Application of aloperine as giant bubble type death inducer in treatment of melanoma

Abstract

The invention discloses application of aloperine as a giant vesicular death inducer in treatment of melanoma. It is verified that aloperine can inhibit growth of melanoma model mouse tumors in vivo and inhibit proliferation, clone formation and migration of B16 cells in vitro. Experiments prove that aloperine can also induce oxidative stress disorder of B16 cells to cause macrovesicular death of the B16 cells, meanwhile, lysosome functions are affected, and aloperine has remarkable application prospects in melanoma drugs.

Description

Technical Field

[0001] This invention relates to the field of antitumor drug technology, specifically the application of strychnine as a macrovesicle death inducer in the treatment of melanoma. Background Technology

[0002] Melanoma is a malignant tumor originating from melanocytes in the skin. It is highly aggressive and metastatic, and has the highest mortality rate among skin cancers, accounting for approximately 3% of all cancers. Malignant melanoma ranks third among malignant skin tumors (approximately 6.8%–20%). It commonly affects adults, with a higher incidence in fair-skinned Caucasians and a lower incidence in dark-skinned Asians and Africans; it is extremely rare in children. Some patients have a familial history of multiple occurrences. Malignant melanoma can develop from congenital or acquired benign melanocytic nevi, or from malignant transformation of dysplastic nevi, or it can be newly diagnosed. In recent years, the incidence and mortality rates of malignant melanoma have been increasing annually, and compared to other solid tumors, its age of onset is lower. Besides early surgical excision, there is a lack of specific treatment for malignant melanoma, resulting in a poor prognosis. Therefore, early diagnosis and treatment of malignant melanoma are extremely important.

[0003] DNA damage to melanocytes is the fundamental cause of malignant melanoma. Against a genetic background, long-term ultraviolet radiation, repeated friction, and trauma can lead to DNA breaks, translocations, mutations, or abnormal methylation in melanocytes. This results in uncontrolled melanocyte proliferation and abnormal differentiation, eventually developing into malignant tumor cells. Melanoma is highly malignant, frequently metastasizes, and has a poor prognosis. Therefore, early diagnosis and timely, appropriate treatment are extremely important. Early local surgical resection is the best method to achieve a cure, with the principle of resection being local non-recurrence. Chemotherapy is only suitable for advanced-stage patients, and immunotherapy is still in the experimental stage with uncertain efficacy. The cost of treatment depends on the chosen method. The prognosis of patients with malignant melanoma depends on the stage at diagnosis, and the overall prognosis is poor. Traditional Chinese medicine (TCM) is a traditional treasure of my country, with advantages such as abundant resources, few side effects, and low cost. In recent years, with the development of modern medical technology, individual TCM herbs have attracted increasing research and attention from scholars.

[0004] Macrovesicular pinocytosis is a process in mammalian cells involving the endocytosis of extracellular fluid, proteins, and nutrients, forming macrovesicular pinocytocytes. These pinocytocytes either return to the cell surface or develop into late-stage endosomes and eventually fuse with lysosomes. Macrovesicular death, first discovered in glioblastoma cells ectopically expressing activated Ras, is a caspase-independent cell death pattern that cannot be reversed by caspase inhibitors such as Z-VAD-FMK. In this death process, the macrovesicular pinocytocytes cannot fuse with lysosomes but instead merge in the cytoplasm to form large vacuoles, leading to decreased cellular metabolic activity, cell membrane rupture, and ultimately cell death. Due to cell expansion and plasma membrane disintegration, the morphological characteristics of macrovesicular death are very similar to those of necrosis. Macrovesicular death is regulated by multiple proteins. Activation of members of the Rac1 protein family enhances macrovesicular pinocytosis, while the absence of Arf6-GTP hinders the circulation of macrovesicular pinocytocytes. Simultaneously, significant vacuolation occurred in the cytoplasm due to the abnormal fusion of macrovesicle pinocytosis bodies. Inducing macrovesicle death in tumor cells has attracted particular attention as a cancer treatment strategy. Currently, the main macrovesicle death inducers include HZX-02-059, CX-5011 free base, MOPIPP, and JH530. However, due to safety concerns, the clinical application of these chemically synthesized drugs still requires careful consideration.

