Rock2 inhibitors and their use in the preparation of medicaments for the treatment of cholangiocarcinoma

Abstract

The application belongs to the technical field of cholangiocarcinoma drugs, and particularly relates to a ROCK2 inhibitor and application thereof in preparation of a drug for treating cholangiocarcinoma. The ROCK2 inhibitor is composed of fasudil hydrochloride and magnesium chloride, and the mass ratio of the two is 100:0.1-0.2. The magnesium chloride improves the effect of fasudil hydrochloride on inhibiting the expression of ROCK2, and the magnesium chloride also enhances the effect of fasudil hydrochloride on inhibiting cholangiocarcinoma cells. The drug of the application has a good inhibiting effect on cholangiocarcinoma cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cholangiocarcinoma drugs, and particularly relates to a ROCK2 inhibitor and application thereof in preparation of a drug for treating cholangiocarcinoma. BACKGROUND

[0002] Cholangiocarcinoma (CCA) is a highly invasive cancer originating from the epithelial cells of the bile duct, which is characterized by insidious onset and poor prognosis. Pemigatinib is a drug for cholangiocarcinoma, but it has been reported that cholangiocarcinoma cells (CCA cells) are resistant to Pemigatinib, so the treatment options for cholangiocarcinoma are still insufficient.

[0003] Ferroptosis is a new form of programmed cell death characterized by iron-dependent lipid peroxide accumulation. It is usually associated with an imbalance in the cellular antioxidant defense system, particularly a decrease in glutathione (GSH) and glutathione peroxidase 4 (GPX4) levels. Cancer cells can resist ferroptosis by upregulating the expression of antioxidant enzymes such as GPX4 and iron regulators such as solute carrier family 7 member 11 (SLC7A11), thereby enhancing their survival ability. In addition, dynamin-related protein 1 (Drp1), a small guanosine triphosphatase, belongs to the dynamin family and is closely related to tumor development and drug resistance; Drp1-mediated mitochondrial fission induces cell ferroptosis. The regulation of Drp1 on ferroptosis is crucial for understanding the mechanism of tumor drug resistance, as drug-resistant tumor cells can inhibit ferroptosis by regulating mitochondrial dynamics to escape the cytotoxic effects of chemotherapy drugs.

[0004] Rho-associated coiled-coil protein kinase 2 (ROCK2) is a key molecule in the Rho / ROCK signaling pathway, and it has been reported that ROCK2 can promote the development of chemotherapy resistance, but the role of ROCK2 in cholangiocarcinoma cells is unclear. SUMMARY

[0005] To solve the above technical problems, the application provides a ROCK2 inhibitor and application thereof in preparation of a drug for treating cholangiocarcinoma.

[0006] The application aims to provide a ROCK2 inhibitor for killing cholangiocarcinoma cells, which is composed of Fasudil Hydrochloride and Magnesium Chloride, and the mass ratio of the two is 100:0.1-0.2. Fasudil Hydrochloride is an organic compound with the chemical formula C 14 H 18 ClN3O2S.

[0007] Preferably, the mass ratio of Fasudil Hydrochloride to Magnesium Chloride is 100:0.15.

[0008] The present invention also provides the use of the ROCK2 inhibitor described above for causing the death of cholangiocarcinoma cells in the preparation of a medicament for treating cholangiocarcinoma.

[0009] Preferably, the drug is composed solely of a ROCK2 inhibitor.

[0010] Preferably, the drug further comprises pharmaceutically acceptable excipients to form a liquid formulation.

[0011] Preferably, the excipient is at least one of a cosolvent and a solvent.

[0012] The cosolvent includes at least one of polyethylene glycol and sodium dodecyl sulfate. The polyethylene glycol constitutes 0.1% to 1% of the liquid formulation by mass, more specifically 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, or 1%; the sodium dodecyl sulfate constitutes 0.1% to 0.2% of the liquid formulation by mass, more specifically 0.1%, 0.3%, 0.6%, 0.9%, 1.2%, 1.5%, or 2%; and the solvent is deionized water.

