Application of (-)-Blebbistatin in preparation of medicine for inducing adipose transdifferentiation of epithelial ovarian cancer cells

Abstract

The invention is applicable to the technical field of biological medicines, and provides application of (-)-Blebbistatin in preparation of a medicine for inducing adipose transdifferentiation of epithelial ovarian cancer cells. The (-)-Blebbistatin regulates and controls a mechanical signal to induce the ovarian cancer cells to be transdifferentiated into fat cells; the (-)-Blebbistatin cooperates with the fat induction factor to induce the ovarian cancer cells to be transdifferentiated into the fat cells. The invention provides a thought for driving ovarian cancer cells to transdifferentiate into fat cells and inhibiting in-vitro proliferation and in-vivo tumorigenesis of the ovarian cancer cells by destroying cytoskeletons through drugs and combining fat induction signals. The composition can reduce damage to healthy tissues and fundamentally eliminate malignant characteristics of tumor cells, so that the risk of relapse and metastasis is reduced, and the composition is independent of a tumor immune microenvironment, so that the composition is also effective to immune cold tumors such as ovarian cancer and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the use of (–)-Blebbistatin in the preparation of a drug for inducing fat transdifferentiation of epithelial ovarian cancer cells. Background Art

[0002] Epithelial ovarian cancer (EOC) is one of the most lethal gynecological malignancies. Current treatment strategies for ovarian cancer mainly include surgical resection, chemotherapy, targeted therapy, and immunotherapy, but these methods all have significant limitations: although surgical intervention is a key means of tumor reduction, it is relatively traumatic, and even if R0 resection (complete resection) is achieved, micrometastatic lesions (<1 cm) may still remain; platinum-based chemotherapy combined with paclitaxel is the main adjuvant or neoadjuvant treatment option, but its application is limited by severe side effects and acquired drug resistance after repeated treatment; targeted therapies such as PARP inhibitors, although promising, are only effective for patients with specific molecular subtypes and have limited clinical applicability; in addition, ovarian cancer is often regarded as an immune "cold" tumor due to insufficient immune cell infiltration in its microenvironment, resulting in a low response rate to immune checkpoint inhibitors. Tumor cell fate reprogramming, as a breakthrough therapeutic strategy, differs from traditional methods (such as surgical resection or chemotherapy to directly kill cancer cells, or indirectly through immune activation) in that it aims to transform malignant cells into benign, non-proliferative cell types. This approach not only reduces damage to healthy tissues, but also fundamentally eliminates the malignant characteristics of tumor cells, thereby reducing the risk of recurrence and metastasis. More importantly, this strategy does not rely on the tumor immune microenvironment, so it is also effective for immune "cold" tumors such as ovarian cancer.

[0003] In addition, mechanical signals of cells can affect genome organization and transcriptional regulation. Regulating cell fate through mechanical force provides a potential new strategy for cell transformation without the need for genetic manipulation. In contrast, mechanical regulation directly acts on the physical microenvironment of cells, which can induce cell fate transformation more efficiently and safely, and has greater clinical translation potential. Summary of the Invention

[0004] The purpose of the embodiments of the present invention is to provide the use of (–)-Blebbistatin in the preparation of a drug for inducing fat transdifferentiation of epithelial ovarian cancer cells, aiming to solve the problems raised in the above background technology.

[0005] The embodiment of the present invention is achieved by using (–)-Blebbistatin in the preparation of a drug for inducing adipose transdifferentiation of epithelial ovarian cancer cells.

[0006] Preferably, the (–)-Blebbistatin regulates mechanical signals to induce transdifferentiation of ovarian cancer cells into adipocytes.

[0007] Preferably, the (–)-Blebbistatin synergizes with adipogenic factors to induce ovarian cancer cells to transdifferentiate into adipocytes.

[0008] Another object of an embodiment of the present invention is to provide a drug for inducing fat transdifferentiation of epithelial ovarian cancer cells, wherein the drug comprises (–)-Blebbistatin.

[0009] Preferably, the drug further comprises fat induction factor.

[0010] Preferably, the fat-inducing factor is BMP4 or a PPARγ agonist.

