Application of a branched-chain amino acid-restricted diet in the preparation of drugs for treating liver metastases of tumors

Abstract

This invention discloses the application of a branched-chain amino acid (BCAA) restricted diet in the preparation of drugs for treating liver metastases of tumors. Through mouse model experiments, this invention found that restricting the intake of BCAAs (valine, isoleucine, and leucine) in the diet, or using the SCD1 inhibitor A939572, can significantly inhibit liver metastases of tumors, especially pancreatic cancer liver metastases. Experimental results show that a BCAA restricted diet or SCD1 inhibitor can reduce the number and volume of liver metastases and inhibit their growth. This invention also reveals its mechanism of action: BCAA restriction reduces the propionylation modification of SREBP1, downregulates the transcriptional expression of its target gene SCD1, and thus reduces lipid synthesis, thereby exerting an anti-tumor liver metastasis effect. This invention provides a novel dietary intervention strategy and drug target for the treatment of liver metastases of tumors.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of a branched-chain amino acid-restricted diet in the preparation of drugs for treating liver metastases of tumors. Background Technology

[0002] Pancreatic cancer is a highly aggressive malignant tumor with a 5-year survival rate of less than 8%. Due to low early diagnosis rates, most patients already have distant metastases at the time of diagnosis, with the liver being the most common target organ for distant metastasis. Approximately 35% of newly diagnosed cases are already in the liver metastasis stage, often with multiple metastatic lesions or locations unsuitable for surgery. Even after primary tumor resection, about half of patients develop liver metastases within 6 months post-surgery. Pancreatic cancer is characterized by dense fibrotic stroma (fibroproliferative response) and commonly exhibits mutations in driver genes such as KRAS, TP53, SMAD4, and CDKN2A. These pathological features collectively constitute physical and immune barriers, hindering the penetration of chemotherapy drugs and the infiltration of effector T cells, and suppressing immune cell activity, leading to primary or acquired resistance to existing chemotherapy, radiotherapy, targeted therapy, and immune checkpoint inhibitors. Liver metastasis is the leading cause of death in pancreatic cancer patients; therefore, there is an urgent clinical need to develop effective treatment strategies targeting the liver metastasis stage.

[0003] To meet the demands of rapid proliferation, tumor cells restructure metabolic pathways involving glucose, fatty acids, and amino acids to provide sufficient energy and biosynthetic raw materials. Amino acids are crucial for protein synthesis and energy supply, and tumor cells exhibit a high dependence on exogenous amino acids; a deficiency of certain amino acids can even lead to tumor growth arrest. Branched-chain amino acids (BCAAs), including valine (Val), isoleucine (Ile), and leucine (Leu), are essential amino acids. Existing research indicates that BCAAs can promote malignant tumor progression by upregulating the expression of lipid synthesis genes such as FASN and ACLY. Research on the effects of BCAAs on pancreatic cancer has primarily focused on the early growth stages of pancreatic cancer. Previous studies have found that a high-BCAA diet promotes the progression of pancreatic intraepithelial neoplasia (PanIN) in transgenic mouse models of pancreatic cancer, while a low-BCAA diet or targeted inhibition of BCAA metabolic enzymes can significantly reduce the formation of pancreatic PanIN in mice (see Chinese Patent Application No. CN201810863766).

[0004] However, pancreatic cancer has an insidious onset, often metastasizing and developing treatment resistance by the time of initial diagnosis. Compared to early-stage non-invasive pancreatic cancer with liver metastasis, advanced stages of invasive pancreatic cancer with liver metastasis exhibit disrupted basal integrity, genetic alterations, fibrosis, and a highly immunosuppressive environment. Most importantly, metabolic reprogramming occurs in the liver metastasis stage, showing significant differences from the early stages. Therefore, restricting branched-chain amino acid (BCAA) intake to inhibit pancreatic cancer progression cannot be considered an effective treatment for advanced liver metastases. To date, it remains unclear whether a low-BCAA diet can inhibit existing pancreatic cancer liver metastases. Furthermore, whether BCAA restriction works through other molecular mechanisms and whether there are drug targets that mimic the effects of this dietary restriction require further investigation.

