Use of cryptoxanthin in the preparation of a combination drug for treating lenalidomide-resistant multiple myeloma
By activating the ferroptosis pathway with cryptoxanthin and inhibiting FSP1 protein expression, the treatment challenge of lenalidomide-resistant multiple myeloma has been solved, significantly improving the treatment effect of LEN and providing a new drug option for LEN-resistant MM.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- AFFILIATED HOSPITAL OF NANTONG UNIV
- Filing Date
- 2025-12-03
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies are unable to effectively inhibit lenalidomide-resistant multiple myeloma cells, resulting in poor treatment outcomes and a 5-year survival rate of only 50%. Drug resistance has become a major obstacle.
By using cryptoxanthin to activate the ferroptosis pathway, ferroptosis was induced by inhibiting FSP1 protein expression levels, thereby reducing the activity of lenalidomide-resistant tumor cells.
It significantly inhibits the activity of LEN-resistant MM cells, improves the therapeutic effect of LEN, overcomes drug resistance, and provides a new treatment strategy.
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Figure CN121265571B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a plant extract, cryptoxanthin, in the preparation of drugs for the prevention and treatment of lenalidomide-resistant multiple myeloma. This invention significantly inhibits the activity of drug-resistant myeloma cells by activating the ferroptosis pathway, providing a new drug option for the clinical treatment of drug-resistant multiple myeloma. Background Technology
[0002] Multiple myeloma (MM) is a malignant hematologic malignancy originating from plasma cells, characterized by the abnormal proliferation of clonal plasma cells in the bone marrow and the secretion of large amounts of monoclonal immunoglobulins (M proteins). MM is the second most common hematologic malignancy, accounting for approximately 1% of all cancers. This disease has a significant tendency for bone destruction, mainly through the activation of osteoclasts and the inhibition of osteoblast activity, leading to osteolytic lesions. Clinically, it often manifests as bone pain, pathological fractures, and hypercalcemia. In addition, MM can cause complications such as renal impairment (commonly light chain tubular nephropathy), anemia (due to bone marrow infiltration and decreased erythropoietin), and recurrent infections due to immunodeficiency. Although treatment methods have been continuously improved in recent years, including proteasome inhibitors (such as bortezomib), immunomodulatory drugs (such as lenalidomide / thalidomide), monoclonal antibodies (such as CD38 monoclonal antibody daratumumab), and autologous hematopoietic stem cell transplantation, which have significantly improved patient prognosis, the vast majority of patients eventually relapse due to acquired drug resistance. As an immunomodulatory drug, almost all patients with multiple myeloma (MM) are exposed to lenalidomide (LEN). Although LEN has effective anti-MM activity, some patients eventually relapse and develop LEN resistance. Drug resistance is one of the greatest challenges facing modern oncology and has become a major cause of cancer treatment failure. With the increasing number of patients receiving LEN, LEN resistance has become a significant obstacle for hematologists worldwide. Currently, the disease is still considered incurable, with a 5-year survival rate of only 50%. Therefore, exploring new therapeutic targets and developing new treatment strategies are of significant clinical importance.
[0003] β-cryptoxanthin (βCRY) is an oxygen-containing carotenoid with the chemical structure 3-hydroxy-β-carotene. In nature, it is mainly found in citrus fruits (such as oranges and grapefruits), persimmons, corn, and peppers. As a provitamin A, it can be converted into retinaldehyde in the human body through central cleavage, thereby participating in physiological processes such as visual circulation, epithelial cell differentiation, and immune regulation. Recent studies have revealed that cryptoxanthin, in addition to its traditional nutritional functions, also possesses a wide range of pharmacological activities: ① Antioxidant effect: reducing oxidative stress damage by scavenging free radicals and activating the Nrf2 / ARE pathway; ② Anti-inflammatory effect: inhibiting the NF-κB and MAPK signaling pathways; ③ Bone protection effect: promoting osteoblast differentiation and inhibiting osteoclast formation; ④ Most importantly, its anticancer activity has been demonstrated in various tumor models, including liver cancer, colon cancer, and breast cancer, through mechanisms such as cell cycle regulation, apoptosis induction, and metastasis inhibition. However, the effects and molecular mechanisms of cryptoxanthin on hematologic malignancies, particularly drug-resistant multiple myeloma, have not yet been systematically studied.