[0011] Preferably, the PPARγ agonist is rosiglitazone.

[0012] The present invention discloses that a cytoskeletal regulator ((–)-Blebbistatin) can cooperate with the fat microenvironment to induce the transdifferentiation of ovarian cancer cells into functional adipocytes. Specifically, (–)-Blebbistatin disrupts the mechanical cytoskeleton, triggering "mechanical-epigenetic remodeling," redistributing heterochromatin and increasing chromatin accessibility, forming an epigenetically permissive state, and promoting the binding of pioneer transcription factors (such as CEBPα / CEBPβ), thereby transforming tumor cells into adipocytes. (–)-Blebbistatin, combined with adipogenic signals (such as BMP4 or PPARγ agonists), establishes a new tumor cell fate intervention strategy of "mechanical regulation, microenvironmental synergy, and epigenetic remodeling";

[0013] This strategy converts ovarian cancer cells into benign, non-proliferative fat cells through tumor cell fate reprogramming rather than cytotoxicity, thereby reducing tumor burden and preventing recurrence of ovarian cancer. It not only reduces damage to healthy tissues, but also fundamentally eliminates the malignant characteristics of tumor cells, thereby reducing the risk of recurrence and metastasis. More importantly, this strategy does not rely on the tumor immune microenvironment, so it is also effective for immune "cold" tumors such as ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A flow chart of the transformation of ovarian cancer cells into fat cells provided by an embodiment of the present invention;

[0015] Figure 2 The experimental results of (–)-Blebbistatin (hereinafter abbreviated as Ble) combined with BMP4 in inducing adipose transdifferentiation of ovarian cancer cells and inhibiting their proliferation in vitro provided in Example 1 of the present invention;

[0016] Figure 3 The experimental results of Example 2 of the present invention show that Ble combined with rosiglitazone induces adipose transdifferentiation of ovarian cancer cells and inhibits proliferation in vitro;

[0017] Figure 4 The experimental results provided in Example 3 of the present invention show that Ble combined with BMP4 or rosiglitazone inhibits the growth of subcutaneous ovarian cancer cells and induces adipose transdifferentiation;

[0018] Figure 5 The experimental results of Example 4 of the present invention showing that Ble inhibits the growth of peritoneal ovarian cancer cells and induces adipose transdifferentiation;

[0019] Figure 6 The experimental results of Example 5 of the present invention, in which the soft matrix combined with BMP4 induced the transformation of mouse fibroblasts into adipocytes;

[0020] Figure 7 This is the analysis result of the molecular mechanism of Ble regulating cell fate transformation provided in Example 6 of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0022] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0023] Example 1. Experiment and analysis of Ble combined with BMP4 to induce adipose transdifferentiation of ovarian cancer cells and inhibit proliferation in vitro:

[0024] Taking a 12-well plate as an example (NEST, 712001), each well was coated with 20 μg / ml human fibronectin solution (MCE, HYP3160) for 12 hours, and the coating solution was discarded before inoculating cells. Human ovarian cancer cell line A2780 (purchased from Zhongqiao Xinzhou) was evenly seeded in each well, with 2×10 cells per well. 4 The cells were cultured with basal culture medium (high glucose DMEM (Gibco, C12430500BT) supplemented with 10% fetal bovine serum (Gibco, 16000-044)) for 24 hours, the culture medium was discarded, and the cells were washed once with PBS.

[0025] The treated A2780 were added to the adipogenic induction medium (N2B27 culture medium: DMEM / F12 (Gibco, 10565018) and Neurobasal (Gibco, 21103-049) 1:1 mixture, and N2 additive (100×, Gibco, 17502048), B27 additive (50×, Gibco, 17504044), 2% bovine serum albumin (Sigma, A8022), β- Mercaptoethanol (1000×, Gibco, 21985023), Glutamax (200×, Gibco, 35050-061), 1 μg / mL insulin (Roche, 11376497001) and double-antibody); 20 μM of the cytoskeleton myosin inhibitor (–)-blebbistatin (MCE, HY-1344) and 20 ng / mL of BMP4 were added, and the cells were cultured for 14 days for identification. The flow chart is shown in the figure. Figure 1 As shown;

