Application of licorice extract in treatment of uveal melanoma

Abstract

The invention provides an application of licorice extracts GL, 18beta-GA and DG in tumor treatment, in particular to an application in treatment of uveal melanoma (UM).

Description

Technical Field

[0001] The present invention relates to the use of licorice extract in the treatment of tumors, especially in the treatment of uveal melanoma (UM). Background Art

[0002] Uveal melanoma is the most common primary intraocular malignancy in adults, with an annual incidence of approximately 0.6 cases per million people per year in the Asian population. Approximately 50% of patients will develop metastatic uveal melanoma (mUM). The most common site of metastasis is the liver, and patients usually die within one year after the diagnosis of metastasis. The high metastasis rate and high mortality of UM bring great physical and mental pressure to patients and seriously affect their quality of life. However, there is currently no effective treatment for mUM in clinical practice. The TCR therapy drug Tebentafusp has become the first new therapy in 40 years to improve the overall survival rate of patients with metastatic UM, but the drug has just completed phase III clinical trials and the follow-up time is short, and it has not been widely used. Therefore, it is necessary to further explore more general clinical treatment options for UM. Summary of the Invention

[0003] On the one hand, the present invention provides the use of licorice extract in the preparation of a drug for treating uveal melanoma in a subject.

[0004] In some embodiments, the treatment includes inhibiting the proliferation / or metastasis of uveal melanoma cells in the subject.

[0005] In some embodiments, the licorice extract includes GL, 18β - GA, DG, or any combination thereof.

[0006] In some embodiments, the subject is a mammal.

[0007] In some embodiments, the subject is a human or a mouse.

[0008] On the other hand, the present invention provides a method for identifying uveal melanoma cells as in - situ tumor cells or metastatic tumor cells for non - diagnostic purposes, including detecting the distribution of HMGB1 in the UM cells.

[0009] In some embodiments, when the nuclear expression level of HMGB1 is higher than the cytoplasmic expression level, the uveal melanoma cells are considered as in - situ tumor cells; when the nuclear expression level of HMGB1 is less than the cytoplasmic expression level, the uveal melanoma cells are considered as metastatic tumor cells. Brief Description of the Drawings

[0010] Figure 1The expression of HMGB1 in UM cells was shown. (A) HMGB1 was highly expressed in tumor cells of UM samples from 11 patients; (B) Differentially expressed genes between tumor cells and non-tumor cells; (C) HMGB1 was widely expressed in UM tumor cells.

[0011] Figure 2 The relationship between HMGB1 and the prognosis of UM patients was shown. (A) Results of Kaplan-Meier survival analysis of different expression levels of HMGB1; (B) Expression levels of HMGB1 in surviving and dead patients; (C) Expression levels of HMGB1 in different pathological types; (D) The expression levels of HMGB1 were positively correlated with those of PD-1, HAVCR2, and VEGFA. * p < 0.05, ** p < 0.01, *** p < 0.001.

[0012] Figure 3 The inhibition of UM cell growth by HMGB1 was shown. (A) Comparison of mRNA expression abundances after HMGB1 gene knockdown; (B) CCK8 showed the cell viability of siCtrl and siHMGB1 in OCM1A and OMM2.3 cells; (C) Results of colony formation assay. ** p < 0.01, *** p < 0.001.

[0013] Figure 4 The apoptosis of UM cells regulated by HMGB1 was shown, and cell apoptosis was analyzed by PI / Annexin V staining. ** p < 0.01, *** p < 0.001.

[0014] Figure 5 The inhibition of migration and invasion of UM cells by HMGB1 was shown. The migration and invasion abilities of C918 and OCM1A cells were measured by Transwell migration and invasion assays, respectively. (A) Migration results of C918 cells; (B) Migration results of OCM1A cells; (C) Invasion results of C918 cells; (D) Invasion results of OCM1A cells.

[0015] Figure 6 The cell activities of UM cell lines treated with different licorice extracts and other drugs (GL, 18β-GA, DG, methotrexate, salicylic acid, ethyl pyruvate, and epigallocatechin gallate) detected by CCK8 assay were shown.

[0016] Figure 7Shows the growth, migration, invasion and apoptosis of UM cells after treatment with different licorice extracts GL, 18β-GA and DG. (A) Cell viability detected by CCK8; (B) Results of colony formation assay; (C) Results of Transwell migration and invasion assays; (D) Cell apoptosis analyzed by PI / Annexin V staining. *** p < 0.001.

[0017] Figure 8 Shows the experimental results of intragastric administration of different concentrations of licorice extract to the C918 animal model. (A) Changes in tumor volume; (B) Comparison of tumor weights; (C) Tumor photos of different groups.

[0018] Figure 9 Shows the results of immunofluorescence staining of HMGB1 in UM in situ tumors (upper panel) and UM liver metastasis tissues (lower panel). The green fluorescence signal is HMGB1.