[0004] Ferroptosis is characterized by the abnormal accumulation of iron-dependent lipid peroxides (lipid ROS), leading to oxidative damage to the cell membrane system. Unlike traditional apoptosis, ferroptosis is morphologically characterized by unique changes such as reduced / absent mitochondrial cristae, mitochondrial membrane shrinkage, and increased membrane density, and is unaffected by caspase inhibitors. Its molecular mechanisms mainly involve: ① Glutathione metabolism disorder: Inhibition of the systemic Xc-transporter leads to reduced cysteine uptake, resulting in glutathione (GSH) depletion; ② Inhibition of the activity of the key antioxidant enzyme GPX4: Directly or indirectly leading to impaired clearance of phospholipid hydrogen peroxide; ③ Iron metabolism disorder: Free iron catalyzes ROS generation through the Fenton reaction. Studies have shown that ferroptosis exhibits unique therapeutic value in the field of oncology, with many tumor cells resistant to conventional therapies (such as cisplatin-resistant ovarian cancer cells and EGFR-TKI-resistant lung cancer cells) being abnormally sensitive to ferroptosis induction. Therefore, targeting and regulating ferroptosis-related pathways (such as directly targeting GPX4 or regulating iron metabolism-related proteins by inhibiting system Xc- or RSL3 via Erastin) has become an emerging strategy to overcome tumor drug resistance, providing a new perspective for solving the problem of drug resistance in multiple myeloma. Summary of the Invention
[0005] Based on the above situation, the present invention aims to solve the technical problem that lenalidomide-resistant multiple myeloma cells are difficult to inhibit by traditional means, and provides a pharmaceutical composition that activates the ferroptosis pathway through cryptoxanthin, effectively reducing the activity of drug-resistant cells and overcoming the drug resistance problem in clinical treatment.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] One of the objectives of this invention is to provide the application of cryptoxanthin in the preparation of drugs for the prevention and treatment of multiple myeloma.
[0008] A second objective of this invention is to provide the use of a pharmaceutical composition containing cryptoxanthin in the preparation of a drug for the prevention and treatment of multiple myeloma.
[0009] Furthermore, the multiple myeloma includes drug-resistant multiple myeloma.
[0010] Furthermore, the drug-resistant multiple myeloma includes lenalidomide-resistant multiple myeloma.
[0011] Furthermore, the dosage form of the drug is any one of capsules, tablets, microcapsules, injections, suppositories, sprays, or ointments.
[0012] Furthermore, the drug can be administered via injection, oral administration, inhalation spray, or transdermal delivery.
[0013] Furthermore, the drug inhibits lenalidomide-resistant multiple myeloma by activating the ferroptosis pathway.
[0014] Furthermore, the drug induces ferroptosis by inhibiting the expression level of FSP1 protein in lenalidomide-resistant tumors, thereby reducing the activity of lenalidomide-resistant tumor cells and ultimately overcoming lenalidomide-resistant tumors.
[0015] The third objective of this invention is to provide the application of cryptoxanthin in the preparation of FSP1 inhibitors.
[0016] The fourth objective of this invention is to provide the application of cryptoxanthin in the preparation of drugs that induce ferroptosis.
[0017] Compared with the prior art, the present invention has the following beneficial effects:
[0018] (1) The present invention found that βCRY can effectively inhibit the in vivo and in vitro activity of drug-resistant MM.
[0019] (2) The drug-resistant MM selected in this invention is LEN-resistant tumor.
[0020] (3) The present invention found that βCRY significantly inhibits cell activity by inducing ferroptosis in LEN-resistant MM tumor cells.
[0021] (4) The present invention found that βCRY can target and inhibit the protein level of FSP1, and is a novel FSP1 inhibitor.
[0022] (5) The present invention found that βCRY can significantly improve the therapeutic effect of LEN on LEN-resistant MM.
[0023] (6) This invention found that βCRY can overcome the problem of LEN resistance by increasing the level of LEN-induced ferroptosis, providing a theoretical basis for the preparation of drugs for LEN-resistant MM.
