Application of raffinose and raffinose derivative in preparation of anti-hepatocellular carcinoma medicine

Abstract

The invention discloses application of raffinose and a derivative thereof in preparation of anti-hepatocellular carcinoma drugs. The main mechanism is that raffinose or the derivative thereof induces hepatocellular carcinoma cells to release senescence-related secretory phenotypes (SASP), inhibits neutrophil recruitment, enhances anti-tumor immune infiltration and realizes an anti-cancer effect. The compound can be used for preparing drugs for inhibiting primary and pulmonary metastatic hepatocellular carcinoma or anti-tumor drugs for research of hepatocellular carcinoma.

Description

Technical Field

[0001] The invention relates to the field of medicines, and in particular to the application of raffinose and its derivatives in the preparation of anti-hepatocellular carcinoma medicines. Background Art

[0002] The liver is the most metabolically active organ in the human body, balancing the uptake and utilization of glucose, lipids, and amino acids. Metabolic imbalance is the cause and characteristic of hepatocellular carcinogenesis. Abnormal metabolic coupling in pathways such as aerobic glycolysis, tricarboxylic acid cycle, and pentose phosphate pathway promotes liver cancer. When liver cancer occurs, chronic necrotic inflammation causes damage, leading to metabolic disorders in liver cells, inhibiting DNA and protein damage repair, affecting stromal cells, immune cells, inflammatory factors, and pH homeostasis, and accelerating liver cancer progression, drug resistance, and adverse reactions. Targeting liver cancer cell metabolism can be synergistic with clinical treatment.

[0003] At present, the treatment of liver cancer is still mainly surgical treatment, supplemented by radiotherapy and chemotherapy. When the surgical opportunity is lost or metastasis occurs, the treatment effect will be worse. In recent years, anti-angiogenic drugs combined with immune checkpoint inhibitors such as PD1 monoclonal antibody, PD-L1 monoclonal antibody, and CTLA4 antibody have been used in clinical practice. Although immunotherapy has low toxicity and side effects and the quality of life of patients has improved, the disease control rate and five-year survival rate are still very low. In addition, the high cost of immunotherapy has become one of the reasons limiting clinical application and reducing the number of patients who benefit. Summary of the invention

[0004] The purpose of the present invention is to provide a plant-derived natural raffinose and its derivatives for use in the preparation of drugs for treating primary and metastatic liver cancer. The main mechanism is that raffinose or its derivatives induce liver cancer cells to release senescence-associated secretory phenotype (SASP), inhibit neutrophil recruitment, enhance anti-tumor immune infiltration, and achieve anti-cancer effects. It can be used as a drug for the preparation of primary and lung metastatic hepatocellular carcinoma inhibitory drugs or anti-tumor drugs for the study of hepatocellular carcinoma.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] Application of raffinose in the preparation of medicine for treating hepatocellular carcinoma.

[0007] Application of raffinose and its derivatives in preparing medicine for treating hepatocellular carcinoma.

[0008] Preferably, the hepatocellular carcinoma comprises primary hepatocellular carcinoma and lung metastatic hepatocellular carcinoma.

[0009] Preferably, raffinose and its derivatives are used to prepare a pharmaceutical composition containing raffinose and its derivatives and a pharmaceutically acceptable carrier.

[0010] Preferably, the pharmaceutical composition contains 0.01%-99.99% raffinose and its derivatives, and the rest is a pharmaceutical carrier.

[0011] Preferably, the raffinose and its derivatives include: raffinose, raffinose hydrate, raffinose trihydrate, raffinose 4.433-hydrate, raffinose pentahydrate, raffinose oleate, raffinose undecaacetate, raffinose leucine, permethyl raffinose, permethyl raffinose, peracetate, hydrochloride or o-raffinose, preferably raffinose, with the structural formula shown in the following table:

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[0013]

[0014]

[0015] The advantages of the present invention are:

[0016] When the inventors studied the liver cancer metabolic network, they discovered a natural metabolite, raffinose, which has anti-liver cancer effects. Raffinose metabolism occurs in lysosomes, which are involved in cellular energy metabolism, material metabolism, organelle quality control, cell survival and death, and immune regulation. Raffinose can regulate tumor cell lysosomes, induce tumor liver cancer cell senescence, reduce the recruitment of immunosuppressive cells, and promote the infiltration of anti-tumor immune cells.

[0017] Raffinose is a trisaccharide with a wide range of plant sources and can be easily prepared into derivatives and compositions. It is currently mainly used as a food additive and cryopreservation solution. There are no reports on its application in the treatment of primary liver cancer and lung metastatic liver cancer.