[0005] Therefore, developing a dietary intervention strategy or drug treatment plan that can effectively inhibit liver metastasis of pancreatic cancer has important clinical value. Summary of the Invention

[0006] The purpose of this invention is to provide the application of a branched-chain amino acid-restricted diet in the preparation of a drug for treating liver metastases of tumors.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In one aspect, this application provides the use of a branched-chain amino acid-restricted diet in the preparation of a drug for treating liver metastases of tumors.

[0008] Secondly, this application provides the use of SCD1 inhibitor A939572 in the preparation of a drug for treating liver metastases of tumors.

[0009] The first aspect of this application provides the use of a branched-chain amino acid-restricted diet in the preparation of a drug for treating liver metastases of tumors.

[0010] Furthermore, the tumor is pancreatic cancer; the treatment of liver metastases includes inhibiting the growth of liver metastases; and / or, inhibiting the number and / or volume of liver metastases.

[0011] Furthermore, a branched-chain amino acid-restricted diet exerts its anti-tumor liver metastasis effect by reducing SREBP1 propionylation modification, downregulating the transcriptional expression of the downstream target gene SCD1, and thereby reducing lipid synthesis.

[0012] Furthermore, the branched-chain amino acid (BCAA) restricted diet is a low-BCAA composition, wherein the total BCAA content in the composition does not exceed 20% of the total BCAA content in a conventional diet.

[0013] Furthermore, a branched-chain amino acid (BCAA) restricted diet includes staple foods that do not contain BCAAs, as well as drinking water containing valine, isoleucine, and leucine.

[0014] Furthermore, the valine content in the drinking water was 0.0792% (w / v), the isoleucine content was 0.0672% (w / v), and the leucine content was 0.1224% (w / v).

[0015] Furthermore, the staple food without branched-chain amino acids is composed of the following components by weight percentage: 18.0% protein, 65.5% carbohydrates, and 7.0% fat; wherein the protein contains L-arginine, L-histidine, L-lysine hydrochloride, L-tyrosine, L-tryptophan, L-phenylalanine, L-methionine, L-cysteine, L-threonine, glycine, L-proline, L-glutamic acid, L-alanine, L-aspartic acid, and L-serine, and does not contain valine, isoleucine, and leucine; the carbohydrates include one or a combination of corn starch, maltodextrin, or sucrose; the fat is soybean oil; and the additives include one or a combination of cellulose, choline tartrate, a mineral mixture, a vitamin mixture, or sodium bicarbonate.

[0016] The second aspect of this application provides the use of the SCD1 inhibitor A939572 in the preparation of a medicament for treating liver metastases of tumors.

[0017] Furthermore, the tumor is pancreatic cancer; the treatment of liver metastases includes inhibiting the growth of liver metastases; and / or, inhibiting the number and / or volume of liver metastases.

[0018] Furthermore, the SCD1 inhibitor A939572 exerts its anti-tumor liver metastasis effect by reducing lipid synthesis.

[0019] Compared with the prior art, the present invention has the following advantages: (1) The invention first proposed a new use of branched-chain amino acid (BCAA) restriction diet for the treatment of liver metastases of tumors: Existing technologies only disclose that a low BCAA diet can be used to prevent and delay the occurrence and development of pancreatic cancer and precancerous lesions (PanIN). However, this invention first discovered through in vitro experiments that BCAA restriction for 24 h significantly inhibited the migration of pancreatic cancer cells without affecting the proliferation of pancreatic cancer cells. More importantly, it first proved through animal model experiments that a low BCAA diet can significantly inhibit existing pancreatic cancer liver metastases, including reducing the number and volume of liver metastases and inhibiting the growth of metastases, providing a new dietary intervention strategy for patients with pancreatic cancer liver metastases.

[0020] (2) First discovery of a novel application of SCD1 inhibitor A939572 in the treatment of liver metastases: This invention proposes for the first time that SCD1 inhibitor A939572 can be used to treat liver metastases, especially pancreatic cancer liver metastases. Experiments have shown that at low concentrations that do not affect cell proliferation, A939572 can significantly inhibit the migration ability of pancreatic cancer cells, and oral administration in animal models can effectively reduce liver metastases, providing a new drug target and small molecule compound for the treatment of liver metastases.