[0024] In summary, this invention validated the efficacy and potential mechanism of βCRY in treating LEN-resistant MM through in vitro and in vivo experiments. Specifically, βCRY can induce ferroptosis by inhibiting the expression level of FSP1 protein in LEN-resistant tumors, thereby significantly reducing the activity of LEN-resistant tumor cells and ultimately overcoming LEN tumor resistance. The findings of this invention confirm the therapeutic effect of a plant extract as a novel FSP1 inhibitor in LEN-resistant MM cells, providing a theoretical basis for further clinical use of βCRY to treat LEN-resistant MM. Therefore, βCRY has promising application prospects in the treatment of drug-resistant MM. Attached Figure Description
[0025] Figure 1 IC50 values of LEN-treated MM.1S and MM.1S / LR cells were determined for CCK8.
[0026] Figure 2 IC50 values of LEN-treated H929 and H929 / LR cells were determined for CCK8.
[0027] Figure 3 To detect the apoptosis level of LEN-treated MM1S and MM1S / LR cells by flow cytometry.
[0028] Figure 4 To detect the apoptosis level of LEN-treated H929 and H929 / LR cells by flow cytometry.
[0029] Figure 5 To detect the cell cycle of LEN-treated MM1S and MM1S / LR cells by flow cytometry.
[0030] Figure 6 To detect the cell cycle of LEN-treated H929 and H929 / LR cells by flow cytometry.
[0031] Figure 7 Cell viability of MM1S / LR cells treated with βCRY and LEN was detected using CCK8 assay.
[0032] Figure 8 The cell viability of H929 / LR cells treated with βCRY and LEN was detected by CCK8 assay.
[0033] Figure 9 IC50 values of MM1S / LR cells treated with βCRY and LEN were determined for CCK8.
[0034] Figure 10 IC50 values of H929 / LR cells treated with βCRY and LEN were determined for CCK8.
[0035] Figure 11 The EdU method was used to detect the proliferation ability of MM1S / LR cells treated with βCRY and LEN.
[0036] Figure 12 Statistical analysis of the proliferation capacity of MM1S / LR cells treated with βCRY and LEN.
[0037] Figure 13 The EdU method was used to detect the proliferation ability of H929 / LR cells treated with βCRY and LEN.
[0038] Figure 14 Statistical analysis of the proliferation capacity of H929 / LR cells treated with βCRY and LEN.
[0039] Figure 15 To detect mitochondrial membrane potential in MM1S / LR cells treated with βCRY and LEN using the JC-1 method.
[0040] Figure 16 Statistical analysis of mitochondrial membrane potential in MM1S / LR cells treated with βCRY and LEN.
[0041] Figure 17 To detect the mitochondrial membrane potential of H929 / LR cells treated with βCRY and LEN using the JC-1 method.
[0042] Figure 18 Statistical analysis of mitochondrial membrane potential in H929 / LR cells treated with βCRY and LEN.
[0043] Figure 19 The levels of ferroptosis markers in MM1S / LR cells before and after treatment with βCRY and LEN were detected by ELISA.
[0044] Figure 20 The levels of ferroptosis markers in H929 / LR cells before and after treatment with βCRY and LEN were detected by ELISA.
[0045] Figure 21 To detect ROS levels in MM1S / LR cells treated with a combination of βCRY and LEN by flow cytometry.
[0046] Figure 22 To detect ROS levels in H929 / LR cells by flow cytometry when βCRY and LEN are used in combination.
[0047] Figure 23To detect lipid peroxidation levels in MM1S / LR cells treated with a combination of βCRY and LEN by flow cytometry.
[0048] Figure 24 To detect lipid peroxidation levels in H929 / LR cells when treated with a combination of βCRY and LEN by flow cytometry.
[0049] Figure 25 Western blot analysis was performed to detect the FSP1 protein level in LEN-resistant cells before and after co-treatment with βCRY and LEN.
[0050] Figure 26 This is a schematic diagram illustrating the molecular docking analysis between βCRY and LEN proteins.
[0051] Figure 27 The results show the tumor volume in mice with myeloma.
[0052] Figure 28 The results show the weight of mice with myeloma.
[0053] Figure 29 To detect the expression level of Ki67 in myeloma mouse tumor tissues by immunohistochemistry.
[0054] Figure 30 The expression level of FSP1 in myeloma mouse tumor tissue was detected by Western blotting.
[0055] Figure 31 To detect the levels of ferroptosis markers in tumor tissues using ELISA. Detailed Implementation
[0056] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.
[0057] The technical solution of the present invention will be further described in detail below with reference to the embodiments.