[0005] Sophora flavescens has been used in traditional Chinese medicine for hundreds of years. Sophora flavescens alkaloids, molecular formula C 15 H 24 N2, CAS No. 56293-29-9, is a colorless prismatic crystal. Melting point 71–73℃. Optical rotation [α]D+86.59°. Derived from the seed of the legume *Sophora flavescens*. It possesses antibacterial, anti-inflammatory, antiarrhythmic, and prostaglandin cyclooxygenase-promoting and cardioprotective effects. Clinically used to treat acute bacillary dysentery. It holds promise for treating arthritis, nephritis, and lupus erythematosus.

[0006] Therefore, finding a new, non-toxic or minimally harmful giant bubble death inducer has significant potential for anti-tumor effects. Summary of the Invention

[0007] This invention addresses the current shortage of drugs for treating melanoma in clinical practice by providing a new application for the treatment of melanoma using strychnine.

[0008] In view of this, the solution of the present invention is as follows: This invention proposes the use of strychnine or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating melanoma.

[0009] Furthermore, the stigmine is used to induce giant vesicular cell death in melanoma tissue.

[0010] Furthermore, the picrophylline is used for at least one of the following purposes: a) Inhibit melanoma volume; b) Increase the expression level of Rac1 protein in the tissue; c) Induces dysregulation of intracellular oxidative stress in melanoma cells; d) Inhibits lysosomal hydrolysis.

[0011] Furthermore, the drug contains an effective dose of strychnine or a pharmaceutically acceptable salt thereof.

[0012] Furthermore, the drug includes pharmaceutically acceptable excipients.

[0013] Preferably, the pharmaceutically acceptable excipients include at least one of diluents, solvents, emulsifiers, cosolvents, solubilizers, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, antioxidants, antibacterial agents, or buffers.

[0014] Furthermore, the drug is a liquid preparation.

[0015] Furthermore, the route of administration of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or transdermal administration; the drug concentration can be selected or adjusted according to the route of administration.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention verifies that strychnine can inhibit tumor growth in a mouse melanoma model in vivo and inhibit the proliferation, colony formation, and migration of B16 cells in vitro. Experiments have shown that strychnine can also induce oxidative stress disorder in B16 cells, leading to macrovesicular cell death, while simultaneously affecting lysosomal function, demonstrating significant potential for application in melanoma drugs.

[0017] In this invention, picrophylline is derived from the traditional Chinese medicine bitter bean (Sophora flavescens) and is a monomeric component extracted from it. Bitter bean has been used as a traditional Chinese medicine for thousands of years, particularly in northwestern my country, where it is used to treat stomach and skin diseases. Therefore, picrophylline offers greater safety in clinical applications.

[0018] Matrine is widely available and inexpensive, making its use as a clinical drug more economically viable. While the specific mechanisms by which other drugs induce giant bubble death are still unclear, matrine has a history of use spanning thousands of years. Therefore, the potential side effects of matrine use can be better assessed and managed. Attached Figure Description

[0019] Figure 1The results of the effect of strychnine on the growth of melanoma in tumor-bearing mice in Example 1 are shown.

[0020] Figure 2 The results of the killing effect of strychnine on melanoma B16 cells in Example 2 are shown.

[0021] Figure 3 The results of the effect of strychnine on intracellular oxidative stress in melanoma cells in Example 2 are shown.

[0022] Figure 4 This is a schematic diagram illustrating the vacuolation of B16 cells caused by administration of strychnine in Example 3.

[0023] Figure 5 This is a verification result of the effect of strychnine on enhancing melanoma cells in Example 3.

[0024] Figure 6 This is a verification result of the upregulation of mRNA levels of giant bubble death-related genes caused by strychnine in Example 3.

[0025] Figure 7 This is a verification result of the increase in proteins related to giant vesicular cell death caused by strychnine in Example 3.

[0026] Figure 8 This is a verification result of the alteration of lysosomal function in melanoma cells caused by strychnine in Example 3. Detailed Implementation

[0027] The technical solution of the present invention will now be clearly and completely described in conjunction with preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] In one embodiment, strychnine was demonstrated to inhibit tumor growth in a mouse melanoma model in vivo and to inhibit the proliferation, colony formation, and migration of B16 cells in vitro. Experiments also showed that strychnine could induce oxidative stress disorders in B16 cells, leading to macrovesicular cell death, while simultaneously affecting lysosomal function. These effects exhibited a concentration-dependent pattern, thus proposing a novel use of strychnine as a drug in the treatment of melanoma.

[0029] In a preferred embodiment, the drug is picrophylline, or a pharmaceutically acceptable salt thereof. It is understood that the pharmaceutically acceptable salt is used to improve its solubility.