[0021] (3) A novel mechanism of branched-chain amino acid restriction against liver metastasis has been revealed: This invention reveals for the first time that branched-chain amino acid restriction reduces the propionylation modification of SREBP1, downregulates the transcriptional expression of SCD1, a key enzyme in fatty acid synthesis of the SREBP1 target gene, and thereby reduces lipid synthesis, thus exerting an anti-tumor liver metastasis effect. The elucidation of this mechanism provides a theoretical basis for the development of anti-metastasis drugs targeting lipid metabolism.

[0022] (4) The present invention provides specific dietary and administration protocols: The present invention provides a clear low-branched-chain amino acid diet formula (a staple food without branched-chain amino acids combined with a specific concentration of valine, isoleucine, and leucine in drinking water) and the dosage and administration method of SCD1 inhibitor A939572 (10 mg / kg, orally), which is operable and repeatable and facilitates clinical application and transformation. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The branched-chain amino acid restriction treatment of this invention for 24 h can significantly reduce the migration ability of mouse pancreatic cancer cells KPC and Panc02, but has limited effect on cell proliferation.

[0025] Figure 2 This invention provides photographs, liver weight statistics, representative HE-stained images, and liver metastasis area statistics of mice bearing KPC and Panc02 tumors, showing how a low-branched-chain amino acid diet can reduce liver metastasis.

[0026] Figure 3 The results of RNA-seq sequencing differential gene enrichment analysis, GSEA analysis of lipid metabolism pathways, and differential gene transcription factor analysis are presented in this invention.

[0027] Figure 4The present invention aims to reduce the propionylation level of SREBP1 protein by restricting branched-chain amino acids.

[0028] Figure 5 The diagram illustrates how branched-chain amino acid restriction affects propionylation of the SREBP1 protein, inhibiting SREBP1 transcription and activating SCD1 expression.

[0029] Figure 6 This diagram illustrates the inhibition of human pancreatic cancer cell migration by the low-concentration SCD1 inhibitor A939572 of this invention.

[0030] Figure 7 The images show photographs, liver weight statistics, HE staining, and representative images of pancreatic cancer xenograft in situ tumor models inhibited by a low-branched-chain amino acid diet or the SCD1 inhibitor A939572, as well as statistics on liver metastasis area. Detailed Implementation

[0031] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0032] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0033] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0034] The first aspect of this application provides the use of a branched-chain amino acid-restricted diet in the preparation of a medicament for treating liver metastases of tumors.

[0035] In some embodiments, the tumor is pancreatic cancer; the treatment of liver metastases includes inhibiting the growth of liver metastases; and / or inhibiting the number and / or volume of liver metastases.

[0036] In some embodiments, a branched-chain amino acid-restricted diet exerts its anti-tumor liver metastasis effect by reducing SREBP1 propionylation modification, downregulating SCD1 transcriptional expression, and thereby reducing lipid synthesis.

[0037] In some embodiments, the branched-chain amino acid (BCAA) restricted diet is a low-BCAA composition in which the total BCAA content does not exceed 20% of the total BCAA content in a conventional diet.

[0038] In some embodiments, a branched-chain amino acid-restricted diet includes staple foods that do not contain branched-chain amino acids, and drinking water containing valine, isoleucine, and leucine.

[0039] In some embodiments, the drinking water contains 0.0792% (w / v) valine, 0.0672% (w / v) isoleucine, and 0.1224% (w / v) leucine.

[0040] In some embodiments, the branched-chain amino acid-free staple food comprises the following components by weight percentage: 18.0% protein, 65.5% carbohydrates, and 7.0% fat; wherein the protein comprises L-arginine, L-histidine, L-lysine hydrochloride, L-tyrosine, L-tryptophan, L-phenylalanine, L-methionine, L-cysteine, L-threonine, glycine, L-proline, L-glutamic acid, L-alanine, L-aspartic acid, and L-serine, and does not contain valine, isoleucine, and leucine; the carbohydrates comprise one or a combination of corn starch, maltodextrin, or sucrose; the fat is soybean oil; and the additives comprise one or a combination of cellulose, choline tartrate, a mineral mixture, a vitamin mixture, or sodium bicarbonate.

[0041] The second aspect of this application provides the use of the SCD1 inhibitor A939572 in the preparation of a medicament for treating liver metastases of tumors.