[0058] Example 1
[0059] I. Experimental Methods
[0060] 1. Constructing a MM drug-resistant cell model
[0061] 1×10 per hole 4MM1S cells (Procell, CL-0614) and H929 cells (Procell, CL-0509) were seeded in 96-well plates and incubated for 24 h with different concentrations (0 µM, 1 µM, 2 µM, 5 µM, 10 µM, 15 µM, 20 µM, and 30 µM) of LEN (MCE, HY-A0003). After incubation with CCK8 reagent (Vazyme, A311-01) for 2 h, cell viability was assessed by measuring absorbance at 450 nm using a microplate reader, and IC50 values were calculated. An induction concentration gradient was established based on the IC50 values, and cells were seeded in T25 culture flasks containing the initial drug concentration and incubated at 37°C. If the cells proliferate stably, the drug concentration is gradually increased (in order to 0.5, 1, 2, 5 and 10 µM), that is, the drug concentration is increased by 50%-100% each time it is passaged. The cells are passaged at the target concentration for more than 3 consecutive times and the growth status is stable.
[0062] 2. CCK8 assay to detect the IC50 value of LEN-treated MM cells
[0063] 1×10 per hole 4 Two types of drug-resistant MM cells (MM1S / LR and H929 / LR cells) induced by LEN were seeded in 96-well plates and incubated with different concentrations (0 µM, 1 µM, 2 µM, 5 µM, 10 µM, 15 µM, 20 µM, and 30 µM) of LEN for 24 h. Two h after incubation with CCK8 reagent, the absorbance at 450 nm was measured using a microplate reader to assess cell viability, and the IC50 value was calculated. Normal MM cells were also tested using the same method at the same LEN treatment time and concentration, with cell viability measured using CCK8 reagent, and the calculated IC50 values were compared with those of the drug-resistant cells.
[0064] Similarly, for MM-resistant cells treated with a combination of βCRY (MCE, HY-108059) and LEN, 1 × 10⁻⁶ cells per well were used. 4 Two types of LEN-induced drug-resistant MM cells (MM1S / LR and H929 / LR cells) were seeded in 96-well plates. Cells were treated with different concentrations of cryptoxanthin (10 µM, 20 µM, and 10 µM) for 24 h, followed by incubation with CCK 8 reagent for 2 h. Cell viability was assessed by measuring absorbance at 450 nm using a microplate reader. βCRY1 and βCRY2 refer to different concentrations of cryptoxanthin, 10 µM and 20 µM, respectively. The corresponding IC50 values were calculated. The molecular formula of βCRY is: C 40 H 56O, CAS number: 472-70-8, its structural formula is as follows:
[0065]
[0066] 3. Apoptosis levels in MM cells before and after LEN treatment
[0067] Two parental MM cell lines and two LEN-resistant MM cell lines were treated with different concentrations (0 µM and 5 µM) of LEN for 24 h. The cells were centrifuged, the pellets were washed twice with PBS, and then incubated with the Annexin V-PE / 7-AAD Apoptosis Detection Kit (Vazyme, A213-01) for 15 min. Finally, the apoptosis levels of MM cells before and after LEN treatment were determined by flow cytometry.
[0068] 4. Changes in the cell cycle of MM cells before and after lenalidomide treatment.
[0069] Two parental MM cell lines and two LEN-resistant MM cell lines were treated with different concentrations (0 µM and 5 µM) of LEN for 24 h. After centrifugation, the precipitate was collected, washed twice with PBS, and the cells were gently vortexed while being fixed with pre-chilled 95% ethanol for 2 h. The cells were then centrifuged again, the precipitate was collected, washed twice with PBS, and incubated at 37°C in the dark for 30 min with propidium iodide staining solution (Vazyme, AC101-01). Finally, the proportion of MM cells at different cell cycle stages before and after LEN treatment was determined by flow cytometry.
[0070] 5. EdU detection for cell proliferation
[0071] LEN-resistant cells were distributed at a rate of 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 10 μL in 24-well plates (pre-placed with glass discs) and treated for 24 h under different conditions (DMSO, LEN, βCRY, βCRY+LEN). After treatment, the cells were centrifuged, and 20 μL of LEdU reagent (Vazyme, A413-01) was added to each well. The cells were incubated at 37°C for 2 h. Cells were fixed with 4% paraformaldehyde (MCE, HY-Y0333) for 15 min, permeabilized with 0.5% Triton X-100 (Beyotime, P0096-100ml) for 20 min, and then stained with Click reaction solution containing VaClick 647-EdU. Simultaneously, the slides were mounted with anti-fluorescence quenching mounting medium (containing DAPI) (Beyotime, P0131-5ml). Red fluorescence in each experimental group was detected by fluorescence microscopy, and the percentage of red fluorescent cells in each group was analyzed. The concentration of βCRY was 10 µM, and the concentration of LEN was 5 µM.