[0013] Preferably, the drug is at least one of the following: (1) a drug that reduces the activity of cholangiocarcinoma cells; (2) a drug that causes ferroptosis in cholangiocarcinoma cells; or (3) a drug that increases the sensitivity to chemotherapy.

[0014] Preferably, the cholangiocarcinoma cells are at least one of human cholangiocarcinoma cells QBC-939, human hepatobiliary cholangiocarcinoma cells RBE, pemetinib-resistant human cholangiocarcinoma cells QBC-939, and pemetinib-resistant human hepatobiliary cholangiocarcinoma cells RBE.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention explores the role of ROCK2 in the development of pemetinib resistance in CCA cells. The study found that ROCK2 expression levels in CCA tissues were significantly higher than in adjacent non-cancerous tissues, indicating a more severe stage of disease progression and lower overall survival. Reducing ROCK2 levels promoted ferroptosis in CCA cells and enhanced sensitivity to pemetinib by decreasing Drp1 expression. Mechanistically, ROCK2 stabilizes Drp1 by competing with UBA52, thereby preventing its degradation. This invention identifies ROCK2 as a promising therapeutic target for improving the therapeutic effect of CCA cells and overcoming drug resistance. Accordingly, this invention develops a ROCK2 inhibitor for inducing cholangiocarcinoma cell death, composed of fasudil hydrochloride and magnesium chloride in a mass ratio of 100:0.1–0.2. Magnesium chloride enhances the inhibitory effect of fasudil hydrochloride on ROCK2 expression, and also enhances the inhibitory effect of fasudil hydrochloride on cholangiocarcinoma cell activity. Attached Figure Description

[0017] Figure 1 This is the result of ROCK2 overexpression in pemetinib-resistant CCA cells.

[0018] In this table, A represents the IC50 values ​​of QBC-939 and R-QBC-939 cells; B represents the IC50 values ​​of RBE and R-RBE cells; C represents the qRT-PCR results of QBC-939, R-QBC-939, RBE, and R-RBE cells (***P<0.001); D represents the Western blot analysis results of ROCK2 in QBC-939, R-QBC-939, RBE, and R-RBE cells; E represents the IHC staining results (***P<0.001); F represents the IHC score results; G represents the qRT-PCR results of CCA tissue and adjacent non-tumor tissue (**P<0.01); H represents the Western blot results of CCA tissue and adjacent non-tumor tissue; and I to K represent the analysis results of perihepatic cholangiocarcinoma (pCCA), distal cholangiocarcinoma (dCCA), and intrahepatic cholangiocarcinoma (iCCA), respectively.

[0019] Figure 2 The results show that ROCK2 inhibits ferroptosis and regulates pemetinib-resistant cholangiocarcinoma cells.

[0020] In this diagram, A represents the staining patterns of QBC-939 and R-QBC-939 cells after the specified treatment; B represents the ROS levels of QBC-939 and R-QBC-939 cells after the specified treatment (***P<0.001); and C represents the Fe levels of QBC-939 and R-QBC-939 cells after the specified treatment. 2+Levels (***P<0.001); D is the protein expression level indicated by Western blot analysis in RBE cells, R-RBE cells, QBC-939 cells, and R-QBC-939 cells; E is the MDA level in QBC-939 cells and R-QBC-939 cells after specified treatment (**P<0.01); F is the GSH / GSSG level in QBC-939 cells and R-QBC-939 cells after specified treatment (**P<0.01); G is the level of R-QBC-939 cells assessed using the CCK-8 assay. Cell viability of cells after specified treatment (**P<0.01, ns, not significant); H represents colony formation assay results; I represents EDU staining results (**P<0.01, ***P<0.001); J is the bar chart corresponding to I; K represents the protein expression level indicated by Western blot in ROCK2 knockdown R-QBC-939 and R-RBE cells; L~O represent ROS, Fe, and other parameters of ROCK2 knockdown R-QBC-939 cells after specified treatment. 2+ The levels of MDA and GSH / GSS were measured (***P<0.001, **P<0.01, *P<0.05); P to R were the results of CCK-8, cell proliferation and EDU assays in ROCK2-knockdown R-QBC-939 cells, respectively (***P<0.001).

