ROCK2 inhibitor and application thereof in preparation of medicine for treating bile duct cancer

Abstract

The invention belongs to the technical field of cholangiocarcinoma medicines, and particularly relates to a ROCK2 inhibitor and application thereof in preparation of a medicine for treating cholangiocarcinoma. The ROCK2 inhibitor is composed of fasudil hydrochloride and magnesium chloride, and the mass ratio of fasudil hydrochloride to magnesium chloride is 100: (0.1-0.2). The magnesium chloride improves the ROCK2 expression inhibition effect of the fasudil hydrochloride, and also enhances the bile duct cancer cell inhibition effect of the fasudil hydrochloride. The medicine disclosed by the invention has a good inhibition effect on bile duct cancer cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of drugs for cholangiocarcinoma, and particularly relates to a ROCK2 inhibitor and its application in the preparation of drugs for treating cholangiocarcinoma. Background Art

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

[0003] Ferroptosis is a new form of programmed cell death, which is characterized by the accumulation of iron-dependent lipid peroxides. It is usually related to the imbalance of the cellular antioxidant defense system, especially the reduction of 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 regulatory proteins 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 belonging to the dynamin family, is closely related to tumor development and drug tolerance; Drp1-mediated mitochondrial fission induces cell ferroptosis. The regulation of ferroptosis by Drp1 is crucial for understanding the mechanism of tumor drug tolerance, because drug-resistant tumor cells can inhibit ferroptosis by regulating mitochondrial dynamics, thereby escaping the cytotoxic effects of chemotherapeutic drugs.

[0004] Rho-associated coiled-coil protein kinase 2 (ROCK2) is a key molecule in the Rho / ROCK signaling pathway. It has been reported that ROCK2 can promote the development of chemoresistance, but the effect of ROCK2 on cholangiocarcinoma cells is not clear. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a ROCK2 inhibitor and its application in the preparation of drugs for treating cholangiocarcinoma.

[0006] The object of the present invention is to provide a ROCK2 inhibitor for inducing the death of 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 an application of the ROCK2 inhibitor for inducing death of cholangiocarcinoma cells in the preparation of a medicament for treating cholangiocarcinoma.

[0009] Preferably, the medicament takes the ROCK2 inhibitor as the sole component.

[0010] Preferably, the medicament further contains pharmaceutically acceptable excipients to prepare a liquid preparation.

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

[0012] The solubilizer includes at least one of polyethylene glycol and sodium dodecyl sulfate. Among them, the mass percentage of polyethylene glycol in the liquid preparation is 0.1% - 1%, more specifically 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%; the mass percentage of sodium dodecyl sulfate in the liquid preparation is 0.1% - 0.2%, more specifically 0.1%, 0.3%, 0.6%, 0.9%, 1.2%, 1.5%, 2%; the solvent is deionized water.

[0013] Preferably, the medicament is at least one of the following: (1) a medicament for reducing the activity of cholangiocarcinoma cells; (2) a medicament for inducing ferroptosis of cholangiocarcinoma cells; (3) a medicament for enhancing chemosensitivity.

[0014] Preferably, the cholangiocarcinoma cells are at least one of human cholangiocarcinoma cells QBC - 939, human hilar cholangiocarcinoma cells RBE, human cholangiocarcinoma cells QBC - 939 with pemigatinib resistance, and human hilar cholangiocarcinoma cells RBE with pemigatinib resistance.

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

[0016] The present invention explores the role of ROCK2 in the development of pemigatinib resistance in CCA cells. The study found that the expression level of ROCK2 in CCA tissues was significantly higher than that in adjacent non - cancerous tissues, indicating a more severe disease progression stage and a lower overall survival rate. Reducing the level of ROCK2 can promote ferroptosis of CCA cells and enhance the sensitivity to pemigatinib by reducing the expression of Drp1. Mechanistically, ROCK2 stabilizes Drp1 by competing with UBA52, thus preventing its degradation. The present invention found that ROCK2 is a promising therapeutic target for improving the treatment effect of CCA cells and overcoming drug resistance. Accordingly, the present invention developed an ROCK2 inhibitor for inducing death of cholangiocarcinoma cells, which is composed of fasudil hydrochloride and magnesium chloride, and the mass ratio of the two is 100:0.1 - 0.2. Magnesium chloride enhances the effect of fasudil hydrochloride in inhibiting ROCK2 expression and also enhances the effect of fasudil hydrochloride in inhibiting the activity of cholangiocarcinoma cells. Description of the Drawings