[0030] In a preferred embodiment, the drug is a liquid preparation, and the route of administration includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or transdermal administration.

[0031] In a preferred embodiment, the dosage of strychnine or a pharmaceutically acceptable salt thereof in the drug is 1-10 mg / kg, preferably 2-5 mg / kg.

[0032] Example 1: Effect of sophoridine on melanoma growth in tumor-bearing mice.

[0033] I. Materials and Methods

[0034] 1. Experimental reagents

[0035] Matrine: Purchased from Chengdu Ruifensi Company.

[0036] PBS, DMEM high-glucose medium, antibiotics, and trypsin were purchased from Wuhan Sewell Pharmaceutical Co., Ltd.

[0037] 2. Cells

[0038] B16 cells were purchased from Wuhan Pronosei Company.

[0039] 3. Laboratory animals

[0040] Animal experiments were conducted using 8-week-old male C57BL / 6J mice (18-24g, SPF grade), purchased from Shulaibao (Wuhan) Biotechnology Co., Ltd. All mice were housed in the animal facility of the Animal Experiment Center at Huazhong University of Science and Technology. Housing conditions included a temperature of 22-24℃, humidity of 45-55%, unlimited water and food, and a 12-hour light / dark cycle. Experiments began after one week of acclimatization. All animal experimental procedures involved in this study strictly adhered to and complied with the relevant ethical regulations for animal experiments issued by Huazhong University of Science and Technology.

[0041] 4. Instruments

[0042] 5. Subcutaneous tumor modeling and drug administration

[0043] Twenty-four C57BL / 6J mice purchased from Shulaibao Biotechnology Co., Ltd. were acclimatized for one week in the animal facility of the Animal Experiment Center of Huazhong University of Science and Technology. B16 cell lines were prepared, digested with trypsin, resuspended, washed three times with PBS, and then serum-free DMEM was added to prepare a cell suspension containing the B16 cell line in 50 mL centrifuge tubes at a concentration of 8 × 10⁻⁶. 6 Cells / mL, then containing 8×10 5100 μL of B16 cell suspension was inoculated into the right axillary skin of C57BL / 6J mice to construct a subcutaneous tumor model. After 7 days, 24 mice were randomly divided into 3 groups of 8 mice each. Matrine was prepared with PBS solution to a concentration of 10 mg / mL before administration. The control group received daily administration of water, the low-dose matrine group received 15 mg / kg daily, and the high-dose group received 30 mg / kg daily, all administered via intraperitoneal injection. The mice's weight and the size of the subcutaneous tumor (tumor volume = length × width × width / 2) were measured every other day. After the expected number of days of intervention, the mice were euthanized by cervical dislocation, and the subcutaneous tumor tissue was harvested for subsequent experiments.

[0044] II. Experimental Results

[0045] Depend on Figure 1 As shown in A, there was no significant difference in body weight between the low-dose and high-dose groups of strychnine compared to the control group. Figure 1 B-HE staining showed diffuse pleomorphic malignant cells in the subcutaneous tumor tissue, either singly or in clusters, with significant atypical or enlarged nuclei. The tumor cells were growing vigorously, indicating that the model was successful. Figure 1 C showed that intraperitoneal injection of styrax significantly inhibited the volume of subcutaneous tumors in mice in a dose-dependent manner, indicating that styrax significantly inhibited the growth of subcutaneous tumors in B16 tumor-bearing mice and had a significant anti-tumor effect. Studies have shown that the expression of Rac1 protein is closely related to giant vesicular cell death. Figure 1 The results showed that intraperitoneal injection of strychnine significantly increased the expression of Rac1 protein in tumor tissue, suggesting the possibility that strychnine can induce giant bubble death in tumor tissue.

[0046] Example 2: Killing effect of physostigmine on melanoma B16 cells

[0047] I. Materials and Methods

[0048] 1. Experimental reagents

[0049] CCK-8 assay, ROS detection kit, and lipid peroxidation detection kit were purchased from Beyotime International Co., Ltd.

[0050] Crystal violet dye and DMSO were purchased from Wuhan Sewell Company.

[0051] GPX4 and HMOX1 primers: purchased from China Qingke Company.

[0052] 2. B16 cells

[0053] B16 cell lines were cultured in DMEM medium containing 10% serum at 37°C in a constant temperature incubator containing 5% carbon dioxide. Experiments were performed on B16 cell lines during their logarithmic growth phase.