[0021] Figure 3 The results show a positive correlation between ROCK2 regulation of dynamin-related protein 1 expression and the expression levels of ROCK2 and Drp1 in CCA tissues.

[0022] In this study, A represents Western blot analysis to detect the expression level of Drp1 in RBE, R-RBE, QBC-939, and R-QBC-939 cells; B represents Western blot analysis of Drp1 knockdown in R-QBC-939 and R-RBE cells. Blot analysis; C shows the cell viability of Drp1-knockdown R-QBC-939 cells detected by CCK-8 assay (**P<0.01); D shows the cell viability of Drp1-knockdown R-RBE cells detected by CCK-8 assay (***P<0.001); E shows the number of Drp1-knockdown R-QBC-939 cells after specified treatment (***P<0.001); F shows the number of Drp1-knockdown R-RBE cells after specified treatment (***P<0.001); G shows the EDU experimental results of Drp1-knockdown R-QBC-939 cells (***P<0.001); H shows the EDU experimental results of Drp1-knockdown R-RBE cells (***P<0.001); I shows the Western blot analysis of Drp1 expression in ROCK2-knockdown R-QBC-939 and R-RBE cells.

[0023] Figure 4 The results show that ROCK2 stabilizes Drp1 expression through competitive binding with UBA52.

[0024] A shows the Western blot results of ROCK2 knockdown of Drp1 expression in R-QBC-939 and R-RBE cells; B shows the expression levels of ROCK2 and Drp1 in R-QBC-939 cells (***P<0.001, ns, not significant); C shows the expression levels of ROCK2 and Drp1 in R-RBE cells (***P<0.001, ns, not significant); D shows the Co-IP and Western blot analysis of ROCK2 knockdown of Ub-Drp1 and ROCK2 in ROCK2-treated R-QBC-939 cells; E shows the co-immunoprecipitation results of ROCK2 and UBA52 or Drp1 and UBA52; F shows the GST dropdown results of ROCK2 and UBA52 or Drp1 and UBA52; G shows the immunofluorescence detection results of ROCK2 and Ub-Drp1. Expression and localization of BA52 in designated cells; H was the expression and localization of Drp1 and UBA52 in designated cells detected by immunofluorescence.

[0025] Figure 5 This is the Western blot analysis result of the ROCK2 inhibitor in Example 2. Detailed Implementation

[0026] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0027] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0028] 1. Materials and Methods

[0029] 1.1 CCA cells, including human cholangiocarcinoma cell line QBC-939 (hereinafter referred to as QBC-939 cells) and human hepatobiliary carcinoma cell line RBE (hereinafter referred to as RBE cells), were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). RBE cells were cultured in modified Durbekoeger medium (DMEM) containing 100 U / mL penicillin / streptomycin (50 U / mL each) and 10% fetal bovine serum. QBC-939 cells were cultured in Los Angeles Memorial Institute 1640 medium (RPMI-1640) containing 100 U / mL penicillin / streptomycin (50 U / mL each) and 10% fetal bovine serum. Both QBC-939 and RBE cells were maintained in an incubator at 37°C and 5% CO2. Detailed information on relevant reagents is provided in Table 1.

[0030] Table 1 Reagent Information

[0031] Reagent name Source Cat# 21645-1-AP Drp1 Proteintech Cat# 12957-1-AP TFR Proteintech Cat# 10084-2-AP SLC7A11 Proteintech Cat# 26864-1-AP GPX4 Proteintech Cat# 67763-1-Ig UBA52 Zenbio Cat# 382648 GST Proteintech Cat# 10000-0-AP β-actin Proteintech Cat# 66009-1-Ig Pemigatinib SelleckChemicals Cat# 1513857-77-6 Protein A / G magnetic beads MCE Cat# HY-K0202 SLC7A11 Proteintech Cat# 26864-1-AP Figure 1 Figure 1 Figure 1

[0032] 1.2 Patients and Specimens: Forty CCA samples and their corresponding adjacent normal tissues were collected from the Department of General Surgery, Jiangxi Cancer Hospital. All cancer tissue specimens were from the CCA-affected area and adjacent tissues. Tissue samples were analyzed using Western blotting and qRT-PCR. For immunohistochemical evaluation, specimens were stored at room temperature in 4% formaldehyde solution.