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

[0018] Among them, A is the IC50 values of QBC-939 cells and R-QBC-939 cells; B is the IC50 values of RBE cells and R-RBE cells; C is the qRT-PCR results of QBC-939, R-QBC-939, RBE and R-RBE (***P<0.001); D is the Western blot analysis results of ROCK2 in QBC-939 cells, R-QBC-939 cells, RBE cells and R-RBE cells; E is the IHC staining results (***P<0.001); F is the IHC Score results; G is the results of qRT-PCR detection of CCA tissues and adjacent non-tumor tissues (**P<0.01); H is the results of western blot detection of CCA tissues and adjacent non-tumor tissues; I-K are the analysis results of perihilar cholangiocarcinoma (pCCA), distal cholangiocarcinoma (dCCA) and intrahepatic cholangiocarcinoma (iCCA) in sequence.

[0019] Figure 2 It is the related result that ROCK2 inhibits ferroptosis and regulates pemigatinib-resistant cholangiocarcinoma cells.

[0020] Among them, A is the staining map of QBC-939 cells and R-QBC-939 cells after specified treatment; B is the ROS levels of QBC-939 cells and R-QBC-939 cells after specified treatment (***P<0.001); C is the Fe of QBC-939 cells and R-QBC-939 cells after specified treatment 2+Horizontal (***P < 0.001); D was Western blot analysis for detecting the indicated protein expression levels in RBE cells, R-RBE cells, QBC-939 cells, and R-QBC-939 cells; E was the MDA levels in QBC-939 cells and R-QBC-939 cells after the indicated treatment (**P < 0.01); F was the GSH / GSSG levels in QBC-939 cells and R-QBC-939 cells after the indicated treatment (**P < 0.01); G was the cell viability of R-QBC-939 cells after the indicated treatment evaluated by the CCK-8 assay (**P < 0.01, ns, not significant); H was the result of the colony formation assay; I was the result of the EDU assay staining (**P < 0.01, ***P < 0.001); J was the bar graph corresponding to I; K was the Western blot detection of the indicated protein expression levels in ROCK2-knockdown R-QBC-939 and R-RBE cells; L to O were the levels of ROS, Fe 2+ , MDA, and GSH / GSS in ROCK2-knockdown R-QBC-939 cells after the indicated treatment (***P < 0.001, **P < 0.01, *P < 0.05); P to R were the results of the CCK-8, cell proliferation, and EDU assays in ROCK2-knockdown R-QBC-939 cells (***P < 0.001).

[0021] Figure 3 were the related results that ROCK2 regulated the expression of dynamin-related protein 1 and the expression levels of ROCK2 and Drp1 were positively correlated in CCA tissues;

[0022] Among them, A was to perform Western blot analysis to detect the expression levels of Drp1 in RBE, R-RBE, QBC-939, and R-QBC-939 cells; B was the Western blot analysis of Drp1 expression knockdown by Drp1 in R-QBC-939 and R-RBE cells; C was to detect cell viability using the CCK-8 method in Drp1-knockdown R-QBC-939 cells (**P<0.01); D was to detect cell viability using the CCK-8 method in Drp1-knockdown R-RBE cells (***P<0.001); E was the cell number of Drp1-knockdown R-QBC-939 cells after specified treatment (***P<0.001); F was the cell number of Drp1-knockdown R-RBE cells after specified treatment (***P<0.001); G was the EDU experimental results of Drp1-knockdown R-QBC-939 cells (***P<0.001); H was the EDU experimental results of Drp1-knockdown R-RBE cells (***P<0.001); I was the Western blot analysis of Drp1 expression knockdown by ROCK2 in R-QBC-939 and R-RBE cells.