[0019] Figure 10 Shows the analysis results of the expression level of HMGB1 in UM cells and subcellular expression after treatment with licorice extract. (A) qRT-PCR and WB analysis results of HMGB1 in UM cells treated with Ctrl (drug formulation buffer), GL, 18β-GA and DG; (B) Nuclear and cytoplasmic expression levels of HMGB1 in the HMGB1 and shHMGB1 groups treated with licorice extract (GL, 18β-GA and DG). Detailed implementation

[0020] Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0021] The term "or" refers to a single element among the listed alternative elements, unless the context clearly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more or all of the listed alternative elements.

[0022] The term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In the text, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the case consisting of the stated elements, integers or steps. For example, when referring to an object "including" a specific element, it is also intended to cover an object consisting of that specific element.

[0023] Glycyrrhizin (GL) is the main triterpenoid compound in licorice extract, which exists in the roots and stems of licorice. GL can be cleaved into glucuronic acid and glycyrrhetinic acid (GA) in the form of aglycone. GA exists in two stereoisomeric forms: 18α-GA and 18β-GA. GL and 18β-GA have the structures shown in the following formulas (I) and (II), respectively.

[0024]

[0025] Diammonium glycyrrhizinate (DG) is the diammonium salt form of glycyrrhizin.

[0026] HMGB1 (High Mobility Group Box 1) is a non-histone nuclear protein widely present in eukaryotes and belongs to the high mobility group protein (HMG protein) family. It regulates DNA structure in the nucleus and, at the same time, acts as a damage-associated molecular pattern (DAMP) molecule outside the cell, participating in pathophysiological processes such as inflammation, immune response, and tumorigenesis.

[0027] When referring to a tumor, "in situ" means that tumor cells are confined to the original location of the tissue of origin, without breaching the basement membrane, invading the surrounding normal tissue, or metastasizing distantly.

[0028] When referring to a tumor, "metastasis" means that the cells of an in situ tumor detach from the original site and migrate to other parts of the body through the blood circulation, lymphatic system, or direct dissemination, etc., and form secondary tumor foci.

[0029] "Treatment" refers to the process of treating a subject to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviating one or more symptoms, reducing the severity of the disease, preventing or delaying the spread of the disease (such as metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the condition of the disease, inhibiting the disease or the progression of the disease, arresting its development, and remission (whether partial or complete). According to the above content, "treatment" does not require complete elimination of all symptoms of the disorder or disease or complete remission.

[0030] "Subject" includes human and non-human mammals. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein. In certain embodiments, the subject is a human.

[0031] In in vitro cytological experiments, we found that licorice extracts, including glycyrrhizin and its derivatives 18β-GA and DG, could inhibit the growth, migration and invasion of UM cells. In mouse animal experiments, it was found that the growth of UM tumors could be significantly inhibited. In addition, we found that for in situ UM tumor cells, HMGB1 was mainly distributed in the nuclei of UM cells; while in UM liver metastasis tissues, HMGB1 was mainly distributed in the cytoplasm. Therefore, the difference in the nuclear-cytoplasmic expression level of HMGB1 can be used to identify whether UM tumor cells are in situ tumor cells or metastatic tumor cells.

[0032] The following specific examples are used to further illustrate the present invention.

[0033] Example 1 HMGB1 is widely expressed in UM tumor cells

[0034] Since August 2014, the inventor has established and managed the Beijing Tongren Ophthalmology Clinical Database (Tongrenophthalmology Research Association of Clinical Evaluation, TRACE), which mainly collects intraocular tumors and rare eye diseases, with UM as the main disease type. So far, more than 2,000 UM patients have been included, and more than 30,000 related biological samples have been stored. The information collected includes clinical diagnosis and treatment data, follow-up information and biological samples. Based on this biobank, we performed single-cell sequencing on UM tissues, and performed unsupervised re-clustering on tumor tissues and the tumor cells therein to detect the gene expression of tumor cells. The results showed that HMGB1 was highly expressed in the tumor cells of UM tissues from different patients ( Figure 1 A); further, differential expression genes were searched for between tumor cells and non-tumor cells, and log2(Fold Change)≥0.25 and P<0.01 were defined as differential genes, and a total of 310 differential expression genes were screened out ( Figure 1 B). HMGB1 was widely expressed in UM tumor cells ( Figure 1 C), suggesting that HMGB1 is related to the occurrence and development of UM.

[0035] Example 2 High expression of HMGB1 in UM is related to poor prognosis of patients

[0036] Based on the resources of this biobank, we performed bulk-mRNA sequencing on 42 UM tissues and 18 normal choroid tissues, and combined with 80 UM samples in TCGA, we performed survival analysis on different expression levels of HMGB1. The results showed that the survival period of patients with high expression of HMGB1 was significantly shortened (p<0.05) ( Figure 2 A). The expression of HMGB1 in the death group was significantly increased (p<0.05) ( Figure 2B). In highly malignant epitheloid cell type UM, the expression of HMGB1 was significantly increased (p<0.01)( Figure 2 C). Significant positive correlations among HMGB1, PD1, HAVCR2 and VEGFA( Figure 2 D). The above results indicate that high expression of HMGB1 is significantly associated with poor prognosis of patients. Combining the results of single-cell sequencing and transcriptome sequencing, we speculate that high expression of HMGB1 in UM cells may promote the malignant potential of UM.