[0033] 1.3 Immunohistochemical staining (IHC): After dewaxing, paraffin-embedded tissue sections were subjected to antigen retrieval in 10 mmol / L sodium citrate buffer, pH 6.0. The tissue sections were incubated overnight at 4°C with primary antibodies ROCK2 (1:200) and Drp1 (1:200). Subsequently, they were incubated with secondary antibody (Zenbio, China) at room temperature for 30 min. Next, the sections were incubated with horseradish peroxidase-streptavidin conjugate. The peroxidase reaction was developed using 3,3-diaminoaniline (DAB) solution (MCE).

[0034] 1.4 RNA Extraction and qRT-PCR: Total RNA was extracted from cultured CCA cells using TRIZOL reagent (Invitrogen, 15596026), quantified on an Evolution 350 UV-Vis spectrophotometer (Thermo), and reverse transcribed using the PrimeScript kit and gDNA Eraser (Takara, RR047A). qRT-PCR was then performed using TB... qRT-PCR was performed using the Ex Taq Quantitative (Tli RNase H Plus) kit (Takara, RR 420A). The expression levels of ROCK2 and Drp1 were determined by 2... -ΔΔCt The assays were performed using a specific method and standardized with their respective controls. β-actin was used as the control. All measurements were repeated three times. Primers were purchased from Genomeditech (Guangzhou, China), and their sequences are shown in Table 2.

[0035] Table 2 Primer Sequence List 1

[0036]

[0037] 1.5 Western Blot: Total protein was extracted from CCA cells using radioimmunoprecipitation lysis buffer, quantified using biuret reagent, and boiled for 10 min after mixing with protein loading buffer. Proteins were separated by 10 g / 100 mL sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% skim milk at room temperature for 60 min, then incubated overnight at 4 °C with the corresponding primary antibody. The membrane was washed three times with 1×Tris-buffered saline containing Tween 20 for 10 min each time. The PVDF membrane was incubated with the corresponding secondary antibody at room temperature for 1 h, washed three more times with 1×Tris-buffered saline containing Tween 20 for 10 min each time, and finally exposed to an enhanced chemiluminescence reagent for imaging.

[0038] 1.6 Cell transfection: All plasmids were purchased from Genechem. Plasmid and primer sequence information is shown in Tables 3 and 4.

[0039] Table 3 Plasmid Information Table

[0040]

[0041] Table 4 Primer Sequence List II

[0042]

[0043]

[0044] 1.7 Cell viability assay: CCA cells in logarithmic growth phase were inoculated at 3×10⁻⁶ cells / year. 3 Inoculate 100 μL of the solution into 96-well plates at a density of 100 μL and incubate for 1 to 4 days. Add 10 μL of CCK-8 reagent (obtained by diluting 10 μL of the stock solution with 90 μL of serum-free medium) every 24 h and incubate at 37 °C for 2 h. Measure the absorbance at 450 nm using a spectrophotometer.

[0045] 1.8 Colony Formation Assay: CCA cells were seeded into 6-well plates (1500 cells per well) and cultured for two weeks in a humid environment containing 5% carbon dioxide. The cells were then stained with 0.1 g / 100 mL crystal violet for 15 min, followed by washing twice with phosphate-buffered saline (PBS). After allowing the wells to dry, colonies were observed and counted.

[0046] 1.9 5-Ethynyl-2'-deoxyuridine (EDU) Proliferation Assay: Cell proliferation was measured using an EDU proliferation assay kit (Beyotime, C0071S, Shanghai, China). Cells were cultured in 48-well plates for 24 h, then incubated with 50 mM EDU solution for 2 h, and fixed with 4% paraformaldehyde. Subsequently, the cells were permeabilized. They were stained with 0.25% Triton X-100 for 15 min, followed by Azide 488 and Hoechst staining sequentially. Finally, they were observed under a fluorescence microscope.