[0018] After surgery, chemoradiotherapy and immunotherapy, the era of metabolic therapy has arrived. The important feature of metabolic therapy is that it has no cytotoxic effect and does not directly induce tumor cell death. Instead, it achieves anti-cancer by regulating the metabolic microenvironment and immune microenvironment, with little side effects on the body. Compared with other tumors such as glioblastoma, raffinose is more targeted to tumor cells in the treatment of hepatocellular carcinoma. Specifically, it is manifested as:

[0019] 1) The inventors have found that raffinose itself has no cytotoxicity. In in vitro experiments, raffinose can only inhibit the proliferation of liver cancer cells at a high concentration of 80mM. The anti-cancer function of raffinose in vivo depends on enhancing anti-tumor immunity;

[0020] 2) The inventors have found that raffinose, under the catalysis of α-galactosidase and aldose reductase, can produce galactitol that is not utilized by mammalian cells. Galactitol accumulates in cells, induces cell senescence, and regulates anti-tumor immunity;

[0021] 3) The inventors have found that α-galactosidase and aldose reductase are not expressed in normal liver cells, but are highly expressed in liver cancer cells. Therefore, raffinose has tumor cell selectivity against liver cancer and has no damage to normal liver cells.

[0022] 4) The inventors found that in liver cancer patients with poor prognosis, the expression of α-galactosidase and aldose reductase was higher, indicating that raffinose is expected to have a better effect on malignant hepatocellular carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :The inhibitory effect of raffinose on hepatocellular carcinoma in situ and lung metastasis in mice. Among them:

[0024] (A) The inventors constructed orthotopic liver transplant tumors by subcapsular injection of Hepa1-6 liver cancer cells, and treated model mice with raffinose and its derivatives numbered as above, and quantitatively compared the weight of tumor-bearing livers.

[0025] (B) The inventors constructed an experimental liver cancer lung metastasis model by tail vein injection of highly metastatic Hepa1-6 liver cancer cells, and treated model mice with raffinose and its derivatives numbered as above, and quantitatively compared the number of liver cancer clone lesions in the lungs.

[0026] (C) The inventors constructed a mouse model of orthotopic liver transplantation with lung metastasis by intrahepatic injection of Hepa1-6 liver cancer cells, treated the mice with raffinose, and qualitatively compared the formation of clonal lesions of liver cancer in the tumor-bearing liver and lung.

[0027] (D) The inventors constructed a mouse model of orthotopic liver transplantation with lung metastasis by intrahepatic injection of Hepa1-6 liver cancer cells, treated the mouse with raffinose, and quantitatively compared the weight of tumor-bearing livers.

[0028] (E) The inventors constructed a mouse model of orthotopic liver transplantation with lung metastasis by intrahepatic injection of Hepa1-6 liver cancer cells, treated the mice with raffinose, and quantitatively compared the liver tumor weights.

[0029] (F) The inventors constructed an experimental liver cancer lung metastasis model by tail vein injection of highly metastatic Hepa1-6 liver cancer cells, gave raffinose treatment, and used H&E staining to qualitatively compare the formation of liver cancer clone lesions in the lungs.

[0030] (G) The inventors constructed an experimental liver cancer lung metastasis model by tail vein injection of highly metastatic Hepa1-6 liver cancer cells, treated the patients with raffinose, and used H&E staining to quantitatively count the number of liver cancer clone lesions formed in lung tissue sections per unit area of ​​view.

[0031] (H) The inventors constructed an experimental liver cancer lung metastasis model by tail vein injection of highly metastatic Hepa1-6 liver cancer cells, gave raffinose treatment, and used immunohistochemical staining to qualitatively compare the expression of Ki67 in lung liver cancer clone lesions.

[0032] (I) The inventors constructed an experimental liver cancer lung metastasis model by tail vein injection of highly metastatic Hepa1-6 liver cancer cells, administered raffinose treatment, and used immunohistochemical staining to quantitatively compare the expression ratio of Ki67 in lung liver cancer clone lesions.

[0033] Figure 2 :The in vitro inhibitory effect of raffinose on mouse liver cancer cells. Among them:

[0034] (A) The inventors used 1 nM to 160 mM concentration of raffinose to treat Hepa1-6 liver cancer cells in vitro, and detected cell viability using CCK8 after 48 hours. The relative cell viability was calculated after measuring OD450.

[0035] (B) The inventors treated Hepa1-6 liver cancer cells with 80 mM raffinose for 7 days and performed β-galactosidase staining to evaluate cell senescence.