[0072] 6. JC-1 detection in cellular mitochondria
[0073] LEN-resistant cells were distributed at a rate of 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 10 μM in 24-well plates and allowed to adhere for 24 hours. Under different treatment conditions (DMSO, LEN, βCRY, βCRY+LEN) for 24 hours, JC-1 reagent (Beyotime, C2006) was added to each well after incubation at 37°C for 2 hours. The fluorescence intensity of red and blue fluorescence in each experimental group was detected by flow cytometry, and the ratio of red to green fluorescence in each group was analyzed. The concentration of βCRY was 10 µM, and the concentration of LEN was 5 µM.
[0074] 7. Detection of ferritin levels
[0075] Two types of LEN-resistant cells were used at a density of 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 μL in 6-well plates and cultured overnight. After adhesion, cells were treated with DMSO, LEN, β-CRY, and β-CRY+LEN for 24 hours, respectively. Cell culture supernatants were collected after treatment, and the levels of ferroptosis-related markers, including MDA (Beyotime, S0131S), 4-HNE (Sangon Biotech, D751041-0048), and GSH (Beyotime, S0053), were detected using ELISA. Simultaneously, cells from the four groups were collected and stained with the DCFH-DA probe (MCE, HY-D0940). Intracellular ROS levels were detected and analyzed using flow cytometry. The levels of lipid peroxidation in cells were detected and analyzed using the BODIPY 581 / 591 C11 probe (MCE, HY-D1301). The concentrations of β-CRY and LEN were 10 µM and 5 µM, respectively.
[0076] 8. Western blot analysis of FSP1 protein levels
[0077] Drug-resistant MM cells were treated with different treatment conditions (DMSO, LEN, βCRY, βCRY+LEN) for 24 hours, and then the cells were collected. The collected cells were mixed with protease and phosphatase inhibitors in RIPA buffer (mixing ratio 100:1), centrifuged, and the supernatant was collected for protein extraction. The protein levels of FSP1 under different treatment conditions were then detected by electrophoresis, transfer, blocking, and antibody coating. For tumor tissue samples, approximately 100 mg of tumor tissue was taken, and total protein extraction and FSP1 protein detection were performed according to the above method. The concentration of βCRY was 10 µM, and the concentration of LEN was 5 µM.
[0078] 9. Establishment of a LEN-resistant multiple myeloma mouse model
[0079] Nude mice aged 6-8 weeks were selected and acclimatized for one week in an SPF-grade animal room at a temperature of 20-24℃ and a humidity of 40%-60% with a 12-hour diurnal cycle. During this period, the mice were randomly divided into four groups of five mice each: a saline group, a LEN treatment group (10 mg / kg), a βCRY treatment group (30 mg / kg), and a combined βCRY (30 mg / kg) and LEN (10 mg / kg) treatment group.
[0080] Establishment of an in vivo tumor model: Balb / c nude mice required for this experiment were purchased from the Experimental Animal Center of Nantong University. The previously constructed drug-resistant cell line H929 / LR was selected and used at a rate of 5 × 10⁻⁶ cells / mL. 5 Cells were subcutaneously inoculated into tumors in nude mice, and the mice were randomly divided into 4 groups after tumor formation. Starting from day 10 after cell infusion, tumor-bearing mice were treated with medication every two days, while the control group received the same volume of physiological saline. Treatment continued until day 22. Mice were euthanized, and tumor tissue was collected from each group for subsequent related indicator testing. Tumor volume detection: Starting from day 10, the tumor volume of mice in each experimental group was measured every two days, and the length and width of the tumor were recorded. The tumor volume was then calculated using the following formula: Tumor volume = length × width × width / 2 mm 3 Statistical analysis was performed. IHC detection of Ki67 in tumor tissue: After the experiment, tumor tissues from each experimental group were collected, and the protein expression of Ki67 in the mouse tumor tissues of each experimental group was detected by immunohistochemistry. Detection of ferroptosis-related indicators in tumor tissue: After the experiment, tumor tissues from each experimental group were collected, ground, centrifuged, and the supernatant was obtained. The relative contents of MDA, 4-HNE, and GSH were detected by ELISA.