[0042] In some embodiments, the tumor is pancreatic cancer; treatment of liver metastases includes inhibiting the growth of liver metastatic lesions; and / or, inhibiting the number and / or volume of liver metastases.

[0043] In some embodiments, the SCD1 inhibitor A939572 exerts its anti-tumor liver metastasis effect by reducing lipid synthesis. Example 1

[0044] The present invention relates to the application of a branched-chain amino acid-restricted diet in the preparation of a drug for treating liver metastases of tumors.

[0045] The tumor is pancreatic cancer; the treatment of liver metastases includes inhibiting the growth of liver metastases; and / or, inhibiting the number and / or volume of liver metastases.

[0046] Branched-chain amino acid restriction diets exert their anti-tumor liver metastasis effect by reducing propionylation modification of SREBP1, downregulating the transcriptional expression of SCD1, and thereby reducing lipid synthesis.

[0047] A branched-chain amino acid (BCAA) restricted diet includes staple foods that do not contain BCAAs, as well as drinking water containing valine, isoleucine, and leucine.

[0048] The branched-chain amino acid (BCAA) restricted diet is a low-BCAA composition, wherein the total BCAA content in the composition does not exceed 20% of the total BCAA content in a regular diet. Example 2

[0049] The difference between Example 2 and Example 1 is that the content of valine in the drinking water is 0.0792% (w / v), the content of isoleucine is 0.0672% (w / v), and the content of leucine is 0.1224% (w / v).

[0050] The staple food without branched-chain amino acids consists of the following components by weight percentage: 18.0% protein, 65.5% carbohydrates, and 7.0% fat. The protein contains L-arginine, L-histidine, L-lysine hydrochloride, L-tyrosine, L-tryptophan, L-phenylalanine, L-methionine, L-cysteine, L-threonine, glycine, L-proline, L-glutamic acid, L-alanine, L-aspartic acid, and L-serine, but does not contain valine, isoleucine, or leucine. The carbohydrates are a combination of three of the following: corn starch, maltodextrin, and sucrose. The fat is soybean oil. The additives are a combination of five of the following: cellulose, choline tartrate, a mineral mixture, a vitamin mixture, and sodium bicarbonate. Example 3

[0051] The difference between Example 3 and Example 2 is that the carbohydrate is corn starch; the fat is soybean oil; and the additive is cellulose. Example 4

[0052] The difference between Example 4 and Example 2 is that the carbohydrate is maltodextrin; the fat is soybean oil; and the additive is a mixture of minerals. Example 5

[0053] The difference between Example 5 and Example 2 is that the carbohydrate is sucrose; the fat is soybean oil; and the additive is choline tartrate. Example 6

[0054] The difference between Example 6 and Example 2 is that the carbohydrate is a combination of two of corn starch and sucrose; the additives are a combination of three of the following: a mineral mixture, a vitamin mixture, and sodium bicarbonate. Example 7

[0055] The difference between Example 7 and Example 2 is that the additive is a mixture of vitamins. Example 8

[0056] The present invention relates to the application of an SCD1 inhibitor, A939572, in the preparation of a drug for treating liver metastases of tumors.

[0057] The tumor is pancreatic cancer; treatment of liver metastases includes inhibiting the growth of liver metastases; and / or, inhibiting the number and / or volume of liver metastases.

[0058] SCD1 inhibitor A939572 exerts its anti-tumor liver metastasis effect by reducing lipid synthesis. Example 9

[0059] The present invention prepares feed lacking branched-chain amino acids according to the following formula, as shown in Table 1: Table 1

[0060] Example 10 This invention prepares branched-chain amino acid-restricted drinking water according to the following formulations: 0.0792% L-valine (w / v) + 0.0672% L-isoleucine (w / v) + 0.1224% L-leucine (w / v); and normal branched-chain amino acid drinking water: 0.396% L-valine (w / v) + 0.336% L-isoleucine (w / v) + 0.612% L-leucine (w / v). Example 11