[0054] 3. Experimental Apparatus

[0055] 4. Detection of cell killing (CCK-8 assay)

[0056] B16 cells in logarithmic growth phase were digested with 0.25% trypsin for 2 min, and the digestion was terminated by adding an equal volume of culture medium containing 10% fetal bovine serum. The cells were centrifuged at 1000 rpm for 3 min. The supernatant was then discarded, and the cells were resuspended in fresh serum-containing culture medium. Cells were seeded at 3000–5000 cells per well in 96-well flat-bottom cell culture plates, with 6 replicates per group, and cultured in an incubator for 24 hours. The culture medium was then discarded, and complete culture medium containing different concentrations of phytic acid (0, 50, 100, 200 μM) was added for intervention stimulation. At the pre-set 24-hour time point, after the cells reached the corresponding time point, the culture medium was discarded, the cells were washed once with sterile PBS, and 100 μL of CCK-8 incubation solution was added to each well. The cells were incubated at 37°C for 1 h. The absorbance was measured at 450 nm using a microplate reader, and the inhibition rate was calculated according to the formula.

[0057] 5. Tumor cell clonogenesis experiment

[0058] B16 cells in logarithmic growth phase were digested with 0.25% trypsin for 2 min, and the digestion was terminated by adding an equal volume of medium containing 10% fetal bovine serum. The cells were centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in fresh serum-containing medium. 500 cells were seeded in 6 cm culture dishes (total volume 5 mL), and then treated with different concentrations of phytic acid (0, 50 μM, 100 μM, and 200 μM phytic acid prepared in PBS). After 24 h, the old medium was removed and replaced with fresh serum-containing medium, and the dishes were cultured in a cell culture incubator for 10 days. The medium was discarded, and the cells were washed three times with PBS, then fixed with 4% paraformaldehyde for 15 min. The paraformaldehyde was discarded, and the cells were washed three times with PBS, then stained with 1 mL of crystal violet dye at room temperature for 15 min. The crystal violet dye was washed off with PBS, and the cells were dried at room temperature before photographing and counting.

[0059] 6. Cell scratch assay

[0060] B16 cells in logarithmic growth phase were digested with 0.25% trypsin for 2 min, and the digestion was terminated by adding an equal volume of culture medium containing 10% fetal bovine serum. The cells were centrifuged at 1000 rpm for 3 min. The supernatant was then discarded, and the cells were resuspended in fresh serum-containing culture medium. 2.5 × 10⁶ cells were then... 5 Cells were seeded into six-well cell culture plates and placed in a cell culture incubator. After the cells had spread to cover the entire bottom of the plate, 200 μL pipette tips were used to streak lines on the bottom of the plates, ensuring even pressure and a generally consistent width of the streaks. After streaking, the cells were washed three times with PBS to remove any detached cells. The PBS was then removed, and 2 mL of serum-free culture medium was added. Different concentration gradients of physostigmine (prepared in PBS at concentrations of 0, 50 μM, 100 μM, and 200 μM) were added for drug intervention stimulation. The plates were then cultured in the cell culture incubator until the corresponding time points, and photographs were taken afterward.

[0061] 7. Detection of cellular reactive oxygen species and lipid peroxidation

[0062] B16 cells in logarithmic growth phase were digested with 0.25% trypsin for 2 min, and the digestion was terminated by adding an equal volume of culture medium containing 10% fetal bovine serum. The cells were centrifuged at 1000 rpm for 3 min. The supernatant was then discarded, and the cells were resuspended in fresh serum-containing culture medium. 2.5 × 10⁶ cells were then... 5 Cells were seeded in six-well cell culture plates and cultured in a cell culture incubator. After reaching the desired cell density, different concentrations of STD (0, 2, 4 mg / mL) were administered for drug stimulation. After 24 h, the culture medium was removed, and the cells were washed three times with PBS. The reactive oxygen species (ROS) detection reagent DCFH-DA or lipid peroxidation detection reagent was then diluted 1:1000 to prepare the ROS fluorescent probe working solution. 1 mL of the working solution was added to each well of the six-well plate, and the plate was incubated in the dark for 30 min. The working solution was then discarded, the cells were washed once with PBS, and then digested with 0.25% trypsin for 2 min. An equal volume of culture medium containing 10% fetal bovine serum was added to terminate the digestion. The cells were collected into new 1.5 mL centrifuge tubes and centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in 1 mL of fresh PBS. Then centrifuge at 1500 rpm for 5 min, discard the supernatant, and repeat this process twice to thoroughly remove any residual fluorescent probe. Finally, remove all PBS from the centrifuge tube using a pipette, add 400 μL of PBS to resuspend the sample, and then transfer it to a flow cytometry tube. Turn on the flow cytometer and analyze the prepared sample.