[0026] Adipocytes were identified by BODIPY staining using BODIPY staining solution (Invitrogen, D3822). Figure 2 As shown, Figure 2 A shows BODIPY staining of adipocytes induced by A2780. Green lipid droplets can be seen. The lipid droplet area ratio is 7.55%, which is significantly higher than the control group (0.87%). The statistical results are shown in Figure 2 B; At the gene expression level, such as Figure 2 As shown in C, fat-specific genes (FABP4, CEBPA, and PPARG) were significantly upregulated in A2780 treated with Ble+BMP4; Figure 2 As shown in D-E, the proportion of CEBPα-positive cells increased significantly, reaching 74.75%; at the functional level, such as Figure 2 As shown in F-G, the number of lipid protein-positive cells in A2780 treated with Ble+BMP4 increased significantly, accounting for 81.3%; Figure 2 As shown in H, A2780 treated with Ble+BMP4 can undergo lipolysis under the stimulation of the β-adrenergic agonist isoproterenol, indicating that it has the physiological function of mature adipocytes;

[0027] In addition, if Figure 2 As shown in Figure 1, CCK8 analysis showed that Ble treatment significantly inhibited the proliferation of A2780 cells, and this anti-proliferative effect was further confirmed by multiple pathways; Figure 2 As shown in J, EdU incorporation experiments showed that DNA synthesis was significantly reduced, and the proportion of EdU-labeled cells in Ble-treated A2780 cells was reduced (18.18%) compared with the control group (29.23%); Figure 2 As shown in K-L, compared with the control group (97.05%), immunofluorescence staining showed that the proportion of Ki67-positive cells was significantly reduced (68.23%); Figure 2 Figure 2 (M) RNA-seq data showed that cell cycle-related genes such as BRCA1, CCNA2, and CCNB1 were significantly downregulated.

[0028] In summary, Ble can induce the transdifferentiation of ovarian cancer cells into functional adipocytes and inhibit their proliferation, supporting its potential as a cancer treatment strategy.

[0029] Example 2: Experiment and analysis of Ble combined with rosiglitazone to induce adipose transdifferentiation of ovarian cancer cells and inhibit proliferation in vitro:

[0030] For ovarian cancer cells, the same induction scheme as in Example 1 was used, except that BMP4 was replaced with 20 μM of the PPARγ agonist rosiglitazone. The experimental results were as follows: Figure 3 As shown, Figure 3 As shown in A, gene expression analysis showed that in A2780 treated with Ble+rosiglitazone, fat-specific genes (FABP4, CEBPA, and PPARG) were significantly upregulated; the proportion of CEBPα-positive cells increased significantly, with an efficiency of 39.77%. Representative figures and statistical results are shown in Figure 3 B~C; Figure 3 As shown in D, A2780 treated with Ble+rosiglitazone can also undergo lipolysis under the stimulation of isoproterenol; Figure 3 As shown in E, the proliferation detection of A2780 cells treated with Ble+rosiglitazone using CCK8 showed that the cell proliferation was slowed down; Figure 3 As shown in FIG. 5F , EdU incorporation assays showed that DNA synthesis in the cells was significantly reduced (18.85%) compared to the control group (31.9%).

[0031] Example 3: Experiment and analysis of Ble combined with BMP4 or rosiglitazone to inhibit the growth of subcutaneous ovarian cancer cells and induce adipose transdifferentiation:

[0032] Human ovarian cancer cell line A2780 was obtained and digested with 0.25% trypsin (Gibco, 25200072) to prepare a single cell suspension. After counting the cells, 5×10 6 10 cells were mixed with an equal volume of Matrigel (BD Biocoat, 354248) to a final volume of 100 μL (cell suspension: Matrigel = 1:1). The mixed suspension was slowly injected subcutaneously into the scapular region of 6-week-old female BALB / c nude mice using a 1-mL sterile syringe to establish an A2780 cell subcutaneous xenograft tumor model.