[0023] Figure 4 are the related results that ROCK2 stabilizes the expression of Drp1 by competitively binding with UBA52

[0024] A is the Western blot results of Drp1 expression knockdown by ROCK2 in R-QBC-939 and R-RBE cells; B is the expression levels of ROCK2 and Drp1 in R-QBC-939 cells (***P<0.001, ns, not significant); C is the expression levels of ROCK2 and Drp1 in R-RBE cells (***P<0.001, ns, not significant); D is the Co-IP and western blot analysis of the detection of Ub-Drp1 and ROCK2 in ROCK2-knockdown R-QBC-939 cells treated with MG 132; E is the co-immunoprecipitation results of detecting ROCK2 and UBA52 or Drp1 and UBA52; F is the GST pull-down results of detecting ROCK2 and UBA52 or Drp1 and UBA52; G is the detection of the expression and localization of ROCK2 and U BA52 in specified cells by immunofluorescence; H is the detection of the expression and localization of Drp1 and UBA52 in specified cells by immunofluorescence.

[0025] Figure 5 are the Western blot analysis results of the ROCK2 inhibitor in Example 2. Detailed implementation manners

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

[0027] In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0028] 1. Materials and Methods

[0029] 1.1. CCA cells include human cholangiocarcinoma cells QBC-939 (hereinafter referred to as QBC-939 cells) and human hepatobiliary duct cancer cells RBE (hereinafter referred to as RBE cells), which are provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). RBE cells are cultured in Dulbecco's Modified Eagle Medium (DMEM medium) containing 100 U / mL penicillin / streptomycin (50 U / mL each of penicillin and streptomycin) and 10% fetal bovine serum by volume. QBC-939 cells are cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640 medium) containing 100 U / mL penicillin / streptomycin (50 U / mL each of penicillin and streptomycin) and 10% fetal bovine serum by volume. Both QBC-939 cells and RBE cells are maintained in an incubator at 37 °C and 5% carbon dioxide by volume. Details of the relevant reagents are shown in Table 1.

[0030] Table 1 Reagent Information

[0031] Reagent Name Source Identification ROCK2 Proteintech 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

[0032] 1.2. Patients and specimens: 40 CCA samples and their corresponding adjacent tissues were collected from the Department of General Surgery of Jiangxi Cancer Hospital. All cancer tissue specimens were from the affected area and adjacent tissues of CCA. Tissue samples were analyzed by immunoblotting and qRT-PCR. When evaluated by immunohistochemistry, the specimens were stored in 4% formaldehyde solution by volume at room temperature.

[0033] 1.3. Immunohistochemical staining (IHC): After dewaxing the paraffin-embedded tissue sections, antigen retrieval was performed in 10 mmol / L sodium citrate buffer at pH 6.0. The tissue sections were incubated overnight at 4 °C with primary antibodies against ROCK2 (1:200) and Drp1 (1:200). Subsequently, the secondary antibody (Zenbio, China) was incubated for 30 min at room temperature. Then, the sections were incubated with horseradish peroxidase streptavidin conjugate. The peroxidase reaction was developed with 3,3-diaminobenzidine (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 ultraviolet-visible spectrophotometer (Thermo), reverse transcribed using a PrimeScript kit and gDNA Eraser (Takara, RR047A), and qRT-PCR was performed using a TB Ex Taq Quantitative (Tli RNase H Plus) kit (Takara, RR 420A). The expression levels of ROCK2 and Drp1 were determined by the 2 -ΔΔCt -method and normalized to their respective controls. The control used was β-actin. All measurements were repeated three times. The 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 by the biuret reagent, and boiled for 10 min after mixing with protein loading buffer. The proteins were separated by 10 g / 100 mL sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a 0.22 μm polyvinylidene fluoride membrane. The polyvinylidene fluoride membrane was blocked with 5% skim milk by volume at room temperature for 60 min and then incubated with the corresponding primary antibody overnight at 4°C. After washing three times with 1× Tris-buffered saline containing Tween 20 for 10 min each time, the polyvinylidene fluoride membrane was incubated with the corresponding secondary antibody for 1 h at room temperature, washed three times again with 1× Tris-buffered saline containing Tween 20 for 10 min each time, and finally exposed to imaging in enhanced chemiluminescence reagent.