[0061] Transwell assays were performed to detect cell migration. KPC and Panco2 cells in logarithmic growth phase were cultured in serum-free medium for 24 hours. After 24 hours, the cells were digested and resuspended in serum-free medium containing 1×10⁻⁶ cells / mL. 4 200 μL of cell suspension was seeded in the upper chamber, and the lower chamber was incubated with 500 μL of normal culture medium and culture medium excluding valine, isoleucine, and leucine (or one of these three) for 24 hours. Cell fixation and staining were performed to assess cell migration, and photographs were taken to count the number of cells that had migrated through the membrane in selected fields. RPMI 1640 medium (valine-, isoleucine-, and leucine-free) was purchased from Shanghai Peiyuan Biotechnology Co., Ltd. Figure 1 As shown in AB, Figure 1 AB is a graph showing that branched-chain amino acid deprivation significantly reduced the migration of mouse pancreatic cancer cells KPC and Panco2 in Example 11 of this invention. The results showed that branched-chain amino acid restriction (-BCAA) could significantly inhibit the migration ability of KPC cells. Among them, valine deprivation alone (-Val) had the most significant inhibitory effect, followed by isoleucine deprivation (-Ile), while leucine deprivation (-Leu) had a relatively limited inhibitory effect. Example 12

[0062] CCK8 assay for cell proliferation. KPC / Panc02 cells with a density of approximately 90% were digested, centrifuged, and the supernatant was removed. Normal culture medium was added and mixed thoroughly to prepare a cell suspension. 100 μL of the cell suspension was added to each well of a 96-well plate, with a cell density of 2000-3000 cells / well (margin wells were filled with sterile PBS), and cultured overnight. The cell culture medium was aspirated, and 100 μL of pre-prepared normal culture medium and a medium excluding valine, isoleucine, and leucine (or one of these three) were added. The cells were cultured for 24 hours, then incubated with CCK8 for 2.5 hours. The absorbance of each well was measured at OD 450 nm using an ELISA reader, and the relative cell viability was calculated. Figure 1 As shown in C, Figure 1 C represents the branched-chain amino acid deprivation in Example 12 of this invention. Within 24 hours, the effect on cell proliferation is limited and has no very significant impact. Example 13

[0063] Twenty-eight 4-6 week old C57BL / 6J mice were randomly divided into four groups: A and B: Normal diet group: Staple food without branched-chain amino acids and free drinking water with 0.396% valine (w / v) + 0.336% isoleucine (w / v) + 0.612% leucine (w / v).

[0064] C and D: Low branched-chain amino acid diet group: staple food without branched-chain amino acids and free drinking water with 0.0792% valine (w / v) + 0.0672% isoleucine + 0.1224% leucine (w / v).

[0065] Mice were fed a normal diet and a low-branched-chain amino acid (BCAA) diet for one week. Groups A and C were inoculated with KPC cells via spleen injection, while groups B and D were inoculated with Panc02 cells via spleen injection. Mice were then continued to be fed a normal diet and a low-BCAA diet. Mouse condition and weight changes were observed. Liver samples were collected 18 days after modeling in the KPC cell liver metastasis model groups (Groups A and C) and 35 days after modeling in the KPC cell liver metastasis model groups (Groups B and D). Liver samples were photographed and weighed. Liver H&E staining was performed, and the liver metastasis area was observed under a 50 μm microscope. The proportion of metastatic liver area was calculated. Figure 2 As shown, Figure 2 The images, liver weight statistics, representative HE-stained images, and liver metastasis area statistics of mice bearing KPC and Panc02 cells in Example 13 of this invention are shown. Figure 2This paper presents the effects of a low-branched-chain amino acid diet on KPC and Panc02 cell mouse liver metastasis models. The results showed that, compared to the normal diet group, the low-branched-chain amino acid diet group exhibited significantly reduced liver metastasis, with significantly decreased liver weight and the proportion of liver metastasis area. Example 14

[0066] Research on the mechanism by which branched-chain amino acid restriction inhibits liver metastasis of pancreatic cancer cells KPC / Panc02 cells in the logarithmic growth phase were cultured for 24 hours in normal medium and in branched-chain amino acid restriction medium (containing valine, isoleucine, and leucine), respectively. Cells were then collected and RNA-seq was performed. The sequencing results were used for differential gene enrichment analysis of Reactome pathways, lipid metabolism pathway enrichment analysis of GSEA, and transcription factor prediction of differential genes. Figure 3 The results are from RNA-seq sequencing Reactome enrichment analysis of differentially expressed lipid metabolism genes due to branched-chain amino acid restriction in Example 14, GSEA analysis of lipid metabolism pathways, and differential transcription factor analysis. The results show that branched-chain amino acid restriction inhibits the expression of lipid metabolism pathway genes, primarily regulated by the transcription factor SREBP1. Example 15

