Use of 1-aminocyclopropanecarboxylic acid in the preparation of a medicament for treating melanoma

Abstract

The application discloses application of 1-aminocyclopropane carboxylic acid in preparation of a melanoma treatment drug and relates to the field of medicinal chemistry. The 1-aminocyclopropane carboxylic acid of the application effectively slows down melanoma cell proliferation by activating a cysteine aspartate protease cascade signal pathway, inhibiting extracellular regulatory protein kinase 1 / 2, phosphorylated protein kinase B and B lymphocyte 2 signal pathways, and significantly reducing tumor tissue volume and mass, thereby playing an obvious curative effect.

Description

Technical Field

[0001] This application relates to the field of oncology drug technology, and in particular to the use of 1-aminocyclopropanecarboxylic acid in the preparation of melanoma treatment drugs. Background Technology

[0002] Melanoma is a highly malignant skin and mucosal tumor originating from melanocytes differentiated from the neural crest. It can occur in various tissues and organs, including the skin, eyes, digestive tract, and reproductive system. Compared to other types of skin cancer, it is more aggressive, metastasizes early, responds poorly to traditional radiotherapy and chemotherapy, has a poor prognosis, and a high recurrence rate. Clinical intervention is particularly ineffective in the late metastatic stage. In recent years, targeted therapy has included inhibitors targeting the BRAF V600E mutation (vemurafenib, dabrafenib), MEK inhibitors (trametinib), and C-KIT inhibitors (imatinib) as standard treatments. While these novel molecularly targeted drugs show significant efficacy in initial treatment, most patients develop resistance after several months to a year, increasing the risk of disease progression or recurrence. Furthermore, while PD-1 / PD-L1 inhibitors and CTLA-4 inhibitors in immunotherapy can significantly prolong progression-free survival in advanced patients, the objective response rate is generally between 30% and 40%, and serious adverse reactions are common.

[0003] Currently, commonly used chemotherapy drugs (dacarbazine, temozolomide) have an objective response rate of <15% in melanoma patients. Radiotherapy is generally only used as a palliative treatment for locally metastatic lesions. Because current treatment regimens have significant limitations for advanced tumors, drug-resistant cases, and certain subtypes, finding a novel and safe therapeutic target and its candidate drugs has become one of the main issues in current melanoma research.

[0004] 1-Aminocyclopropanecarboxylic acid (ACC) is a unique cyclic non-protein amino acid with a highly strained three-membered ring and both amino and carboxyl functional groups. It is chemically reactive and highly water-soluble. In vivo, ACC is not only a direct precursor in the biosynthesis of the plant hormone ethylene, participating in the regulation of plant growth, development, and stress responses, but it can also act as an independent signaling molecule, participating in the regulation of metabolic homeostasis and signaling networks in multiple species. In the mammalian nervous system, ACC possesses anticonvulsant, neuroprotective, anti-anxiety, anti-addictive, antidepressant, and cognitive-enhancing functions, showing promise for applications in neuroprotection, mental illness and cognitive impairment, and cardiovascular protection. However, no research on the anti-tumor effects of ACC, especially its anti-melanoma properties, has been found to date. Summary of the Invention

[0005] The purpose of this application is to provide the application of drugs with better efficacy in the treatment of melanoma.

[0006] To achieve the above objectives, this application utilizes the use of 1-aminocyclopropanecarboxylic acid in the preparation of melanoma treatment drugs.

[0007] Preferably, the application of 1-aminocyclopropanecarboxylic acid in the preparation of melanoma therapeutic drugs is characterized in that: the 1-aminocyclopropanecarboxylic acid downregulates the expression of extracellular signal-regulated kinase 1 / 2, B-cell lymphoma-2 protein and microphthalmia-associated transcription factor protein and the phosphorylated protein kinase B / protein kinase B ratio, and upregulates the expression of cleaved cysteine ​​aspartate protease protein 3 and the cleaved cysteine ​​aspartate protease 3 / cysteine ​​aspartate protease 3 ratio.

[0008] Preferably, the 1-aminocyclopropanecarboxylic acid activates the cysteine-aspartic protease cascade signaling pathway and inhibits the extracellular regulatory protein kinase 1 / 2, phosphorylated protein kinase B, and B lymphoma-2 signaling pathways.

[0009] Preferably, the recommended dose of 1-aminocyclopropanecarboxylic acid is 800 μg / kg.

[0010] Preferably, the drug dosage form is an injection.