[0038] 1.6, Cell transfection: All plasmids were purchased from Genechem. The plasmid and primer sequence information are shown in Table 3 and Table 4.

[0039] Table 3 Plasmid information table

[0040]

[0041] Table 4 Primer sequence list 2

[0042]

[0043]

[0044] 1.7, Cell viability assay: CCA cells in the logarithmic growth phase were seeded in 96-well plates at a density of 3×10 3 cells / 100 μL and incubated for 1 to 4 days. Every 24 h, 10 μL of CCK-8 reagent (obtained by diluting 10 μL of the stock solution with 90 μL of serum-free medium) was added and incubated at 37 °C for 2 h. The absorbance at 450 nm was detected using a spectrophotometer.

[0045] 1.8, Colony formation assay: CCA cells were seeded in 6-well plates (1500 cells per well) and cultured in a humid environment containing 5% carbon dioxide by volume for two weeks. Stained with 0.1 g / 100 mL crystal violet for 15 min and then washed twice with phosphate buffer (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 determined using an EDU proliferation detection kit (Beyotime, C0071S, Shanghai, China). Cells were cultured in 48-well plates for 24 h, then incubated with a 50 mM EDU solution for 2 h and fixed with 4% paraformaldehyde by mass. Subsequently, the cells were permeabilized. Stained with 0.25% Triton X-100 by volume for 15 min, and then stained with Azide 488 and Hoechst in sequence. Finally, observed under a fluorescence microscope.

[0047] 1.10, Immunofluorescence: Differentiated CCA cells were seeded in 24-well plates at a density of 1.5×10 4 cells / mL. After culturing for 1 day, fixed with 4% paraformaldehyde by mass for 15 min and then washed three times with PBS, 3 min each time. Subsequently, 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 non-specific binding. Then the cells were incubated overnight at 4 °C with antibodies against ROCK2, Drp1, and UBA52, washed three times with PBS containing Tween 20, incubated with a fluorescent secondary antibody for 1 h, stained with Hoechst (Beyotime, 33342, Shanghai, China) in sequence to observe the cell nuclei, washed three times with PBS containing Tween 20 again, and photographed using a confocal microscope.

[0048] 1.11, Biochemical assays: The concentrations or activities of MDA (Beyotime, S0131S, Shanghai, China) and GSH / GSSG (Beyotime, S0053, Shanghai, China) in the samples were determined using commercially available detection kits.

[0049] 1.12, ROS and Fe2+ Imaging: Cells were seeded in 6-well chamber slides (5×10 5 cells per well) and cultured for 24 h. After the slides were washed with PBS, they were incubated with PBS containing 2 mM DCFH-DA (Beyotime, S0033S, Shanghai, China) or 1 μM FerroOrange (Dojindo, F374, Japan) for 20 min in sequence. Subsequently, the cell nuclei were stained with Hoechst stain (Beyotime, 33342, Shanghai, China). Finally, observations were made 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), and glutathione agarose 4B beads (sigma) were incubated with GST, GST-UBA52, and purified His-ROCK2 or His-Drp1 in incubation buffer (20 mmol / L Tris-HCl, pH 7.4.0, 1 wt% Triton X-100) overnight at 4 °C, 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 subjected to immunoblot analysis.

[0051] 1.14 Co-immunoprecipitation: Protein samples were prepared in the same way as before immunoblotting, before adding the loading buffer. For the interactions between UBA52 and ROCK2 / Drp1, and between ROCK2 and Drp1, each 1 mg of protein sample was incubated with 2 μg of primary antibody overnight at 4 °C. Protein A / G magnetic beads (MCE) were added and incubated at 4 °C for 2 h. Then, the precipitate was collected by centrifugation at 14,000 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: The results were expressed as the mean ± standard deviation of three independent experiments. Fisher's test was used to evaluate the relationship between the clinicopathological characteristics of cholangiocarcinoma patients and the expression of ROCK2 and Drp1. Statistical analysis and the construction of all charts 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 pemigatinib-resistant cholangiocarcinoma cells, leading to poor prognosis