[0067] Research on the regulatory mechanism of branched-chain amino acid restriction on SREBP1 KPC / Panc02 cells in the logarithmic growth phase were cultured for 24 hours in normal medium and in branched-chain amino acid deprivation medium (containing valine, isoleucine, and leucine), respectively. Cells were collected, and panpropionylation modification was detected by Western blotting. KPC cells were then collected for propionylation modification proteomics sequencing to detect propionylation modification sites of SREBP1. Figure 4 Example 15 showed that branched-chain amino acid restriction reduced SREBP1 propionylation modification. The results showed that branched-chain amino acid restriction inhibited the level of SREBP1 propionylation modification, and that K373 and K386 of SREBP1 are propionylation modification sites, which are located in the leucine zipper region of SREBP1 and regulate transcriptional activity. Example 16

[0068] Mechanism of branched-chain amino acid restriction in downregulating lipid synthesis via SREBP1 propionylation modification KPC, Panc02, Capan1, and SW1990 cells in logarithmic growth phase were cultured in serum-free medium for 24 hours. After 24 hours, they were cultured in normal medium and branched-chain amino acid restriction medium (containing valine, isoleucine, and leucine) for 24 hours, respectively. Cells were then collected and analyzed by RT-PCR. Fasn , Scd1 , Acly, Acaca mRNA expression was measured, with β-actin used as an internal control; SCD1 protein expression was detected by Western blotting, with β-actin used as an internal control. Wild-type Srebp1, Srebp1K373R, and Srebp1K386R plasmids and a Scd1 promoter reporter plasmid were constructed. Dual-luciferase activity was measured after transfection of 293T cells. KPC cells were transfected with wild-type Srebp1, Srebp1K373R, and Srebp1K386R plasmids, and RT-PCR was performed to detect... Scd1 mRNA expression was measured using β-actin as an internal control; SCD1 protein expression was detected by Western blotting using β-actin as an internal control. Figure 5 Example 16 of this invention demonstrates how branched-chain amino acid restriction (-BCAA) reduces SREBP1 propionylation modification, thereby inhibiting SCD1 expression. Results showed that after branched-chain amino acid restriction (-BCAA), the mRNA level of SCD1, a key gene in fatty acid synthesis, was significantly reduced, and protein expression also decreased accordingly. SREBP1 propionylation modification directly regulates the transcriptional expression of SCD1. Example 17

[0069] KPC, Panc02, Capan1, and SW1990 cells in logarithmic growth phase were seeded into 96-well plates at a density of 2000-5000 cells / well (margin wells were filled with sterile PBS) and cultured overnight. The cell culture medium was aspirated, and pre-prepared medium containing a gradient of SCD1 inhibitor A939572 was added to each well, with five replicates per group. Forty-eight hours after drug administration, the supernatant was aspirated, and 100 μL of freshly prepared CCK8 mixture in serum-free medium was added to each well. The plates were incubated at 37°C for 1.5-2.5 hours. The absorbance of each well was measured at OD 450 nm using an ELISA reader to calculate cell viability.

[0070] Transwell assays were performed to detect cell migration. KPC, Panco2, Capan1, and SW1990 cells in logarithmic growth phase were cultured in serum-free medium for 24 hours. After 24 hours, the cells were digested and resuspended in normal medium containing 1×10⁻⁶ cells / mL. 4 200 μL of cell suspension was seeded in the upper chamber, and the lower chamber was incubated with 500 μL of medium containing 0.5 μM A939572 for 24 or 48 hours. Cells were fixed, stained and their migration was detected. The number of cells that had migrated through the membrane in the selected field of view was counted. Figure 6 Example 17 of this invention describes the use of a low-concentration SCD1 inhibitor, A939572, to inhibit pancreatic cancer cell migration. Results showed that using A939572 (0.5 μM), which does not affect cell proliferation, significantly inhibited pancreatic cancer cell migration. Example 18

[0071] Forty-two nude mice aged 4-6 weeks were randomly divided into 6 groups: A and B: Normal diet group: Staple food without branched-chain amino acids and free drinking water with 0.396% valine (w / v) + 0.336% isoleucine (w / v) + 0.612% leucine (w / v).