[0033] The dosing regimens were as follows: ① Ble + BMP4 group: 3 mg / kg body weight of (–)-Blebbistatin (dissolved in 2% dimethyl sulfoxide, 40% polyethylene glycol 400, 2% Tween 80, and 56% saline) + 10 μg / kg body weight of BMP4 (dissolved in PBS buffer containing trehalose); ② Ble + rosiglitazone group: 3 mg / kg body weight of (–)-Blebbistatin + 20 mg / kg body weight of rosiglitazone (dissolved in the above-mentioned vehicle); ③ Control group: injection of an equal volume of vehicle solution (without drug). Dosing began the day after cell inoculation, using a combination of intratumoral and peritumoral injections using a 30G insulin needle. 20 μL of drug or control were injected daily at multiple sites (50% of the drug volume was injected into the center and edge of the tumor). During the dosing period, the long diameter (L) and short diameter (W) of the tumor were measured daily with a vernier caliper, calculated as volume = 0.5 × L × W. 2 Calculated by the formula; 14 days after administration, the mice were killed by cervical dislocation, and the tumor tissues were completely removed, weighed and recorded;

[0034] The results are as follows Figure 4 As shown, Ble treatment significantly reduced tumor volume and weight, representative figures and statistical figures are shown in Figure 4 A~C; Figure 4 As shown in D, gene expression analysis of subcutaneous tumors showed that adipose markers (FABP4, CEBPA, and PPARG) were significantly upregulated in the Ble+BMP4 group and the Ble+rosiglitazone group; Figure 4 As shown in E, RNA-seq also found that fat-related genes (CD36, LPL, and ACSL5) were significantly upregulated; Figure 4 As shown in F, gene enrichment analysis showed that the upregulated genes were involved in pathways such as adipose differentiation and fatty acid metabolism; Figure 4 As shown in G, Oil Red O staining showed lipid deposition at the tumor edge and injection site; Figure 4 As described by HJ, immunofluorescence staining of paraffin sections showed colocalization of human nuclear antigen and lipoprotein. The colocalization area accounted for 31.69% and 48.02% in the Ble+BMP4 group and the Ble+rosiglitazone group, respectively, indicating that subcutaneously inoculated human ovarian cancer cells A2780 transdifferentiated into adipocytes after Ble treatment.

[0035] Example 4: Experiment and analysis of Ble inhibiting the growth of peritoneal ovarian cancer cells and inducing adipose transdifferentiation:

[0036] Human ovarian cancer cell line SKOV3 stably expressing luciferase was selected and digested to prepare single cell suspension. The concentration was adjusted to 2×10 7 cells / ml, 500 μl of cell suspension (containing 1×107 cells) to establish an ovarian cancer peritoneal metastasis model;

[0037] The dosing regimens were as follows: ① Ble group: 3 mg / kg body weight of (–)-Blebbistatin (the vector formulation was the same as in Example 3); ② control group: an equal volume of vector solution; dosing began the day after cell inoculation by intraperitoneal injection, with 200 μL of drug or control injected daily for 10 consecutive days; on the 5th day, tumor burden was detected by small animal in vivo imaging, as follows: D-luciferin potassium salt (15 mg / mL, 200 μL / animal) was injected intraperitoneally before imaging, and signals were collected 10 minutes later using the IVISLuminaXR system (exposure time was automatically adjusted from 1 second to 1 minute), and the total photon flux (unit: p / s / cm) was quantified using Living Image software. 2 / sr), representative figures and statistical results are shown in Figure 5 A-B, it can be seen that the tumor burden in the Ble group was much lower than that in the control group (P < 0.0001); after the treatment, the mice were killed, the abdominal cavity was opened, the metastatic tumor nodules were collected, and the paraffin-embedded tumors were subjected to immunofluorescence staining. Figure 5 As shown in C to D, the results show the co-localization of human nuclear antigen and lipid protein, and the co-localization area accounts for 70.54%, indicating that ovarian cancer cells SKOV3 inoculated into the peritoneal cavity are transdifferentiated into adipocytes after Ble treatment; Figure 5 As shown in E to F, the proportion of Ki67-positive cells in the human nuclear antigen and lipid protein double-positive cells was significantly reduced, indicating that the tumor cells that underwent adipose transdifferentiation had stopped proliferating. This result was consistent with the reduction in tumor burden in the Ble-treated group shown by in vivo imaging.