[0056] To characterize the pemigatinib resistance of CCA cells, we established two pemigatinib-resistant CCA cell lines, namely R-RBE cells and R-QBC-939 cells, by exposing CCA cells to gradually increasing concentrations of pemigatinib for a long time. Compared with their parental cell lines (RBE cells and QBC-939 cells), R-RBE cells and R-QBC-939 cells showed lower sensitivity to pemigatinib, and the results are shown in Figure 1 A and B. In addition, the results of Western blot and qRT-PCR analysis showed that ROCK2 was highly expressed in pemigatinib-resistant R-QBC-939 cells and R-RBE cells, as shown in Figure 1 C and D. This indicates that ROCK2 plays a key role in the development of pemigatinib resistance in CCA cells. The present invention also examined the expression of ROCK2 in CCA patients. Immunohistochemistry (IHC) results showed that compared with adjacent non-tumor tissues (Non-tumor), the frequency of ROCK2-positive cells was higher and the staining intensity was stronger in CCA tissues (tumor) of patients with poor response to pemigatinib, as shown in Figure 1 E and F. The results of qRT-PCR and western blot analysis showed that the expression level of ROCK2 in CCA tissues was significantly increased compared with adjacent non-tumor tissues, as shown in Figure 1 G and H. The present invention analyzed the expression level of ROCK2 in CCA subtypes. High ROCK2 expression levels were associated with poor prognosis in patients with iCCA, pCCA, and dCCA, as shown in Figure 1 I-K. In summary, ROCK2 is strongly expressed in pemigatinib-resistant cholangiocarcinoma cells, leading to poor prognosis, and has the value of a prognostic biomarker and treatment for CCA.

[0057] 2.2. ROCK2 inhibits ferroptosis, leading to pemigatinib resistance in cholangiocarcinoma cells

[0058] The present invention performed 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 lowly expressed, as shown in Figure 2 D. The levels of reactive oxygen species (ROS) and Fe in parental cells and pemigatinib-resistant CCA cells 2+, Analysis of the levels of malondialdehyde (MDA) and glutathione disulfide (GSSG) showed that ferroptosis was significantly inhibited. Staining maps of QBC-939 and R-QBC-939 cells after pemigatinib treatment under the specified treatments, as well as ROS, Fe 2+ , MDA and GSH / GSSG levels are shown in Figure 2 A - C, E and F of. The present invention studied the effect of the ferroptosis inducer fasudil hydrochloride on CCA cells resistant to pemigatinib. Cell Counting Kit-8 (CCK-8), colony formation and EDU assays; the results showed that treatment with fasudil hydrochloride significantly enhanced the sensitivity of CCA cells resistant to pemigatinib to pemigatinib, see Figure 2 G - J of. These findings indicate that fasudil hydrochloride not only induces ferroptosis but also plays a key role in reversing the resistance of CCA cells to pemigatinib.

[0059] The present invention studied the effect of ROCK2 knockdown on the expression levels of ferroptosis-related proteins in R-RBE and R-QBC-939 cells by immunoblot analysis. ROCK2 knockdown increased the expression level of TFR and significantly decreased the levels of GPX4 and SLC7A11, see Figure 2 K of. To further evaluate the effect of ROCK2 knockdown on ferroptosis, we measured ROS, Fe 2+ , MDA, and the levels of GSH / GSSG decreased in CCA cells of shNC+Pemigatinib (blank control), shROCK2#1+Pemigatinib (ROCK2 knockout group 1) and shROCK2#2+Pemigatinib (ROCK2 knockout group 2). After pemigatnib treatment, ferroptosis significantly increased in CCA cells of ROCK2 knockout group 1 and ROCK2 knockout group 2, see Figure 2 L - O of. These results indicate that downregulation of ROCK2 enhances ferroptosis in pemigatinib-resistant CCA cells. Knockdown of ROCK2 in R-RBE and R-QBC-939 cells significantly increased the sensitivity of these cells to pemigatinib, see Figure 2 P - R of.

[0060] In summary, these findings indicate that ROCK2 plays a key role in inhibiting ferroptosis, thereby affecting the resistance of CCA cells to pemigatinib.