[0036] Figure 3 :Raffinose inhibits the distribution of neutrophils and macrophages in mouse lung metastatic hepatocellular carcinoma. Wherein: (A) The inventors constructed an experimental liver cancer lung metastasis model by tail vein injection of highly metastatic Hepa1-6 liver cancer cells, gave raffinose treatment, and used immunohistochemical staining to qualitatively compare the expression of Ly6G in lung liver cancer clone lesions.

[0037] (B) The inventors constructed an experimental liver cancer lung metastasis model by tail vein injection of highly metastatic Hepa1-6 liver cancer cells, gave raffinose treatment, and used immunohistochemical staining to quantitatively compare the expression of Ly6G in lung liver cancer clone lesions.

[0038] (C) The inventors constructed an experimental liver cancer lung metastasis model by tail vein injection of highly metastatic Hepa1-6 liver cancer cells, gave raffinose treatment, and used immunohistochemical staining to quantitatively compare the relative expression of Ly6G in lung liver cancer clone lesions.

[0039] (D) The inventors constructed an experimental liver cancer lung metastasis model by tail vein injection of highly metastatic Hepa1-6 liver cancer cells, gave raffinose treatment, and used immunohistochemical staining to qualitatively compare the relative expression of F4 / 80 in lung liver cancer clone lesions.

[0040] Figure 4 :Raffinose has an effect on the CD8 metastatic hepatocellular carcinoma in mice + The promoting effect of T cell distribution. Among them: (A) The inventors constructed an experimental liver cancer lung metastasis model by tail vein injection of highly metastatic Hepa1-6 liver cancer cells, gave raffinose treatment, and used immunohistochemical staining to qualitatively compare the relative expression of CD8a in lung liver cancer clone lesions.

[0041] (B) The inventors constructed an experimental liver cancer lung metastasis model by tail vein injection of highly metastatic Hepa1-6 liver cancer cells, gave raffinose treatment, and used immunohistochemical staining to quantitatively compare the CD8a in the lung liver cancer clone lesions. + T cell tumor infiltration.

[0042] Figure 5 :Raffinose decomposition enzymes are not expressed in liver cells, but highly expressed in liver cancer cells. Raffinose has tumor cell selectivity in anti-liver cancer. Among them:

[0043] (A) The inventors used the TCGA database to analyze the expression of α-galactosidase (encoded by the GLA gene), an enzyme involved in the degradation of raffinose, in normal liver tissue (normal) and primary tumor tissue (primary tumor).

[0044] (B) The inventors used the TCGA database to analyze the expression of α-galactosidase (encoded by the GLA gene) along with the progression of liver cancer.

[0045] (C) The inventors used the TCGA database to analyze the expression of α-galactosidase (encoded by the GLA gene) as patients gained weight.

[0046] (D) The inventors used the TCGA database to analyze the expression of α-galactosidase (encoded by the GLA gene) associated with the occurrence of liver cancer metastasis.

[0047] (E) The inventors used the TCGA database to analyze the expression of aldose reductase (encoded by the AKR1B1 gene), an enzyme involved in the decomposition of raffinose, in normal liver tissue (normal) and primary tumor tissue (primary tumor).

[0048] (F) The inventors used the TCGA database to analyze the expression of aldose reductase (encoded by the AKR1B1 gene) along with the progression of liver cancer.

[0049] Figure 6 :Patients with high expression of raffinose decomposition enzymes α-galactosidase and aldose reductase have poor prognosis. Among them: (A) The inventors used the TCGA database to analyze the relationship between the expression of α-galactosidase (encoded by the GLA gene) and the prognosis of liver cancer patients.

[0050] (B) The inventors used the TCGA database to analyze the relationship between the expression of aldose reductase (encoded by the AKR1B1 gene) and the prognosis of liver cancer patients. DETAILED DESCRIPTION

[0051] In order to make the above features and advantages of the present invention more clearly understood, the following embodiments are given for detailed description. The methods of the present invention are all conventional methods in the art unless otherwise specified.

[0052] Example 1 The inhibitory effect of raffinose on hepatocellular carcinoma in situ and metastatic to the lung in mice.