[0038] Example 5: Experiment and analysis of soft matrix combined with BMP4 to induce mouse fibroblasts to transform into adipocytes:

[0039] Cytosoft with an elastic modulus of 64 kPa D was used. TM Elastic matrix culture plates (Sigma, 5145) were prepared by adding 40 μg / mL human fibronectin solution to each well of the Cytosoft plate (2 mL / well for 6-well plates). The plates were allowed to stand at room temperature for 1 hour, and the human fibronectin solution was discarded. The plates were gently washed twice with pre-cooled PBS (2 mL / well each time). Mouse embryonic fibroblasts were collected by 0.25% trypsin digestion and counted to adjust the density to 5 × 10 4 After the cells adhered, the culture medium was changed to the same adipogenic medium as in Example 1, and 20 ng / ml of BMP4 was added. The medium was changed every two days. Figure 6 As shown in A; BODIPY staining was performed after 14 days of culture, as shown in Example 1. Figure 6As shown in B-C, the results showed that the accumulation of lipid droplets in the soft matrix group was significantly higher than that in the control group; Figure 6 As shown in D, fat-specific genes (Cebpb, Cebpd, and Pparg) were upregulated; Figure 6 As shown in E–G, Western blotting detected that the expression of adipose transcription factor CEBPα and lipin was much higher than that in the control group and the hard matrix group; Figure 6 As shown in H~I, these cells exhibit the characteristics of functional adipocytes, including the secretion of adiponectin and the ability to decompose fat under the action of isoproterenol, confirming that the cells obtained by regulating fat transdifferentiation through mechanical signals have the physiological functions of adipocytes. In addition, the substitutability of soft matrix culture for drug effects is also verified.

[0040] Example 6: Analysis of the molecular mechanism of Ble regulating cell fate transformation:

[0041] According to the induction method of Example 1, immunofluorescence staining of heterochromatin HP1 was performed on the second day after Ble treatment of mouse embryonic fibroblasts, and its distribution in the nucleus was three-dimensionally reconstructed. The results are as follows: Figure 7 As shown in A and B, in untreated cells, HP1 is mainly located at the top, but in Ble-treated cells, HP1 begins to move to the bottom, indicating heterochromatin organization remodeling; Subsequently, an in situ DNase I-TUNEL assay was performed, that is, the TUNEL method was used to detect double-strand breaks induced by DNase I treatment, as shown in Figure 3. Figure 7 As shown in C, the fluorescence intensity measured by TUNEL can be used as a direct indicator of DNase I sensitivity and thus as a direct indicator of chromatin accessibility; Figure 7 As shown in D-E, compared with the control cells, Ble treatment significantly increased the TUNEL fluorescence intensity, indicating chromatin decondensation; after 24 hours of Ble treatment, as shown in Figure 7 As shown in Figures F to G, the binding of adipose transcription factors (Cebpb and Pparg) to heterochromatin marker protein HP1 and nuclear lamin protein LMNA / C was significantly reduced, indicating that the transcriptional silencing state maintained by the nuclear lamina was released, thereby activating the expression of adipose-related genes; Figure 7 As shown in Figures H to I, epigenetic modifications of adipose transcription factors (Cebpa, Cebpb, Cebpd, and Pparg) were altered, with a decrease in the repressive marker H3K9me3 and an increase in the activating marker H3K27ac. Figure 7 As shown in J, the binding of these transcription factors to RNA polymerase II was significantly enhanced, indicating that their transcriptional activity was enhanced; Figure 7 As shown in Figure 3, its mRNA expression level was also significantly increased, further confirming the activation of the adipose transcriptional network.

[0042] In summary, the data and results indicate that Ble-mediated cytoskeletal disruption changes heterochromatin distribution and increases chromatin accessibility, further enhancing the activity of pioneer transcription factors in an adipogenic environment, thereby promoting the onset of adipose transdifferentiation.