[0047] 1.10 Immunofluorescence: Differentiated CCA cells were screened at 1.5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of [number] cells / mL in 24-well plates and cultured for 1 day. Afterward, they were fixed with 4% paraformaldehyde for 15 min, followed by washing three times with PBS for 3 min each time. Subsequently, the cells were permeabilized with 0.5 g / 100 mL Triton X-100 at room temperature for 20 min. After washing three times with PBS again, 500 μL of 5% (v / v) goat serum was added to each well for blocking for 30 min to prevent nonspecific binding. The cells were then incubated overnight at 4°C, washed three times with PBS containing Tween 20 using antibodies against ROCK2, Drp1, and UBA52, and incubated with fluorescent secondary antibody for 1 h. Cell nuclei were observed by Hoechst staining (Beyotime, 33342, Shanghai, China), followed by washing three times with PBS containing Tween 20. Images were then taken using a confocal microscope.

[0048] 1.11 Biochemical test: The concentration or activity of MDA (Beyotime, S0131S, Shanghai, China) and GSH / GSSG (Beyotime, S0053, Shanghai, China) in the sample was determined using a commercially available test kit.

[0049] 1.12, ROS and Fe2+ Imaging: Cells were seeded in 6-well slides (5 × 10⁶ cells per well). 5 Cells were cultured for 24 hours. After washing with PBS, the slides were incubated sequentially with PBS containing 2 mM DCFH-DA (Beyotime, S0033S, Shanghai, China) or 1 μM FerroOrange (Tatsuta, F374, Japan) for 20 min. The nuclei were then stained with Hoechst stain (Beyotime, 33342, Shanghai, China). Finally, the cells were observed under a fluorescence microscope.

[0050] 1.13. GST pull-down assay: Purified proteins (GST, GST-UBA52, His-ROCK2, His-Drp1) purchased from AtaGenix (Wuhan, Hubei, China), along with glutathione agarose 4B beads (Sigma), were incubated overnight at 4°C with GST, GST-UBA52, and purified His-ROCK2 or His-Drp1 in incubation buffer (20 mmol / L Tris-hydrochloric acid, pH 7.4.0, 1 wt% Triton X-100), and washed five times with incubation buffer. The proteins were boiled at 95°C for 5 min, eluted with SDS buffer, separated by SDS-PAGE, and then analyzed by Western blotting.

[0051] 1.14. Co-immunoprecipitation: Protein sample preparation was the same as before immunoblotting, except for the addition of loading buffer. For the interactions between UBA52 and ROCK2 / Drp1, and ROCK2 and Drp1, 1 mg of protein sample was incubated overnight at 4°C with 2 μg of primary antibody. Protein A / G magnetic beads (MCE) were added, and incubation was carried out at 4°C for 2 h. Then, the precipitate was collected by centrifugation at 14000 rpm for 1 min at 4°C and washed three times with RIPA lysis buffer. The Protein A / G magnetic beads were washed with 1× loading buffer to remove unbound proteins. The precipitated proteins were analyzed by immunoblotting.

[0052] 1.15. Cell knockout: The UBA52 gene knockout cholangiocarcinoma cell line was constructed by Nanchang Fosun Biotechnology Co., Ltd.

[0053] 1.16 Statistical Analysis: Results are expressed as mean ± standard deviation of three independent trials. Fisher's test was used to assess the relationship between clinicopathological features and ROCK2 and Drp1 expression in patients with cholangiocarcinoma. Statistical analysis and the construction of all figures were performed using SPSS 21.0 and GraphPad Prism 8.0, respectively. P < 0.05 was considered statistically significant.

[0054] 2. Experimental Results

[0055] 2.1. ROCK2 is strongly expressed in pemetinib-resistant cholangiocarcinoma cells, leading to poor prognosis.