[0053] For the intrahepatic transplantation tumor model, C57BL6 / J male 7-8 week old mice were purchased from Jicui Yaokang (hereinafter the same), anesthetized with chloral hydrate, and a 5 mm long window was opened on the right side of the mouse abdomen near the xiphoid process using ophthalmic scissors to expose the left liver lobe of the mouse. An insulin syringe was used to inject 1×10 6 Hepa1-6 liver cancer cells were placed on the surface of the mice, and then pressed with a cotton swab for 2-5 seconds to prevent tumor cells from spilling out of the liver. The surgical incision was sutured with medical sutures. On the 5th day after surgery, raffinose or its derivatives were given intraperitoneally at a dose of 10 mg / time, every other day. After 11 days, the mice were euthanized, and the tumor-bearing livers of the mice were collected, weighed, and counted.

[0054] For the lung metastatic liver cancer xenograft model, 2 × 10 6 The highly metastatic Hepa1-6 liver cancer cells were treated with raffinose or its derivatives through intraperitoneal administration on the second day after modeling, with a dose of 10 mg / time, and the drugs were given every day. After 15 days, the mice were euthanized, the tumor-bearing lungs of the mice were collected, and the number of tumor lesions was counted.

[0055] For the intrahepatic tumor lung metastasis model, mice were anesthetized with chloral hydrate, and a 5 mm long window was opened on the right side of the mouse abdomen near the xiphoid process using ophthalmic scissors to expose the left liver lobe of the mouse. 1×10 6 The highly metastatic Hepa1-6 liver cancer cells were then pressed with a cotton swab for 2-5 seconds to prevent tumor cells from spilling out of the liver, and the surgical incision was sutured with medical sutures. Raffinose treatment was given intraperitoneally on the 5th day after surgery, with a dose of 10 mg / time, and was given every other day. The mice were euthanized 11 days later, and the tumor-bearing livers of the mice were collected, weighed and counted, and the tumor-bearing lungs of the mice were collected and the number of tumor lesions was counted.

[0056] For the above experimental model, lung tissues were collected 15 days after raffinose treatment, dehydrated with graded ethanol and embedded in paraffin. The lung tissue sections were stained with hematoxylin-eosin, and the number of metastatic foci per unit area of ​​the sections was counted.

[0057] For the above experimental model, 15 days after modeling, the lung tissues were collected, dehydrated with gradient ethanol and then embedded in paraffin. The lung tissue sections were stained using immunohistochemistry, and the area ratio of Ki67-positive cells to the tumor area was counted.

[0058] Example 2 In vitro inhibitory effect of raffinose on mouse liver cancer cells.

[0059] Hepa1-6 liver cancer cells were plated in a 96-well plate at a density of 2000 cells / well. After the cells adhered to the plate, raffinose at final concentrations of 1nM, 10nM, 100nM, 1μM, 10μM, 100μM, 1mM, 10mM, 80mM, and 160mM was added. After 48 hours of treatment, the absorbance of the cell culture fluid at OD450 was detected by CCK8 to calculate the relative cell viability. For Hepa1-6 liver cancer cells treated with 80mM raffinose for 7 days, cell senescence was evaluated by β-galactosidase staining.

[0060] Example 3 The inhibitory effect of raffinose on the distribution of neutrophils and macrophages in mouse lung metastatic hepatocellular carcinoma.

[0061] For the lung metastatic liver cancer xenograft model, 1 × 10 6 The highly metastatic Hepa1-6 liver cancer cells were treated with raffinose or its derivatives through the peritoneal cavity starting from the second day after modeling, with a dose of 10 mg / time and administration every other day. The mice were euthanized 15 days later, and the tumor-bearing lungs of the mice were collected and paraffin-embedded after dehydration with gradient ethanol. The lung tissue sections were stained with immunohistochemistry, and the number of Ly6G-positive neutrophils per unit area was counted.

[0062] For the intrahepatic transplantation tumor model, mice were anesthetized with chloral hydrate, and a 5 mm long window was opened on the right side of the mouse abdomen near the xiphoid process using ophthalmic scissors to expose the left liver lobe. 1 × 10 6 The highly metastatic Hepa1-6 liver cancer cells were then pressed with a cotton swab for 2-5 seconds to prevent tumor cells from spilling out of the liver, and the surgical incision was sutured with medical sutures. Raffinose treatment was given intraperitoneally on the 5th day after surgery, with a dose of 10 mg / time, and the medication was given every other day. After 11 days, the mice were euthanized, and the tumor-bearing livers of the mice were collected and paraffin-embedded after gradient ethanol dehydration. The lung tissue sections were stained using immunohistochemistry, and typical photos of the distribution of F4 / 80 positive macrophages in cancer tissues and adjacent tissues were taken.

[0063] Example 4 Effect of raffinose on CD8 hepatocellular carcinoma metastasis in mice + Promotion of T cell distribution.

