liver cancer cells ( Figure 7 CD). Therefore, the above experimental results prove that low-sugar culture conditions and aerobic glycolysis inhibitors are conducive to the application of 5-CQA and its derivatives in the treatment of liver cancer.

[0134] For those skilled in the art, the present invention can be improved or transformed according to the above embodiments, and all these improvements or transformations should fall within the protection scope of the claims of the present invention.

Claims

1. A pharmaceutical composition for reducing drug resistance of liver cancer cells, characterized in that: The pharmaceutical composition comprises component A and component B: Wherein component A is 5-caffeoylquinic acid; Component B is an aerobic glycolysis inhibitor.

2. The pharmaceutical composition according to claim 1, characterized in that The aerobic glycolysis inhibitor is a PKM2 kinase inhibitor.

3. The pharmaceutical composition according to claim 2, characterized in that The PKM2 kinase inhibitor is Compound 3k.

4. The pharmaceutical composition according to claim 3, characterized in that The molar concentration of Compound 3k is 40-100 nM.

5. The pharmaceutical composition according to claim 1, characterized in that The substance concentration of the 5-caffeoylquinic acid is greater than 200 and less than 400 μM.

6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a drug for enhancing the sensitivity of liver cancer cells to 5-caffeoylquinic acid and its derivatives and delaying their drug resistance.

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