[0072] C and D: Normal diet + A939572 administration group: Staple food without branched-chain amino acids and free drinking water with 0.396% valine (w / v) + 0.336% isoleucine (w / v) + 0.612% leucine (w / v), and A939572 was administered by gavage (10 mg / kg) daily.

[0073] E and F: Low branched-chain amino acid diet group: staple food without branched-chain amino acids and free access to drinking water containing 0.0792% valine (w / v) + 0.0672% isoleucine (w / v) + 0.1224% leucine (w / v).

[0074] One week after mouse rearing, groups A, C, and E were inoculated with PANC1 cells via spleen injection, while groups B, D, and F were inoculated with SW1990 cells via spleen injection. Forty-five days after modeling, mouse livers were collected, photographed, and weighed. Liver staining with H&E and panCK was performed, and the livers were observed under a 50 μm microscope to determine the proportion of liver metastases. Figure 7 These images, along with liver weight statistics, HE staining, representative images of pancreatic cancer cell xenograft in situ, and statistics of liver metastasis area, are from Example 18 of this invention and show the inhibition of liver metastasis in a human pancreatic cancer cell xenograft in situ model by a low-branched-chain amino acid diet or oral administration of the SCD1 inhibitor A939572. The results show that both a low-branched-chain amino acid diet and oral administration of the SCD1 inhibitor A939572 significantly inhibited liver metastasis of pancreatic cancer cells, reduced liver weight, and significantly decreased the area of ​​liver metastasis.

[0075] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. Application of a branched-chain amino acid-restricted diet in the preparation of drugs for treating liver metastases of tumors.

2. The application according to claim 1, characterized in that: The tumor is pancreatic cancer; the treatment of liver metastases includes inhibiting the growth of liver metastases; and / or inhibiting the number and / or volume of liver metastases.

3. The application according to claim 1 or 2, characterized in that: The branched-chain amino acid-restricted diet exerts its anti-tumor liver metastasis effect by reducing SREBP1 propionylation modification, downregulating the transcriptional expression of the downstream target gene SCD1, and thereby reducing lipid synthesis.

4. The application according to claim 1, characterized in that: The branched-chain amino acid (BCAA) restricted diet is a low-BCAA composition, and the total BCAA content in the composition does not exceed 20% of the total BCAA content in a regular diet.

5. The application according to claim 1 or 2, characterized in that: The branched-chain amino acid restricted diet includes staple foods that do not contain branched-chain amino acids, and drinking water containing valine, isoleucine and leucine; the drinking water contains 0.0792% (w / v) valine, 0.0672% (w / v) isoleucine and 0.1224% (w / v) leucine.

6. The application according to claim 4, characterized in that: The staple food, which is free of branched-chain amino acids, is composed of the following components by weight percentage: 18.0% protein, 65.5% carbohydrates, and 7.0% fat; wherein the protein contains L-arginine, L-histidine, L-lysine hydrochloride, L-tyrosine, L-tryptophan, L-phenylalanine, L-methionine, L-cysteine, L-threonine, glycine, L-proline, L-glutamic acid, L-alanine, L-aspartic acid, and L-serine, and does not contain valine, isoleucine, and leucine; the carbohydrates include one or a combination of corn starch, maltodextrin, or sucrose; the fat is soybean oil; and the additives include one or a combination of cellulose, choline tartrate, a mineral mixture, a vitamin mixture, or sodium bicarbonate.

7. The application of an SCD1 inhibitor, A939572, in the preparation of a drug for treating liver metastases of tumors.

8. The application according to claim 7, characterized in that: The tumor is pancreatic cancer; the treatment of liver metastases includes inhibiting the growth of liver metastases; and / or inhibiting the number and / or volume of liver metastases.

9. The application according to claim 7, characterized in that: The SCD1 inhibitor A939572 was administered orally at a dose of 10 mg / kg to a mouse model of pancreatic cancer liver metastasis.

10. The application according to claim 7, characterized in that: The SCD1 inhibitor A939572 exerts its anti-tumor liver metastasis effect by reducing lipid synthesis.

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