[0081] 10. Molecular docking of dendritic oxalis and GPX4 protein
[0082] To assess the binding energy and interaction mode between βCRY and the FSP1 protein, we used Autodock Vina 1.2.2 protein-ligand docking software. The molecular structure of βCRY was obtained from the PubChem compound database (https: / / pubchem.ncbi.nlm.nih.gov / ), and the 3D coordinates of the FSP1 protein were downloaded from the PDB (http: / / www.rcsb.org / ). Molecular docking analysis was performed using Autodock Vina 1.2.2 (http: / / autodock.scripps.edu / ) to visualize the molecular docking model.
[0083] II. Experimental Results
[0084] This invention first compared the effects of LEN treatment and βCRY treatment on cell viability, apoptosis levels, cell cycle, and ferroptosis-related indicators in in vitro experiments using CCK8, flow cytometry, and ELISA on normal MM cells and LEN-resistant MM cells. Then, in vivo experiments verified the antitumor effect of βCRY on LEN-resistant MM cells. Specific experimental results are as follows:
[0085] 1. In in vitro experiments, this invention first constructed two LEN-resistant multiple myeloma (MM) cell models: LEN-resistant MM1S cells (MM1S / LR) and LEN-resistant H929 cells (H929 / LR). Cell viability was detected using CCK8 cells. The LEN treatment concentrations were set at 0 µM, 1 µM, 2 µM, 5 µM, 10 µM, 15 µM, 20 µM, and 30 µM. The experimental results showed that the IC50 value of LEN on normal MM1S cells was 5.61 µM, while the IC50 value on LEN-resistant MM1S cells was 13.98 µM. Figure 1 Similarly, for H929 cells, the IC50 value of LEN against normal H929 cells was 5.99 µM, while the IC50 value against drug-resistant H929 cells was 16.47 µM. Figure 2 The above results all indicate that the IC50 values of drug-resistant MM cells were significantly higher than those of the corresponding parental cell lines, demonstrating the successful construction of drug-resistant cell lines in this invention. To further investigate the drug resistance mechanism, flow cytometry was used to detect the apoptosis levels of normal and drug-resistant MM1S and H929 cells before and after LEN (5 µM) treatment. The results showed that with LEN treatment, the apoptosis rate of MM1S cells was 16.9%, significantly higher than the 5.17% apoptosis rate of MM1S / LR cells. Figure 3 A completely consistent phenomenon was observed in H929 cells, namely, the apoptosis rate of H929 cells was 27.6%, significantly higher than the apoptosis rate of 13.0% in MM1S / LR cells, confirming the resistance of drug-resistant cells to LEN-induced apoptosis. Figure 4 Furthermore, flow cytometry analysis of the cell cycle revealed that, with LEN treatment, the proportion of normal MM1S cells in the G2 / M phase significantly increased (14.3%) compared to MM1S / LR cells (5.8%). Figure 5 This indicates that LEN does not significantly arrest cell cycle in MM1S / LR cells. A similar trend was observed in H929 cells, with 19.4% of normal H929 cells in the G2 / M phase, compared to only 6.4% in H929 / LR cells. Figure 6The above experiments demonstrate that the drug-resistant cell lines MM1S / LR and H929 / LR required by this invention have been successfully constructed, providing conditions for subsequent verification of the therapeutic effect of βCRY and its potential mechanisms.