[0063] 8. Detect the mRNA expression level of the target gene.

[0064] 1) Cell sample extraction: B16 cells in logarithmic growth phase were digested with 0.25% trypsin for 2 min, and then an equal volume of culture medium containing 10% fetal bovine serum was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 3 min. The supernatant was then discarded, and the cells were resuspended in fresh serum-containing culture medium. 2.5 × 10⁶ cells were then collected. 5 Cells were seeded in six-well cell culture plates and cultured in a cell culture incubator. After reaching the desired cell density, different concentrations of physostigmine (0, 50, 100, 200 μM) were administered for drug stimulation. After 24 h, the culture medium was removed, and the cells were washed three times with PBS. 1 mL of Trizol was added to each well, and the cells were lysed on ice for 15 min. The Trizol solution containing lysed cells was then transferred to 1.5 mL EP tubes, and 200 μL of chloroform was added. The mixture was inverted and incubated on ice for 5 min. The EP tubes were then centrifuged at 15000 rpm at 4 °C for 15 min. The EP tubes were gently removed from the centrifuge and placed on a 1.5 mL centrifuge rack. 400 μL of the supernatant was transferred to a new 1.5 mL EP tube, and 400 μL of isopropanol was added to each tube. The mixture was inverted and incubated on ice for 10 min. Centrifuge the 1.5 mL EP tube containing the sample at 15000 rpm for 15 min. After centrifugation, discard the supernatant; a small amount of white precipitate will be visible at the bottom of the tube. Add 1 mL of 75% ethanol pre-chilled at -20 °C. Mix well with a pipette and centrifuge again at 15000 rpm for 15 min at 4 °C. Repeat the ethanol washing step twice. Then, remove any remaining liquid from the tube with a pipette and place the EP tube on ice in a fume hood to dry for 20 min. Once the precipitate at the bottom of the tube is completely dry, add 50 μL of DEPC water to dissolve the precipitate. Finally, measure the concentration of the extracted mRNA using a micro spectrophotometer.

[0065] 2) cDNA synthesis: Based on the concentration of the extracted mRNA on the spectrophotometer obtained in the previous steps, calculate the corresponding volume required to aspirate 1 μg of mRNA. Prepare the mixed reaction system in a 1.5 mL RNase-free centrifuge tube according to the recipe in Table 1-1.

[0066] Table 1-1: Reverse Transcription Reaction System

[0067] Then, mix the liquid with a pipette and set the program in the PCR instrument according to Table 1-2.

[0068] Table 1-2: PCR Program Settings

[0069] The obtained reverse transcription product (RT reaction solution) can be immediately used for subsequent qPCR reactions, or stored at -20°C for up to six months; for long-term storage, it should be stored at -80°C. Repeated freeze-thaw cycles should be avoided for the reverse transcription product cDNA.

[0070] 3) qRT-PCR reaction to detect the expression of related genes

[0071] The primer sequences used in this experiment were all provided by Beijing Qingke Biotechnology Co., Ltd., and their primer sequences are shown in Table 1-3.

[0072] Table 1-3: Primer sequences of related genes

[0073] The reaction mixture was prepared using the 2×Universal SYBR Green Fast qPCR Mix kit, as shown in Table 1-4.

[0074] Table 1-4: qPCR reaction system

[0075] After preparing the reaction mixture, use a pipette to mix it thoroughly. Then place it in the PCR instrument and set the corresponding program according to Table 1-5: Table 1-5: qPCR reaction procedures

[0076] II. Experimental Results

[0077] See the experimental results figure. Figures 2-3 .

[0078] 1. Matrine can kill B16 cells and inhibit the proliferation and migration of B16 melanoma cells.

[0079] CCK8 assays demonstrated that different concentrations of strychnine could kill melanoma B16 cells in a dose-dependent manner. Figure 2 A). Colony formation assays and scratch assays demonstrated that strychnine can simultaneously inhibit the proliferation and migration of melanoma cell lines. Figure 2 B, 2C).

[0080] 2. Sophora flavescens can cause oxidative stress disorders in melanoma cells.