[0043] The present invention analyzes and studies the successful conversion of ovarian cancer cells into adipocytes by regulating mechanical signals, providing a new strategy for the treatment of ovarian cancer. Ovarian cancer cells have the characteristic of metastasizing to the fat-rich peritoneal tissue, which provides a favorable microenvironment for adipose transdifferentiation. Compared with previous methods of achieving adipose transdifferentiation of breast cancer cells through classical signaling pathways, the strategy provided by the present invention is not limited by the epithelial-mesenchymal transition (EMT) process of cells, and the transdifferentiation efficiency can reach approximately 70% in a peritoneal metastasis mouse model. Moreover, by enhancing the pro-adipogenic microenvironment through exogenous cytokines or small molecule compounds, the conversion efficiency is expected to be further improved. This has important therapeutic potential for tumor recurrence caused by residual tumor cells after surgical resection or cells that escape immunotherapy.

[0044] Current ovarian cancer treatments face challenges, including the difficulty of completely eradicating micrometastases through surgery, the susceptibility of chemotherapy to drug resistance, a limited population for targeted therapy, and suppression of the tumor immune microenvironment. However, therapeutic strategies that regulate tumor cell fate and target the fundamental mechanisms of tumor cell survival can reduce the toxicity of traditional chemotherapy and radiotherapy to normal cells, overcome drug resistance, and are independent of the tumor immune microenvironment, making them equally effective against "cold tumors" such as ovarian cancer.

[0045] In the cell fate regulation strategy, the research on transcription factors and epigenetic regulation is more in-depth. Previous studies have used methods such as gene editing, epigenetic regulation and cell reprogramming to change the fate of tumor cells, but these methods have limitations such as off-target effects, non-specific effects and low efficiency. The embodiments of the present invention achieve tumor cell fate transformation through mechanical force regulation, providing a new strategy for cell reprogramming that does not rely on gene manipulation. Drugs that destroy the cytoskeleton can induce heterochromatin redistribution, increase chromatin accessibility, promote the binding of adipogenic transcription factors, and thereby activate the expression of adipogenic-related genes; simulating the mechanical environment of cytoskeleton destruction through soft matrix culture can also induce fat transdifferentiation, confirming the key role of mechanical signals in regulating cell fate. This shows that in addition to transcription factors and epigenetic regulation, mechanical regulation may be the third important dimension that determines cell fate;

[0046] The embodiments of the present invention combine mechanical regulation with microenvironment induction, successfully achieving the fate transformation of tumor cells in the fat-enriched peritoneal cavity, and providing a new malignant cell reprogramming method to overcome treatment resistance and immunosuppressive barriers.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of (–)-Blebbistatin in the preparation of drugs for inducing adipose transdifferentiation of epithelial ovarian cancer cells.

2. The use of (–)-Blebbistatin in the preparation of a drug for inducing adipose transdifferentiation of epithelial ovarian cancer cells according to claim 1, characterized in that: The (–)-Blebbistatin regulates mechanical signals to induce transdifferentiation of ovarian cancer cells into adipocytes.

3. The use of (–)-Blebbistatin in the preparation of a drug for inducing adipose transdifferentiation of epithelial ovarian cancer cells according to claim 1, characterized in that: The (–)-Blebbistatin synergizes with adipogenic factors to induce transdifferentiation of ovarian cancer cells into adipocytes.

4. A drug for inducing adipose transdifferentiation of epithelial ovarian cancer cells, characterized in that: The drugs include (–)-Blebbistatin.

5. The drug for inducing adipose transdifferentiation of epithelial ovarian cancer cells according to claim 4, characterized in that The drug also includes fat induction factor.

6. The drug for inducing adipose transdifferentiation of epithelial ovarian cancer cells according to claim 5, characterized in that The fat induction factor is BMP4 or PPARγ agonist.

7. The drug for inducing fat transdifferentiation of epithelial ovarian cancer cells according to claim 6, characterized in that The PPARγ agonist is rosiglitazone.

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