[0056] To characterize pemetinib resistance in CCA cells, we established two pemetinib-resistant CCA cell lines, R-RBE cells and R-QBC-939 cells, by long-term exposure of CCA cells to gradually increasing concentrations of pemetinib. Compared with their parental cell lines (RBE cells and QBC-939 cells), R-RBE cells and R-QBC-939 cells showed lower sensitivity to pemetinib. (See attached results). Figure 1 A and B. Furthermore, Western blot and qRT-PCR analyses showed that ROCK2 was highly expressed in pemetinib-resistant R-QBC-939 and R-RBE cells. See [link to relevant documentation]. Figure 1 C and D. This indicates that ROCK2 plays a crucial role in the development of pemetinib resistance in CCA cells. This invention also examined ROCK2 expression in CCA patients. Immunohistochemical (IHC) results showed that, compared to adjacent non-tumor tissue, the frequency and staining intensity of ROCK2-positive cells were higher in CCA tissue (tumor) from patients with poor response to pemetinib. See [link to relevant documentation]. Figure 2 E and F. qRT-PCR and western blot analysis showed that ROCK2 expression levels were significantly increased in CCA tissues compared to adjacent non-tumor tissues. See [link to relevant documentation]. Figure 2 The G and H levels were analyzed in this study. High ROCK2 expression levels were associated with poor prognosis in iCCA, pCCA, and dCCA patients. (See also: G and H). Figure 2 In conclusion, ROCK2 is strongly expressed in pemetinib-resistant cholangiocarcinoma cells, leading to poor prognosis and serving as a prognostic biomarker and therapeutic value for CCA.

[0057] 2.2 ROCK2 inhibits ferroptosis, leading to resistance of cholangiocarcinoma cells to pemetinib.

[0058] This invention used Western blot analysis to determine the expression levels of transferrin receptor (TFR), SLC7A11, and GPX4 in RBE cells, R-RBE cells, QBC-939 cells, and R-QBC-939 cells. SLC7A11 and GPX4 were highly expressed in R-RBE and R-QBC-939 cells, while TFR was expressed at low levels. (See [link to previous section]). Figure 2 D. Reactive oxygen species (ROS) and Fe in parental cells and pemetinib-resistant CCA cells. 2+Analysis of malondialdehyde (MDA) and glutathione disulfide (GSSG) levels showed that ferroptosis was significantly inhibited. Staining images of QBC-939 and R-QBC-939 cells after specified treatment with pemetinib, as well as ROS, Fe... 2+ See MDA and GSH / GSSG levels. Figure 2 A through C, E, and F. This invention investigated the effect of the ferroptosis inducer fasudil hydrochloride on pemetinib-resistant CCA cells. Cell counting kit-8 (CCK-8), colony formation, and EDU assays were used; the results showed that fasudil hydrochloride treatment significantly enhanced the sensitivity of pemetinib-resistant CCA cells to pemetinib. See A through F. This invention investigated the effect of the ferroptosis inducer fasudil hydrochloride on pemetinib-resistant CCA cells. Figure 2 These findings suggest that fasudil hydrochloride not only induces ferroptosis but also plays a crucial role in reversing CCA cell resistance to pemetinib.

[0059] This invention investigated the effect of ROCK2 knockdown on the expression levels of ferroptosis-related proteins in R-RBE and R-QBC-939 cells using Western blotting analysis. ROCK2 knockdown increased the expression level of TFR and significantly decreased the levels of GPX4 and SLC7A11. (See [link to relevant documentation]). Figure 3 To further evaluate the effect of ROCK2 knockdown on ferroptosis, we measured ROS, Fe... 2+ PEmetagatinib treatment significantly reduced ferroptosis in CCA cells of shNC+Pemigatinib (blank control), shROCK2#1+Pemigatinib (ROCK2 knockout group 1), and shROCK2#2+Pemigatinib (ROCK2 knockout group 2). Ferropyplasty was also significantly increased in CCA cells of ROCK2 knockout group 1 and ROCK2 knockout group 2 after pemetagatinib treatment. (See [link to relevant documentation]). Figure 3 L~O. These results indicate that downregulation of ROCK2 enhances ferroptosis in pemetatinib-resistant CCA cells. Knockdown of ROCK2 in R-RBE and R-QBC-939 cells significantly increased the sensitivity of these cells to pemetatinib, see [link to relevant documentation]. Figure 3 P~R.