[0081] Flow cytometry analysis of intracellular oxidative stress levels revealed that strychnine significantly increased intracellular reactive oxygen species (ROS) in a concentration-dependent manner. Figure 3 A, 3B). Next, we used flow cytometry to detect cell membrane lipid peroxidation, and the results showed that strychnine significantly increased the level of cell membrane lipid peroxidation in melanoma B16 cells. Figure 3 C, 3D). Simultaneously, we examined the mRNA expression levels of oxidative stress-related molecules GPX4 and HMOX1. The results showed that physostigmine reduced GPX4 mRNA levels and increased HMOX1 mRNA levels in B16 cells. Figure 3 (E, 3F). This demonstrates that strychnine can cause dysregulation of oxidative stress in melanoma cells.

[0082] Example 3: Matrine can induce macrovesicular death in melanoma cells.

[0083] I. Materials and Methods

[0084] 1. Experimental reagents

[0085] Dextran, Luciferox, and DQ-BSA were purchased from MCE.

[0086] Caveolin-1, TFRC, EGFR, Rac1, and Rab7 mRNA primers: purchased from China Qingke Company.

[0087] Caveolin-1, TFRC, EGFR, Rac1, and Rab7 protein antibodies: purchased from Abcam.

[0088] Lysosomal receptor dye: purchased from Yisheng Biotechnology Co., Ltd., China.

[0089] 2. B16 cells

[0090] B16 cell lines were cultured in DMEM medium containing 10% serum at 37°C in a constant temperature incubator containing 5% carbon dioxide. Experiments were performed on B16 cell lines during their logarithmic growth phase.

[0091] 3. Experimental Apparatus

[0092] 4. Cell morphology observation

[0093] B16 cells in logarithmic growth phase were digested with 0.25% trypsin for 2 min, and the digestion was terminated by adding an equal volume of culture medium containing 10% fetal bovine serum. The cells were centrifuged at 1000 rpm for 3 min. The supernatant was then discarded, and the cells were resuspended in fresh serum-containing medium at a density of 2 × 10⁶ cells / well.5 Cells were seeded into 6-well cell culture plates and cultured in an incubator. After 24 hours, the culture medium was discarded, and then complete culture medium containing phytic acid was added. After culturing for another 24 hours, images of the morphological changes of the cells were obtained using an optical microscope.

[0094] 5. Dextran, luciferin, lysosomal receptors, and DQ-BSA immunofluorescence detection

[0095] B16 cells in logarithmic growth phase were digested with 0.25% trypsin, and digestion was terminated by adding an equal volume of medium containing 10% fetal bovine serum after 2 min. The cells were centrifuged at 1000 rpm for 3 min. The supernatant was then discarded, and the cells were resuspended in fresh serum-containing medium. 5000 cells were seeded in confocal culture dishes and cultured in a cell culture incubator. After reaching the desired cell density, the cells were stimulated with picrophylline. After 24 hours, the medium was removed, and the cells were washed three times with PBS. Then, 1 mL of complete medium containing dextran, luciferin, lysosomal receptor, or DQ-BSA (all 1:1000) was added to each well, and the cells were incubated in the dark for 45 min. The working solution was then discarded, the cells were washed once with PBS, and the cells were observed and photographed under a confocal microscope.

[0096] 6. Detection of mRNA expression (same as in Example 2)

[0097] The primer sequences used were all provided by Beijing Qingke Biotechnology Co., Ltd., and the primer sequences are shown in Table 2-1 below.

[0098] Table 2-1: Primer sequences of related genes

[0099] 7. Detection of protein expression

[0100] 1) Total protein extraction: B16 cells in logarithmic growth phase were digested with 0.25% trypsin for 2 min, and the digestion was terminated by adding an equal volume of culture medium containing 10% fetal bovine serum. The cells were centrifuged at 1000 rpm for 3 min. The supernatant was then discarded, and the cells were resuspended in fresh serum-containing culture medium. 2.5 × 10⁶ cells were then... 5Cells were seeded into six-well cell culture plates and cultured in a cell culture incubator. After reaching the desired cell density, the cells were stimulated with phytic acid. After 24 h, the old culture medium was discarded, and the cells were washed twice with PBS. Then, approximately 160 μL of protein lysis buffer was added to each well (10 μL of PMSF, 10 μL of phosphatase inhibitor A, and 10 μL of phosphatase inhibitor B were added to each 1 mL of RIPA lysis buffer). The plate was shaken to ensure that all cells were fully submerged in the lysis buffer, and then placed on ice for 30 min for lysis. Afterward, the lysis buffer was transferred to 1.5 mL centrifuge tubes.