[0064] For the lung metastatic liver cancer xenograft model, 2 × 10 6 The highly metastatic Hepa1-6 liver cancer cells were treated with raffinose or its derivatives through intraperitoneal administration on the second day after modeling, with a dose of 10 mg / time, and the drugs were given every day. After 15 days, the mice were euthanized, and the tumor-bearing lungs of the mice were collected, dehydrated with gradient ethanol, and embedded in paraffin. The lung tissue sections were stained with immunohistochemistry, and the distribution ratio of CD8a-positive T cells in the tumor area was counted.

[0065] Combination Figure 1-Figure 4 The above-mentioned raffinose and its derivatives significantly inhibited the growth of liver orthotopic transplanted tumors and the growth of liver cancer in lung tissue. The inhibitory effect of raffinose on tumors was the best at the corresponding dose ( Figure 1 Raffinose significantly inhibited the formation of pre-metastasis foci of liver cancer cells in lung tissue ( Figure 1 For established lung metastases, raffinose significantly reduced tumor cell proliferation, as reflected by a decrease in the proportion of Ki67-positive cells ( Figure 1 However, in in vitro experiments, raffinose needs to reach a concentration of 80mM to show a significant inhibitory effect on the proliferation of liver cancer cells, reflecting enhanced cell senescence ( Figure 2 ), which shows that raffinose does not simply act on liver cancer cells to achieve anti-tumor effects, nor does it simply rely on cytotoxic effects to achieve anti-tumor effects. Therefore, by detecting the infiltration of immunosuppressive cells, it was found that raffinose significantly reduced the distribution of neutrophils and macrophages in tumor tissues ( Figure 3), indicating that raffinose breaks the anti-cancer immunosuppression barrier. Furthermore, the infiltration of anti-tumor immune cells was detected, and it was found that raffinose significantly increased the distribution of CD8a-positive T cells in tumor tissues ( Figure 4 ), indicating that raffinose promotes anti-cancer immunity. In summary, raffinose induces liver cancer cell senescence, breaks immunosuppression, enhances the infiltration of anti-tumor immune cells into tumors, and exerts an anti-liver cancer effect.

[0066] Example 5 Raffinose has tumor cell selectivity against liver cancer.

[0067] The gene expression characteristics of hepatocellular carcinoma patients were analyzed using the TCGA database, and it was found that hepatocytes did not express the raffinose decomposition enzyme α-galactosidase (encoded by the GLA gene) and aldose reductase (encoded by the AKR1B1 gene), while hepatoma cells highly expressed ( Figure 5 A and E).

[0068] Raffinose can be decomposed into galactitol by α-galactosidase and aldose reductase. Galactitol is not used by mammalian cells and accumulates in liver cancer cells, inducing liver cancer cell senescence ( Figure 2 Middle B). Raffinose is not decomposed in normal liver cells and does not cause senescence of normal liver cells. Raffinose has liver cancer cell selectivity in anti-liver cancer. Raffinose enhances anti-liver cancer immunity by regulating the release of senescence-related secretory phenotypes in liver cancer cells.

[0069] The expression of α-galactosidase in tumor samples from patients with hepatocellular carcinoma increases with tumor progression, patient weight, and tumor metastasis ( Figure 5 In BD), aldose reductase expression increases with tumorigenesis and progression ( Figure 5 In HCC patients with poor prognosis, α-galactosidase and aldose reductase expression were higher ( Figure 6 AB), indicating that raffinose is expected to have a better effect on malignant hepatocellular carcinoma.

[0070] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Application of raffinose and its derivatives in the preparation of drugs for treating hepatocellular carcinoma.

2. The use according to claim 1, characterized in that: The hepatocellular carcinoma includes primary hepatocellular carcinoma and lung metastatic hepatocellular carcinoma.

3. The use according to claim 1, characterized in that: The raffinose and its derivatives are selected from one or more of Raffinose, Raffinose hydrate, Raffinose trihydrate, Raffinose 4.433-hydrate, Raffinose pentahydrate, Raffinose oleate, Raffinose undecaacetate, Raffinose leucine, Raffinose, permethyl, Raffinose peracetate, Raffinose hydrochloride, and o-Raffinose.

4. The use according to any one of claims 1 to 3, characterized in that: Raffinose and its derivatives are used to prepare a pharmaceutical composition containing raffinose and its derivatives and a pharmaceutical carrier.

5. The use according to claim 4, characterized in that: The pharmaceutical composition contains 0.01%-99.99% of raffinose and its derivatives, and the rest is a pharmaceutical carrier.

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