[0060] In summary, these findings suggest that ROCK2 plays a crucial role in inhibiting ferroptosis, thereby influencing CCA cell resistance to pemetretinib.

[0061] 2.3 ROCK2 regulates Drp1 expression; ROCK2 and Drp1 expression levels are positively correlated in CCA tissues.

[0062] To further investigate the mechanism by which ROCK2 regulates ferroptosis in CCA cells, Drp1 is a key protein that regulates mitochondrial fission, is closely related to ROS generation, and is involved in the process of ferroptosis. Based on these findings, we compared R-RBE and R-QBC-939 cells with their parental cells (RBE and QBC-939) and found that drug-resistant cells exhibited high levels of Drp1, as shown in Figure 3 A of Figure 4 The reduction of Drp1 level did not result in a significant change in the ROCK2 level, indicating that Drp1 is a downstream target of ROCK2, as shown in Figure 4 B of

[0063] Next, we analyzed the relationship between the expression levels of ROCK2 and Drp1. The results of western blot analysis showed that the reduction of ROCK2 level led to a decrease in Drp1 level in R-RBE and R-QBC-939 cells, as shown in Figure 4 I of

[0064] 2.4. ROCK2 stabilizes the expression of Drp1 by competitively binding to UBA52

[0065] ROCK2 interacts with multiple substrates to exert its effects, including inhibiting substrate ubiquitination and degradation. To clarify how ROCK2 regulates the expression of Drp1 in CCA cells, the present invention first examined whether ROCK2 and Drp1 directly interacted. The results of co-immunoprecipitation analysis showed that there was no direct interaction between these two proteins, as shown in Figure 4 A of Figure 4 In addition, the qRT-PCR results showed that the expression of ROCK2 did not affect the mRNA level of Drp1, as shown in Figure 4 B - C of However, the ectopic expression of ROCK2 led to a significant reduction in Drp1 polyubiquitination, while the knockdown of ROCK2 increased Drp1 polyubiquitination, as shown in Figure 5 D of Co-immunoprecipitation analysis showed that there was an interaction between ROCK2 and UBA52, as well as between Drp1 and UBA52, as shown in ​ E of ​ The purified GST-UBA52 complex bound to His-tagged ROCK2 and Drp1 in vitro, as shown in F of The results of confocal microscopy analysis showed that UBA52 co-localized with ROCK2 and Drp1 in the cytoplasm, as shown in

[0066] G - H of The results indicate that ROCK2 regulates the expression of Drp1 through UBA52. 3. Discussion

[0067]

[0067] This invention elucidates the following mechanism of resistance to pemetinib:

[0068] ROCK2 plays a crucial role in the development of pemetinib resistance in ferroptosis (CCA) by regulating ferroptosis, with particular emphasis on the involvement of the UBA52-Drp1 axis. Our results suggest that ROCK2 is not only a biomarker for poor prognosis in CCA but also a therapeutic target for enhancing the efficacy of pemetinib.

[0069] ROCK2 is a key signaling molecule in the Rho / ROCK signaling pathway. This invention observes ROCK2 overexpression in CCA cells, and high ROCK2 expression is associated with poor prognosis in CCA patients. ROCK2 is highly expressed in pemetinib-resistant CCA cell lines, and its expression is positively correlated with pemetinib resistance. Decreased ROCK2 expression promotes ferroptosis in CCA cells, making them more sensitive to pemetinib treatment. Furthermore, the ROCK2 inhibitor fasudil significantly improved the sensitivity of CCA cells to pemetinib. This finding confirms previous research results, indicating that ROCK2 plays a crucial role in inhibiting ferroptosis, thereby influencing drug resistance.

[0070] This invention also found that inhibiting Drp1 expression reversed ferroptosis in pemetinib-resistant CCA cell lines; ROCK2 affects ferroptosis and leads to Drp1-dependent pemetinib resistance in CCA cells. Drp1 is a key factor in ROCK2-mediated inhibition of ferroptosis in CCA cells. Therefore, targeting the ROCK2-Drp1 axis is a therapeutic strategy to overcome chemoresistance in CCA cells.