[0061] 2.3, ROCK2 regulates the expression of Drp1, and the expression levels of ROCK2 and Drp1 are positively correlated in CCA tissues

[0062] To further investigate the mechanism by which ROCK2 regulates ferroptosis in CCA cells, Drp1, a key protein that regulates mitochondrial fission and is closely associated with ROS generation, was found to be involved in the ferroptosis process. Based on these findings, we compared R-RBE and R-QBC-939 cells with their parental cells (RBE and QBC-939) and found that the resistant cells exhibited high levels of Drp1, see Figure 3 A. The reduction of Drp1 levels did not lead to significant changes in ROCK2 levels, indicating that Drp1 is a downstream target of ROCK2, see Figure 3 B. Knockdown of Drp1 in R-RBE and R-QBC-939 cells enhances the sensitivity of these cells to pemigatinib treatment, see Figure 3 C~H.

[0063] Next, we analyzed the relationship between ROCK2 and Drp1 expression levels. Western blot analysis showed that decreased ROCK2 levels led to decreased Drp1 levels in R-RBE and R-QBC-939 cells, see Figure 3 I.

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

[0065] ROCK2 interacts with a variety of substrates to exert its effects, including inhibition of substrate ubiquitination and degradation. To elucidate how ROCK2 regulates the expression of Drp1 in CCA cells, the present invention first examined whether ROCK2 and Drp1 interact directly. The results of co-immunoprecipitation analysis showed that there was no direct interaction between the two proteins, see Figure 4 In addition, qRT-PCR results showed that the expression of ROCK2 did not affect the mRNA level of Drp1, see Figure 4 However, ectopic expression of ROCK2 led to a significant decrease in Drp1 polyubiquitination, while ROCK2 knockdown increased Drp1 polyubiquitination, see Figure 4 D. Co-immunoprecipitation analysis showed that there is interaction between ROCK2 and UBA52, as well as between Drp1 and UBA52, see Figure 4 The purified GST-UBA52 complex binds to His-tagged ROCK2 and Drp1 in vitro, see Figure 4 F. Confocal microscopy analysis showed that UBA52 co-localized with ROCK2 and Drp1 in the cytoplasm, see Figure 4 G~H, the results showed that ROCK2 regulated the expression of Drp1 through UBA52.

[0066] 3. Discussion

[0067] The drug resistance mechanism of pemigatinib of the present invention is described as follows:

[0068] ROCK2 plays a key role in the development of pemigatinib resistance by regulating ferroptosis in CCA, with particular emphasis on the involvement of the UBA52-Drp1 axis. Our research results show that ROCK2 is not only a biomarker for poor prognosis in CCA, but also a therapeutic target to enhance the efficacy of pemigatinib.

[0069] ROCK2 is a key signaling molecule in the Rho / ROCK signaling pathway. The present invention observes that ROCK2 is overexpressed in CCA cells, and the highly expressed ROCK2 is associated with poor prognosis of CCA patients. ROCK2 is highly expressed in pemigatinib-resistant CCA cell lines, and its expression is positively correlated with pemigatinib resistance. The reduction of ROCK2 expression level promotes ferroptosis of CCA cells, making them more sensitive to pemigatinib treatment. In addition, fasudil, a ROCK2 inhibitor, significantly increases the sensitivity of CCA cells to pemigatinib. This finding confirms the results of previous studies, indicating that ROCK2 plays a key role in inhibiting ferroptosis, thereby affecting drug resistance.

[0070] The present invention also finds that inhibiting Drp1 expression reverses ferroptosis in pemigatinib-resistant CCA cell lines; ROCK2 affects ferroptosis and leads to Drp1-dependent pemigatinib 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 chemotherapy resistance in CCA cells.

[0071] Based on the above research results, the present invention provides a ROCK2 inhibitor for inducing death of cholangiocarcinoma cells, which is composed of fasudil hydrochloride and magnesium chloride, and the mass ratio of the two is 100:0.1-0.2. The following embodiments are included.

[0072] Example 1

[0073] A ROCK2 inhibitor for inducing death of cholangiocarcinoma cells, which is composed of fasudil hydrochloride and magnesium chloride, and the mass ratio of the two is 100:0.1.

[0074] Example 2

[0075] A ROCK2 inhibitor for inducing death of cholangiocarcinoma cells, which is composed of fasudil hydrochloride and magnesium chloride, and the mass ratio of the two is 100:0.15.