[0101] 2) Protein denaturation of samples: Add 1 / 4 volume of 5×SDS-PAGE Sample Loading Buffer to a 1.5mL centrifuge tube containing the protein sample. Mix thoroughly on a vortex mixer, heat in a 100℃ metal bath for 15 minutes, then remove and store at -20℃ for later use. For long-term storage, store in a -80℃ freezer.

[0102] 3) SDS-PAGE gel preparation: The concentrations of the SDS-PAGE gels were selected according to the molecular weight of the target protein, namely 10%, 12%, and 15%. Each set of gel plates contained 7.5 mL of separating gel, and the formulation of the separating gel is shown in Table 2-2.

[0103] Table 2-2: Preparation Table for Glue Release (10mL System)

[0104] Add the reagents listed in the table above to a 50 mL centrifuge tube in the order listed. After adding TEMED, invert the tube thoroughly to mix well. Then, add 7.5 mL of the separating gel between the assembled glass plates. Add 1 mL of anhydrous ethanol to flatten any foam on the surface of the separating gel. Let the tube stand at room temperature for 1 hour to allow the separating gel to solidify. While waiting, prepare a 4% stacking gel according to Table 2-3.

[0105] Table 2-3: Preparation of Stacking Gel (5mL system)

[0106] After 1 hour, once the lower separating gel has solidified, discard the upper anhydrous ethanol and pour it clean. Add each component in the order shown in the table above, and finally add TEMED. Invert the container to mix the system thoroughly. Then, use a 1 mL pipette to add the prepared stacking gel to the top of the separating gel, and slowly insert the comb, being careful not to leave any air bubbles. Let it stand for about 45 minutes until the gel is completely solidified.

[0107] 4) Sample Loading: Rinse the prepared SDS-PAGE gel thoroughly under running water, then fix the gel onto the holder. Fill both glass plates with electrophoresis buffer, then remove the comb and add the appropriate volume of protein sample to each well using a 10 μL pipette. Simultaneously, add protein markers to both sides. After loading the samples, place the gel holder into the electrophoresis tank and fill the tank with the appropriate volume of electrophoresis buffer. Prepare the electrophoresis buffer according to Table 2-4.

[0108] Table 2-4: Electrophoresis Solution Formulation

[0109] 5) Electrophoresis: After adding the sample, close the lid of the electrophoresis tank, turn on the power, set the total current to 20mA for each gel, and then perform constant current electrophoresis. The electrophoresis time is generally 1.5h to 2h.

[0110] 6) Transfer: After electrophoresis, wash the glass plate thoroughly and then immerse it in pre-cooled transfer buffer. The transfer buffer is prepared according to Table 2-5.

[0111] Table 2-5: Electro-conversion fluid formulation

[0112] Cut off the stacking gel, measure the length of the separating gel with a ruler, and then cut a PVDF membrane of the corresponding size. Pre-excite the PVDF membrane in methanol. Place the white transfer clamp on top and the black clamp on the bottom. Then, place the sponge, transfer filter paper, gel strip, PVDF membrane, transfer filter paper, and sponge in sequence on the black side. Finally, clamp the white and black sides tightly, ensuring no air bubbles are left. Transfer the transfer clamp into the transfer tank and replenish the transfer buffer. Then, set the appropriate transfer time and transfer current according to the molecular size of the target protein.

[0113] 7) Blocking: After the transfer is complete, put the PVDF membrane into the prepared blocking solution (5% skim milk powder, prepared with TBST) and block it on a shaker for about 2 hours.

[0114] 8) Primary antibody incubation: After blocking, discard the blocking buffer, then add an appropriate amount of TBST and wash for 10 min, repeating 3 times. Prepare diluted primary antibody according to the ratio in the antibody instructions, then place the PVDF membrane in the diluted primary antibody and incubate overnight on a shaker at 4°C. On the second day, remove the PVDF membrane and wash it three times with TBST at room temperature, 10 min each time.

[0115] 10) Secondary antibody incubation: Dilute the horseradish peroxidase-labeled secondary antibody corresponding to the primary antibody species with TBST at a ratio of 1:4000 to prepare the secondary antibody reaction solution. Then, place the PVDF membrane into the secondary antibody and incubate at room temperature for 1.5 h. Then, remove the PVDF membrane from the secondary antibody and wash it three times with TBST at room temperature for 10 min each time.

[0116] 11) WB strip exposure: Immerse the PVDF film in ECL ultrasensitive exposure solution prepared in a 1:1 ratio. After immersion for 10 seconds, place it in an imager for exposure to obtain a WB image.