[0011] Due to the adoption of the above technical solutions, the beneficial effects of this application are as follows:

[0012] In vitro cell experiments showed that 1-aminocyclopropanecarboxylic acid (ACC) inhibited the proliferation of B16 cells. Further Western blot results indicated that ACC upregulated the expression of cleaved caspase3 protein and the cleaved caspase3 / caspase3 ratio, while downregulating the expression of ERK1 / 2, Bcl2, and MITF proteins and the P-AKT / AKT ratio and P-ERK1 / 2 / ERK1 / 2 ratio. In vivo experiments using a subcutaneous melanoma xenograft model showed a significant downregulation of the P-ERK1 / 2 / ERK1 / 2 ratio (*), indicating that ACC effectively slowed the proliferation of melanoma tissue by inhibiting the activation of the ERK1 / 2 signaling pathway, thereby significantly reducing tumor tissue volume and mass and demonstrating a significant therapeutic effect. Attached Figure Description

[0013] Figure 1 The results show the effect of 1-aminocyclopropanecarboxylic acid (ACC) on the survival rate of B16 melanoma cells, where ns represents no significant difference, and ****P < 0.0001.

[0014] Figure 2The results of ACC regulation of protein expression and modification in B16 melanoma cells are shown. A is the protein immunoblotting map; B is the relative expression level of ERK1 / 2, Akt, P-Akt, cleaved Caspase3, Caspase3, MITF, and Bcl2 proteins. *P<0.05, **P<0.01; ERK1 / 2: extracellular signal-regulated kinase 1 / 2; Akt: protein kinase B; P-Akt: phosphorylated protein kinase B; cleaved Caspase3: cleaved cysteine ​​aspartate protease 3; Caspase3: cysteine ​​aspartate protease 3; MITF: microphthalmia-associated transcription factor; Bcl2: B-cell lymphoma-2 protein; β-Tubulin: β-tubulin.

[0015] Figure 3 The results of ACC treatment for subcutaneous melanoma xenografts are shown in Figure A, where A is an actual image of a subcutaneous melanoma xenograft in nude mice; B is a growth curve of the surface area of ​​the xenograft in nude mice; C is a comparison of the surface area of ​​the xenograft in nude mice (day 12 after drug administration); and D is the weight of the xenograft in nude mice. *P<0.05.

[0016] Figure 4 HE staining results for ACC-treated subcutaneous melanoma xenografts, where A is the control group (20% dimethyl sulfoxide); B is the ACC-treated group (800 μg / kg).

[0017] Figure 5 The results show the effects of ACC on protein expression and modification in subcutaneous melanoma xenografts. A represents the protein immunoblotting map; B represents the relative expression levels of ERK1 / 2, P-ERK1 / 2, and β-Tubulin proteins. The control group value was defined as 1 for quantitative analysis. *P<0.05, **P<0.01, ERK1 / 2: extracellular signal-regulated kinase 1 / 2; P-ERK1 / 2: phosphorylated extracellular signal-regulated kinase 1 / 2; β-Tubulin: β-tubulin. Detailed Implementation

[0018] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.

[0019] The 1-aminocyclopropanecarboxylic acid (ACC) used in the following examples was purchased from Maclean's, and the B16 mouse melanoma cells were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0020] Example 1: Experimental study on the effect of 1-aminocyclopropanecarboxylic acid (ACC) on the proliferation of malignant melanoma cells in vitro.

[0021] (1) The effect of ACC on the growth and proliferation of B16 melanoma cells was detected by the CCK-8 assay.

[0022] The recovery, culture, and cryopreservation of B16 mouse melanoma cells: Cryopreservation tubes were rapidly thawed in a 37°C water bath, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in pre-warmed complete culture medium. The cell suspension was transferred to a 5 mL culture flask containing 4 mL of fresh culture medium, thoroughly mixed, and incubated at 37°C with 5% CO2. The cell suspension from the cryopreservation tubes was thawed in a water bath to restore viability, and then aliquoted into DMEM medium containing penicillin 100 μg / mL and streptomycin 100 μg / mL, with the addition of 10% fetal bovine serum to provide a suitable growth environment. Once the cells were passaged for the first time and entered the logarithmic growth phase (density approximately 80%), they were immediately cryopreserved for long-term preservation. Cells in the logarithmic growth phase were prepared into a single-cell suspension, centrifuged at 1000 r / min for 5 min to remove non-adherent cells, and the pellet was collected. The pellet was resuspended in a special cryopreservation solution to 1 mL, transferred to a cryovial, sealed, and labeled. The cryovial was pre-cooled at the mouth of a liquid nitrogen tank for 5 min, and then slowly immersed in liquid nitrogen at -196℃ for long-term storage.