[0086] 2. Subsequently, to further investigate the therapeutic effect of βCRY, the activity of LEN-resistant MM cells under different treatment conditions was detected using CCK8 assay. Treatment groups included DMSO, LEN (5 µM), βCRY1 (10 µM), βCRY2 (20 µM), βCRY1+LEN, and βCRY2+LEN. For MM1S / LR cells, CCK8 assay results showed that LEN had no significant effect on MM1S / LR cell activity, but βCRY reduced MM1S / LR cell activity and showed a synergistic effect when combined with LEN, indicating that βCRY has a cytotoxic effect on MM1S / LR cells. Figure 7 Similar results were also detected in H929 / LR cells, showing the therapeutic effect of βCRY. Figure 8 Next, eight different concentrations of LEN (0 µM, 1 µM, 2 µM, 5 µM, 10 µM, 15 µM, 20 µM, and 30 µM) and βCRY (10 µM) were used to treat two different drug-resistant MM cell lines for 24 h. The IC50 values of different groups were measured using the CCK8 assay. The results showed that the IC50 values of MM1S / LR and H929 / LR cells were significantly reduced under the action of βCRY. Specifically, the IC50 value of LEN in MM1S / LR was 5.56 µM under the combined treatment with βCRY, and the IC50 value of LEN in H929 / LR was 5.86 µM, which were similar to the IC50 values of the corresponding sensitive cell lines. This preliminarily indicates that βCRY can significantly increase the sensitivity of LEN to inhibit the activity of MM1S / LR and H929 cells. Figure 9-10 Then, the proliferation capacity of cells in different treatment groups was detected by EdU assay. The experimental groups were DMSO, LEN, βCRY, and βCRY+LEN treatment groups. The results showed that LEN had no effect on the proliferation capacity of MM1S / LR cells, but βCRY treatment significantly inhibited the proliferation capacity of MM1S / LR cells. Further investigation revealed that the cell proliferation capacity of the βCRY and LEN combined treatment group was the lowest. Figure 11-12 Similarly, a similar phenomenon was observed in H929 / LR cells. Figure 13-14The above results preliminarily indicate that βCRY can significantly inhibit the proliferation of MM drug-resistant cells and significantly improve the therapeutic efficacy of LEN. Subsequently, the mitochondrial membrane potential of MM drug-resistant cells in various treatments was detected by flow cytometry. The experimental groups were DMSO, LEN, βCRY, and βCRY+LEN treatment groups. The results showed that for MM1S / LR cells, the ratio of JC-1 multimer to JC-1 monomer in the DMSO treatment group was approximately 4.8, in the LEN treatment group it was 6.8, in the βCRY treatment group it was 1.7, and in the βCRY+LEN treatment group it was 1.1. Figure 15-16 For H929 / LR cells, the ratios of JC-1 multimers to JC-1 monomers in the four experimental groups were 5.7, 8.4, 2.2, and 1.8, respectively. Figure 17-18 The above results indicate that as the ratio of JC-1 polymer to JC-1 monomer decreases significantly, mitochondrial membrane potential decreases significantly, and cell function is significantly inhibited.
[0087] 3. To further investigate whether the inhibitory activity of β-CRY on MM cells and MM-resistant cells is related to ferroptosis, ferroptosis-related indicators, including MDA, 4-HNE, and GSH, were detected by ELISA. The experimental setup was the same as above. The results showed that, compared to MM1S / LR and H929 / LR cells, the above-mentioned ferroptosis indicators did not change significantly after LEN treatment alone. However, with β-CRY treatment, we found that MDA and 4-HNE levels increased significantly, while GSH levels decreased significantly. Furthermore, the changes in these indicators were more significant in the β-CRY+LEN treatment group. These results preliminarily suggest that β-CRY can promote LEN drug sensitivity by inducing ferroptosis in MM-resistant cells, ultimately achieving the goal of treating MM resistance. Figures 19-20 Subsequently, we used flow cytometry to detect the intracellular ROS levels in each experimental group. The results showed that in MM-resistant cells, LEN treatment alone did not cause a significant increase in ROS levels. However, β-CRY treatment significantly increased the ROS levels, and further combined treatment with LEN and β-CRY further increased the ROS levels. Figure 21 Similar experimental results were also observed in H929 / LR cells. Figure 22 Lipid peroxidation levels can directly characterize the level of cellular ferroptosis. Flow cytometry analysis of lipid peroxidation levels in individual cells showed that LEN treatment alone did not significantly increase lipid peroxidation levels. However, β-CRY treatment significantly increased lipid peroxidation levels, further increasing LEN-induced lipid peroxidation levels. Figure 23-24Ferroplasm inhibitor FSP1 significantly inhibits ferroptosis. Therefore, we detected the intracellular FSP1 protein level in various cells by Western blotting. The results showed that in MM-resistant cells, LEN treatment alone did not cause a change in FSP1 protein level. However, β-CRY treatment significantly inhibited FSP1 protein level (…). Figure 25 We further analyzed the interaction between βCRY and FSP1 proteins using molecular docking technology. The results showed that βCRY can form strong hydrogen bonds with residues of the FSP1 protein. Figure 26 The above results indicate that βCRY can induce ferroptosis by targeting and inhibiting FSP1 protein levels, thereby achieving the goal of treating LEN-resistant MM.