[0117] II. Experimental results are shown below. Figures 4-8 .

[0118] 1. Matrine causes vacuoles to appear in melanoma cells.

[0119] Next, we investigated the specific mechanism by which picrophylline kills melanoma B16 cells. After administering picrophylline (200 μM picrophylline prepared in PBS solution), we observed the cells using an optical microscope. The results showed that picrophylline administration caused obvious, visible vacuoles to appear within B16 cells. Figure 4 This is very similar to a newly discovered cell death mechanism—macrovesicular death. Therefore, we hypothesize that styrax exerts its killing effect by inducing macrovesicular death in B16 cells.

[0120] 1. Matrine enhances endocytosis in melanoma cells.

[0121] To verify whether strychnine exerts its therapeutic effect on melanoma through macrovesicle death, we used immunofluorescence to detect it. Dextran and luciferin are fluorescent molecules used to detect the intensity of macrovesicle death in cells. The detection results after strychnine administration are as follows: Figure 5 As shown, Figure 5 A and 5B represent the effects of strychnine on the ability of B16 cells to take up dextran. Figure 5 C Figure 5 D represents the effect of strychnine on luciferin uptake in B16 cells. We found that the fluorescence intensity of luciferin and dextran in the treated group was significantly higher than that in the control group. This indicates that strychnine can significantly promote macrovesicle engulfing in tumor cells, and excessive macrovesicle engulfing is one of the causes of macrovesicle death.

[0122] 3. Matrine upregulates the mRNA levels of genes associated with giant bubble cell death.

[0123] Subsequently, we examined the alterations in the transcriptional levels of giant vesicular death-related genes in melanoma cells after administration of styraxine. The results of qPCR analysis are as follows: Figure 6 As shown, Figure 6 A, 6B, Figure 6 C Figure 6 D、 Figure 6 E corresponds to the fact that the mRNA levels of Caveolin1, Rac1, TFRC, EGFR and Rab7 all increased significantly after administration of styraxine, indicating the occurrence of giant vesicle death.

[0124] 4. Matrine leads to an increase in proteins associated with giant bubble cell death.

[0125] mRNA levels predicted an increase in the transcriptional levels of related genes. Next, we investigated whether these changes remained consistent at the protein level. We used Western blotting (WB) to detect the expression of Rac1, Rab7, EGFR, TFRC, and Caveolin-1 proteins to observe changes in protein levels. The results showed that after administration of styraxine, proteins related to macrovesicular death significantly increased, indicating that styraxine can induce macrovesicular death in melanoma cells. Figure 7 ).

[0126] 5. Sophora flavescens causes alterations in lysosomal function within melanoma cells.

[0127] The occurrence of giant vesicular cell death is accompanied by the inhibition of endosome and lysosomal fusion. Therefore, we next examined the related functions of lysosomes. We used a lysosomal pH-sensing dye to examine the effect of phytidine on lysosomal pH. The results showed that phytidine did not affect the pH of lysosomes in B16 cells, which contrasted with the significant effect of Baf A1 on lysosomal pH. Figure 8 A, 8B). Subsequently, we used DQ-BSA dye to detect the lysosomal hydrolytic capacity. The results showed that the fluorescence intensity of DQ-BSA decreased significantly after administration of styraxine, indicating that the lysosomal hydrolytic capacity of B16 cells was inhibited. Figure 8 C, 8D).

[0128] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The use of strychnine or its pharmaceutically acceptable salts in the preparation of drugs for treating melanoma.

2. The application according to claim 1, characterized in that, The choledochine is used to induce vesicular cell death in melanoma tissue.

3. The application according to claim 1, characterized in that, The choline is used for at least one of the following purposes: a) Inhibit melanoma volume; b) Increase the expression level of Rac1 protein in the tissue; c) Induces dysregulation of intracellular oxidative stress in melanoma cells; d) Inhibits lysosomal hydrolysis.

4. The application according to claim 1, characterized in that, The drug contains an effective dose of strychnine or a pharmaceutically acceptable salt thereof.

5. The application according to claim 1, characterized in that, The drug includes pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable excipients include at least one of the following: diluents, solvents, emulsifiers, cosolvents, solubilizers, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, antioxidants, antibacterial agents, or buffers.

7. The application according to claim 1, characterized in that, The drug is a liquid preparation.

8. The application according to claim 1, characterized in that, The route of administration of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or transdermal administration.

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