[0071] Based on the above research results, this invention provides a ROCK2 inhibitor for inducing death in cholangiocarcinoma cells, composed of fasudil hydrochloride and magnesium chloride in a mass ratio of 100:0.1-0.2. The invention includes the following examples.

[0072] Example 1

[0073] The ROCK2 inhibitor used to kill bile duct cancer cells consists of fasudil hydrochloride and magnesium chloride in a mass ratio of 100:0.1.

[0074] Example 2

[0075] The ROCK2 inhibitor, used to induce death in cholangiocarcinoma cells, consists of fasudil hydrochloride and magnesium chloride in a mass ratio of 100:0.15.

[0076] Example 3

[0077] The ROCK2 inhibitor used to kill cholangiocarcinoma cells consists of fasudil hydrochloride and magnesium chloride in a mass ratio of 100:0.2.

[0078] Each hole is 1.5×10 4 CCA cells were seeded at a density of cells / mL in 48-well plates and then mixed with the ROCK2 inhibitor for inducing bile duct cancer cell death as described in Example 2. The final concentration of the ROCK2 inhibitor for inducing bile duct cancer cell death in each well was 10 μL / 100 μL. After culturing for 24 h, ROCK2 expression levels were measured, and the results are shown below. ​ The results showed that the expression levels of ROCK2 were decreased in QBC-939 cells, R-QBC-939 cells, RBE cells, and R-RBE cells.

[0079] Each hole is 1.5×10 4 CCA cells were seeded in 48-well plates at a density of cells / mL and then mixed with the ROCK2 inhibitor for cholangiocarcinoma cell death from Example 2, with a final concentration of 10 μL / 100 μL per well. Cells were cultured for 24 h, then incubated with 50 mM EDU solution for 2 h and fixed with 4% paraformaldehyde. Subsequently, the cells were permeabilized. They were stained with 0.25% Triton X-100 for 15 min, followed by Azide 488 and Hoechst staining. Finally, they were observed under a fluorescence microscope. An equal volume of fasudil hydrochloride was used instead of the ROCK2 inhibitor for cholangiocarcinoma cell death as a control group. The results showed that the EDU staining positivity rates of QBC-939 cells, R-QBC-939 cells, RBE cells, and R-RBE cells were 12%, 18%, 9%, and 12%, respectively, while the EDU staining positivity rates of the control group were 35%, 49%, 31%, and 36%, respectively, indicating that the ROCK2 inhibitor of the present invention has potential for CCA treatment. Furthermore, comparison with the control group showed that magnesium chloride enhanced the inhibitory effect of fasudil hydrochloride on ROCK2 expression, and magnesium chloride also enhanced the inhibitory effect of fasudil hydrochloride on cholangiocarcinoma cells.

[0080] It should be noted that the ROCK2 inhibitors in Examples 1 and 3 also have similar CCA inhibition effects as those in Example 2.

[0081] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0082] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. The use of a ROCK2 inhibitor for inducing the death of cholangiocarcinoma cells in the preparation of a medicament for treating pemetinib-resistant cholangiocarcinoma, characterized in that, The ROCK2 inhibitor used to induce the death of cholangiocarcinoma cells consists of fasudil hydrochloride and magnesium chloride in a mass ratio of 100:0.1~0.

2.

2. The application according to claim 1, characterized in that, The mass ratio of fasudil hydrochloride to magnesium chloride is 100:0.

15.

3. The application according to claim 1, characterized in that, The drug has ROCK2 inhibitors, which are used to kill bile duct cancer cells, as its sole active ingredient.

4. The application according to claim 3, characterized in that, The drug also contains pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The excipient is at least one of a cosolvent and a solvent.

6. The application according to claim 5, characterized in that, The co-solvent includes at least one of polyethylene glycol and sodium dodecyl sulfate; The solvent is deionized water.

7. The application according to claim 1, characterized in that, The cholangiocarcinoma cells are at least one of pemetinib-resistant human cholangiocarcinoma cells QBC-939 and pemetinib-resistant human hepatobiliary carcinoma cells RBE.

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