[0023] Preparation of ACC solution: Weigh 1.01 mg of ACC and dissolve it in 40 mL of DMEM medium containing triple antibodies to prepare a 250 μM stock solution. Before use, filter the solution through a 0.22 μm filter for sterilization and dilute it with DMEM medium containing triple antibodies to working concentrations of 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM. Store at 4°C for later use.

[0024] CCK-8 assay for cell viability: B16 cells were seeded in 96-well plates at 100 μL per well, with a density of approximately 70% after adhesion. Cells were treated with 0, 10, 20, 30, 40, and 50 μM ACC solution, with replicates for each group. After culturing for 24 h, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated in the dark for 1 h. The absorbance (A) of each well was measured at 450 nm using a microplate reader. Cell viability was calculated using the formula: Cell viability (%) = [(A-treated group - A-blank group) / (A-control group - A-blank group)] × 100%, and the half-inhibitory concentration (IC50) was calculated accordingly. 50 The blank group contained no cells, only culture medium and CCK-8 reagent, while the control group contained cells, culture medium and CCK-8 reagent.

[0025] like Figure 1The results showed that within the concentration range of 0–50 μM, the survival rate of B16 cells decreased with increasing ACC concentration; except for the 10 μM group, the differences in all other groups were statistically significant. The IC50 was calculated using a GraphPad Prism curve. 50 = 35 μM. This indicates that ACC can inhibit the proliferation of mouse melanoma cells, and the higher the concentration, the more significant the inhibitory effect.

[0026] (2) The effect of ACC on protein expression in B16 melanoma cells was studied by Western blotting.

[0027] Extraction buffer containing protease inhibitor, phosphatase inhibitor, and radioimmunoprecipitation lysis buffer was prepared at a volume ratio of 1:1:50. After culturing for 24 h in a control group (cells + culture medium) and an ACC-treated group (cells + culture medium + 35 μM ACC), B16 melanoma cells were collected from the culture medium of both groups by centrifugation. 1 mL of extraction buffer was added, and the cells were lysed in an ice bath for 20 min, followed by centrifugation for 30 min (4℃, 12000 rpm) to collect the clear supernatant. The protein concentration in the supernatant was determined using the diquinoline formate protein quantification method. Based on the results, the samples from the control and ACC-treated groups were standardized by dilution or concentration to ensure consistent protein concentrations across groups. The protein solution was mixed with 5× loading buffer at a 4:1 ratio and heated in a 100℃ water bath for 10 min to fully denature the protein.

[0028] SDS-PAGE gel electrophoresis: Gel preparation: Select an appropriate separating gel concentration based on the molecular weight of the target protein. Sample loading: Correctly install the prepared gel plate into the electrophoresis tank (lower end inward, higher end outward), add electrophoresis buffer to submerge the electrodes, connect the electrodes black to black and red to red, and electrophoresis at a constant voltage of 220 V for 40 min. Transfer: Prepare the transfer equipment in advance, placing a sponge and 3 sheets of filter paper on both sides of the transfer clamp. After removing the stacking gel, place the gel on the filter paper against the black background, cover it with the PVDF membrane and filter paper in sequence, remove air bubbles with a roller, and assemble the transfer apparatus. Place the transfer clamp with the black side facing the negative electrode and the red side facing the positive electrode into the transfer tank, add an appropriate amount of transfer buffer, and transfer at a constant current of 400 mA on ice for 1 h. Blocking: After transfer, place the PVDF membrane in 5% skim milk powder and block on a shaker at room temperature for 2 h. After blocking, wash three times with TBST for 10 min each time. Antibody incubation: Incubate the membrane with the corresponding primary antibody overnight at 4℃. The primary antibodies used included: ERK1 / 2 (1:1000), P-ERK1 / 2 (1:1000), Akt (1:1000), P-Akt (1:1000), P-Caspase3 (1:3000), Caspase3 (1:3000), MITF (1:2000), Bcl2 (1:1000), and internal control antibodies β-actin and β-tubulin (both 1:10000). After incubation, the membrane was washed three times with TBST for 10 min each time. It was then incubated with the corresponding secondary antibody (1:10000) at room temperature for 2 h, followed by three more washes with TBST for 10 min each time. Development: ECL developing solution was prepared 2 min in advance. The PVDF membrane was immersed in the developing solution and allowed to stand for 10 s before being immediately placed in the imaging system for exposure and image recording. ImageJ software was used for quantitative analysis of the band gray values.