[0088] 4. Finally, we further validated the efficacy and potential mechanism of β-CRY in treating LEN-resistant MM through in vivo experiments. H929 / LR cells were selected as the research subject, and tumors (approximately 100 mm²) were formed in nude mice. 3 To further verify the antitumor effect of βCRY on LEN-resistant tumors, four groups were set up in the experiment: control group (saline group), LEN treatment group, βCRY treatment group, and βCRY+LEN combined treatment group. First, starting from day 10, we measured changes in tumor volume every two days. The results showed that there was no significant difference in tumor volume between the LEN treatment group and the control group, indicating that LEN was ineffective in treating LEN-resistant MM. However, in the βCRY treatment group, the tumor volume was significantly reduced, showing a significant difference from the control group. Further investigation revealed that the combined treatment group of βCRY and LEN achieved a higher antitumor effect, indicating that βCRY can significantly promote LEN's inhibition of LEN-resistant tumor growth, meaning that βCRY can be used to enhance the treatment sensitivity of LEN-resistant tumors. Figure 27 Subsequently, we examined the weight change curves of mice in each experimental group. The results showed that there was no significant difference in weight among the four groups of mice, indicating that the concentrations of βCRY and LEN used in the experiment did not cause biological toxicity. Figure 28 Next, immunohistochemical experiments were used to further detect the expression of Ki67 in the tumor tissues of mice in each experimental group. The results showed that there was no significant difference in the Ki67 positivity rate between the LEN-treated group and the control group. However, βCRY treatment significantly reduced Ki67 expression, which preliminarily indicates that βCRY can significantly inhibit the growth of LEN-resistant tumors in vivo. Figure 29 Western blot analysis showed that βCRY significantly inhibited FSP1 protein levels, thereby inducing ferroptosis. Figure 30To further investigate the relationship between β-CRY treatment and ferroptosis in LEN-resistant tumors in vivo, we used ELISA to detect the levels of ferroptosis-related markers in tumor tissues of various experimental mice. The results showed no significant changes in ferroptosis markers between the LEN-treated group and the control group, indicating that LEN alone cannot induce ferroptosis to achieve the goal of tumor treatment. Simultaneously, we found that after β-CRY pretreatment, the levels of MDA and 4-HNE in tumor tissues significantly increased, while the level of GSH significantly decreased. This suggests that β-CRY can significantly induce ferroptosis in LEN-resistant tumors, and that combined treatment with LEN can achieve even higher levels of ferroptosis, ultimately resulting in a better anti-tumor therapeutic effect. Figure 31 ).
[0089] In summary, this invention validated the efficacy and potential mechanism of βCRY in treating LEN-resistant MM through in vitro and in vivo experiments. Specifically, βCRY can induce ferroptosis by inhibiting the expression level of FSP1 protein in LEN-resistant tumors, thereby significantly reducing the activity of LEN-resistant tumor cells and ultimately overcoming LEN tumor resistance. The findings of this invention confirm the therapeutic effect of a plant extract as a novel FSP1 inhibitor in LEN-resistant MM cells, providing a theoretical basis for further clinical use of βCRY to treat LEN-resistant MM. Therefore, βCRY has promising application prospects in the treatment of drug-resistant MM.
[0090] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. Use of cryptoxanthin for the preparation of a medicament for the prevention and treatment of multiple myeloma, characterized in that, The multiple myeloma includes lenalidomide-resistant multiple myeloma.
2. Use of a pharmaceutical composition comprising cryptoxanthin for the manufacture of a medicament for the prevention and treatment of multiple myeloma, characterized in that, The multiple myeloma includes lenalidomide-resistant multiple myeloma.
3. The use according to any one of claims 1 to 2, characterized in that, The dosage form of the drug is any one of a capsule, a tablet or an injection.
4. Use according to claim 3, characterized in that, The administration mode of the drug is injection or oral administration.
5. Use according to claim 4, characterized in that, The drug inhibits lenalidomide-resistant multiple myeloma by activating the ferroptosis pathway.
6. Use according to claim 5, characterized in that, The drug induces the occurrence of ferroptosis by inhibiting the expression level of FSP1 protein in lenalidomide-resistant tumors, thereby reducing the activity of lenalidomide-resistant tumor cells, and ultimately achieving the purpose of overcoming lenalidomide tumor drug resistance.