[0037] Example 3 HMGB1 promotes the proliferation, migration and invasion of UM cells and inhibits apoptosis

[0038] To clarify the role of HMGB1 in UM, we conducted in vitro functional studies on the effects of HMGB1 on the proliferation, invasion and metastasis of UM. HMGB1 gene knockout was performed using siRNA in three UM cell lines OCM1A, OMM2.3 and C918, and the knockout efficiency was verified by qPCR. Figure 3 A). The results of CCK8 showed that interfering with the HMGB1 gene inhibited the growth of OCM1A cells and OMM2.3 cells. Figure 3 B). In addition, the number of colony formations decreased in OCM1A and C918 cells with HMGB1 silencing. Figure 3 C). HMGB1 knockout significantly promoted tumor cell apoptosis. Figure 4 ). The Transwell assay confirmed that the migration and invasion abilities of UM cells in the siHMGB1 group were significantly reduced. Figure 5 A-5D). The above results suggest that HMGB1 promotes the proliferation, migration and invasion of UM cells and inhibits apoptosis.

[0039] Example 4 Effects of licorice extracts on the growth and migration of UM cells

[0040] Based on the preliminary drug screening results( Figure 6 ), we studied three different types of licorice extracts (GL, 18β-GA and DG) and observed their effects on UM cells. The results of CCK-8 and colony formation assays showed that GL, 18β-GA and DG could significantly inhibit the growth of C918, OCM1A and OMM2.3 cells (7A and 7B). The migration and invasion abilities of UM cells in the GL, 18β-GA and DG treatment groups were significantly reduced. Figure 7 C), and apoptosis increased significantly. Figure 7 D).

[0041] Example 5 Animal experiments prove that licorice extracts effectively inhibit the growth of UM tumors

[0042] The C918 cell line was used to establish a subcutaneous tumor model in BALB / c nude mice. Administration was by gavage, and the effects of different concentrations of licorice extract (control group, 18β-GA 20 mg / kg, 18β-GA 50 mg / kg, DG 10 mg / kg, DG 20 mg / kg, GL 50 mg / kg, GL 100 mg / kg) on tumor growth were compared. The results showed that intragastric administration of 18β-GA 50 mg / kg, DG 10 mg / kg, DG 20 mg / kg, and GL 100 mg / kg could effectively inhibit the growth of UM tumors ( Figure 8 A-C).

[0043] Example 6 Localization of HMGB1 in UM cells suggests that its nucleocytoplasmic translocation may be related to UM invasion and metastasis

[0044] To further explore the mechanism of action of HMGB1, we first performed immunofluorescence staining to determine the subcellular localization of HMGB1. The results showed that in UM intraocular in situ tumors, HMGB1 was mainly distributed in the nuclei of UM cells; while in UM liver metastatic tissues, HMGB1 was mainly distributed in the cytoplasm ( Figure 9 ). This suggests that changes in the subcellular localization of HMGB1 may be related to the increased malignant potential of UM.

[0045] Example 7 Effects of licorice extract on the nucleocytoplasmic translocation of HMGB1 in UM cells

[0046] We further analyzed the changes in the expression levels of HMGB1 in UM cells and its subcellular expression after treatment with licorice extract. After treatment with GL, 18β-GA, and DG, the total expression level of HMGB1 remained unchanged ( Figure 10 A); while the expression of HMGB1 decreased in the cytoplasm and increased in the nucleus ( Figure 10 B). The above results suggest that GL, 18β-GA, and DG may inhibit cell proliferation, migration, and invasion and promote apoptosis by inhibiting the nucleocytoplasmic translocation of HMGB1.

Claims

1. Use of a licorice extract in the preparation of a medicament for treating uveal melanoma in a subject.

2. The use according to claim 1, wherein the treatment comprises inhibiting the proliferation and / or metastasis of uveal melanoma cells in the subject.

3. The use according to claim 1 or 2, wherein the liquorice extract comprises GL, 18β-GA, DG or any combination thereof.

4. The use according to claim 1 or 2, wherein the subject is a mammal.

5. The use according to claim 1 or 2, wherein the subject is a human or a mouse.

6. A method for identifying uveal melanoma cells as in situ tumor cells or metastatic tumor cells for non-diagnostic purposes, comprising detecting the distribution of HMGB1 in the UM cells.

7. The method of claim 6, wherein when the nuclear expression level of HMGB1 is higher than the cytoplasmic expression level, the uveal melanoma cells are considered to be in situ tumor cells; when the nuclear expression level of HMGB1 is lower than the cytoplasmic expression level, the uveal melanoma cells are considered to be metastatic tumor cells.

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