[0029] ERK1 / 2, AKT, and Bcl2 proteins are involved in the regulation of tumor growth; Caspase3, as an apoptosis executive protein, initiates apoptosis through a cascade reaction; MITF is a melanoma marker, and its expression level reflects the growth status of melanoma. Figure 2The results in section AB show that ACC significantly induced apoptosis in B16 cells. Western blotting confirmed that ACC treatment decreased the P-AKT / AKT ratio (**) in B16 cells, while increasing the cleaved Caspase3 / Caspase3 ratio (*); ERK1 / 2 (*), MITF (*), and Bcl2 (*) expression was downregulated, while cleaved Caspase3 (*) modification was upregulated. These results indicate that ACC inhibits cell growth-related signaling pathways by downregulating the expression of ERK1 / 2, Bcl2, and MITF proteins and the P-AKT / AKT ratio; simultaneously, it upregulates the expression of cleaved Caspase3 protein and the cleaved Caspase3 / Caspase3 ratio, inducing apoptosis signaling pathways. Through these two effects, it inhibits the proliferation and growth of B16 cells.

[0030] Example 2: Experimental study on the effect of 1-aminocyclopropanecarboxylic acid (ACC) on subcutaneous xenografts in nude mice

[0031] (1) Effects of ACC on tumor tissue quality and volume in a subcutaneous nude mouse melanoma model

[0032] Construction of a subcutaneous xenograft model in nude mice: Three-week-old athymic nude mice were selected as experimental animals and randomly divided into a control group and an ACC experimental group (400 μg / kg and 800 μg / kg) according to their body weight. Before the experiment, B16 cells were washed twice with phosphate-buffered saline, digested with 0.25% trypsin at 37°C for 2 min, and digestion was terminated with complete culture medium. The cell suspension was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the pellet was collected. The cells were resuspended in diluted matrix gel to a total volume of 2 mL and stored on ice.

[0033] ACC preparation: Accurately weigh 2 mg of ACC powder and place it in a 15 mL centrifuge tube. Sterilize by UV irradiation for 30 min. Add 7 mL of deionized water and 2 mL of dimethyl sulfoxide (DMSO) sequentially, and adjust the pH to 8.43 with sodium hydroxide solution. Incubate the mixture in a 50°C water bath with shaking until completely dissolved. Filter for sterilization and use later.

[0034] Drug treatment and tumor growth curve plotting: Before inoculation, the skin of the right axilla of mice was disinfected with 75% ethanol. 100 μL of cell suspension was accurately drawn using a microsyringe and injected into the subcutaneous tissue on the dorsal side of the ipsilateral axilla, with the needle slowly withdrawn to prevent leakage. The long and short diameters of the tumor were measured daily using calipers after inoculation, and the tumor size was recorded. Drug intervention was initiated when the tumor area reached 50–150 mm². Preliminary results showed that a dose of 400 μg / kg had a weak tumor-suppressing effect; therefore, a dose of 800 μg / kg was used in subsequent experiments. The drug solution was prepared at 800 μg / kg, and the dosage volume was calculated based on the actual body weight of each mouse. The drug was slowly and evenly injected from the center of the tumor using a microsyringe, with the injection and withdrawal speed controlled to prevent leakage. Administration was given every other day for 2 weeks. The long and short diameters of the tumor were measured daily, and the tumor surface area (long diameter × short diameter) was calculated. On day 13, all nude mice were euthanized, and the subcutaneous tumor tissue was dissected, and the surface area and mass of the tumor were measured. Figure 3 As shown in section AB. Further as... Figure 3 The results of the CD section showed that, compared with the control group (DMSO solution), the ACC-treated group had a smaller tumor surface area (mean 313.022 mm² vs 272.957 mm², P<0.05) and a smaller tumor mass (mean 4.865 g vs 3.705 g, P<0.05), indicating that ACC can inhibit the proliferation of melanoma tissue.

[0035] (2) Observe the morphological changes of tumor tissue by HE staining.

[0036] After tumor tissue was collected, it was flash-frozen in liquid nitrogen and stained with hematoxylin and eosin (HE). Figure 4 As shown in Part A, the tumor cells in the control group were densely packed, with large, deeply stained nuclei and a high nucleocytic ratio, exhibiting a typical proliferative state; Figure 4 As shown in Part B, the ACC-treated group showed a significant decrease in cell density, the appearance of numerous lightly stained areas, altered cell nucleus morphology, and disruption of the crowded structure of tumor cells, indicating that ACC has an inhibitory effect on melanoma cells.

[0037] (3) Western Blot detection of protein expression

[0038] This embodiment prepares a total protein extract from tumor tissue:

[0039] Extraction buffer containing protease inhibitor, phosphatase inhibitor, and radioimmunoprecipitation lysis buffer was prepared at a volume ratio of 1:1:50. 100 mg of tumor tissue was added to 1 mL of extraction buffer and homogenized for 20 min on ice. The mixture was then centrifuged at 12000 r / min for 30 min at 4°C, and the supernatant was collected. The total protein concentration in the supernatant was determined using the diquinoline formate protein quantification method. An equal volume of protein sample was then subjected to SDS-PAGE electrophoresis for separation, and the proteins in the gel were transferred to a PVDF membrane. Blocking was performed at room temperature for 1.5 h using 5% skim milk as the blocking buffer to prevent non-specific binding. Primary antibodies were then added at the following dilutions: ERK1 / 2 (1:1000), p-ERK1 / 2 (1:1000), and the internal control antibody β-tubulin (1:10000). After incubation with the primary antibodies, the membrane was washed thoroughly, and then the corresponding secondary antibody (1:10000) was added and incubated at 37°C for 2 h. The signal was detected using an ECL chemiluminescence system, and the gray values ​​of the target band were standardized and quantitatively analyzed using ImageJ software. The results are as follows: Figure 5 As shown in section AB: the P-ERK1 / 2 / ERK1 / 2 ratio (*) was significantly downregulated, indicating that ACC inhibits tumor cell proliferation and migration by suppressing the activation of the ERK1 / 2 signaling pathway.

[0040] In summary: CCK-8 assay results showed that 24 h after drug administration, the survival rate of B16 cells decreased with increasing ACC concentration, and the IC50 concentration decreased. 50 The concentration was 35 μM. Western blot results showed that ACC upregulated the expression of cleaved caspase3 protein and the cleaved caspase3 / caspase3 ratio, and downregulated the expression of ERK1 / 2, Bcl2, and MITF proteins and the P-AKT / AKT ratio and P-ERK1 / 2 / ERK1 / 2 ratio. HE staining showed that numerous lightly stained areas appeared in the tumor tissue after ACC treatment, and the tumor cell density was significantly reduced. The above results indicate that the 1-aminocyclopropanecarboxylic acid of the present invention inhibits the proliferation of B16 cells by activating the cysteine-aspartate protease cascade signaling pathway, inhibiting the extracellular regulated protein kinase 1 / 2, phosphorylated protein kinase B, and B lymphoma-2 signaling pathways, activating the apoptosis signaling pathway, and simultaneously inhibiting cell proliferation-related signaling pathways.

[0041] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. Application of 1-Aminocyclopropanecarboxylic acid in the preparation of melanoma treatment drugs.

2. The application of 1-aminocyclopropanecarboxylic acid according to claim 1 in the preparation of melanoma treatment drugs, characterized in that: The 1-aminocyclopropanecarboxylic acid downregulated the expression levels of extracellular signal-regulated kinase 1 / 2, B-cell lymphoma-2 protein, and microphthalmia-associated transcription factor protein, as well as the phosphorylated protein kinase B / protein kinase B ratio, and upregulated the expression level of cleaved cysteine ​​aspartate protease protein 3 and the cleaved cysteine ​​aspartate protease 3 / cysteine ​​aspartate protease 3 ratio.

3. The application of 1-aminocyclopropanecarboxylic acid according to claim 1 in the preparation of melanoma treatment drugs, characterized in that: The 1-aminocyclopropanecarboxylic acid activates the cysteine-aspartate protease cascade signaling pathway and inhibits extracellular regulatory protein kinase 1 / 2, phosphorylated protein kinase B, and B-lymphoma-2 signaling pathways.

4. The use of 1-aminocyclopropanecarboxylic acid according to claim 1 in the preparation of melanoma treatment drugs, characterized in that: The dosage of the 1-aminocyclopropane carboxylic acid is 800 μg / kg.

5. The use of 1-aminocyclopropanecarboxylic acid according to claim 1 in the preparation of melanoma treatment drugs, characterized in that: The drug dosage form is an injection.

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