[0076] Example 3

[0077] A ROCK2 inhibitor for inducing death of cholangiocarcinoma cells, which is composed of fasudil hydrochloride and magnesium chloride, and the mass ratio of the two is 100:0.2.

[0078] CCA cells were seeded in a 48-well plate at a density of 1.5×10 4 cells / mL, and then mixed with the ROCK2 inhibitor for inducing death of cholangiocarcinoma cells in Example 2. The final concentration of the ROCK2 inhibitor for inducing death of cholangiocarcinoma cells in each well was 10 μL / 100 μL. After culturing for 24 h, the ROCK2 expression level was measured. The results are shown in Figure 5 . The results showed that the ROCK2 expression levels of QBC-939 cells, R-QBC-939 cells, RBE cells, and R-RBE cells were all decreased.

[0079] CCA cells were seeded in a 48-well plate at a density of 1.5×10 4 cells / mL, and then mixed with the ROCK2 inhibitor for inducing death of cholangiocarcinoma cells in Example 2. The final concentration of the ROCK2 inhibitor for inducing death of cholangiocarcinoma cells in each well was 10 μL / 100 μL. After culturing for 24 h, the cells were incubated with 50 mM EDU solution for 2 h and fixed with 4% paraformaldehyde by mass fraction. Subsequently, the cells were permeabilized. They were stained with 0.25% Triton X-100 by volume fraction for 15 min, and then stained with Azide 488 and Hoechst successively. Finally, they were observed under a fluorescence microscope. At the same time, an equal amount of fasudil hydrochloride was used to replace the ROCK2 inhibitor for inducing death of cholangiocarcinoma cells as a control group. The results showed that the positive rates of EDU staining of QBC-939 cells, R-QBC-939 cells, RBE cells, and R-RBE cells were 12%, 18%, 9%, and 12% respectively, while the positive rates of EDU staining of QBC-939 cells, R-QBC-939 cells, RBE cells, and R-RBE cells in the control group were 35%, 49%, 31%, and 36% respectively, indicating that the ROCK2 inhibitor of the present invention has the prospect of treating CCA. In addition, through comparison with the control group, the results showed that magnesium chloride enhanced the effect of fasudil hydrochloride in inhibiting ROCK2 expression, and magnesium chloride also enhanced the effect of fasudil hydrochloride in inhibiting cholangiocarcinoma cells.

[0080] It should be noted that the ROCK2 inhibitors in Example 1 and Example 3 also have a CCA inhibitory effect similar to that in Example 2.

[0081] It should be noted that when the present invention involves a numerical range, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the adopted step method is the same as that of the embodiment, in order to prevent repetition, the present invention describes the preferred embodiments. Although the preferred embodiments of the present invention have been described, once those skilled in the art learn the creative concept of the present invention, additional changes and modifications can be made to these embodiments, and these changes and modifications all fall within the scope of the present invention.

[0082] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of equivalent technologies of the present invention, the present invention also intends to include these changes and modifications.

Claims

1. A ROCK2 inhibitor for causing cholangiocarcinoma cell death, characterized in that: The invention is composed of fasudil hydrochloride and magnesium chloride, and the mass ratio of the two is 100:0.1-0.

2.

2. The ROCK2 inhibitor for causing cholangiocarcinoma cell death according to claim 1, characterized in that The mass ratio of fasudil hydrochloride to magnesium chloride is 100:0.

15.

3. Use of the ROCK2 inhibitor for causing cholangiocarcinoma cell death according to claim 1 in the preparation of a drug for treating cholangiocarcinoma.

4. The use according to claim 3, characterized in that: The drug contains a ROCK2 inhibitor as its only active ingredient, which is used to cause the death of bile duct cancer cells.

5. The use according to claim 4, characterized in that: The drug also contains pharmaceutically acceptable excipients.

6. The use according to claim 5, characterized in that: The auxiliary material is at least one of a co-solvent and a solvent.

7. The use according to claim 6, characterized in that: The cosolvent includes at least one of polyethylene glycol and sodium lauryl sulfate; The solvent is deionized water.

8. The use according to claim 3, characterized in that: The drug is at least one of the following: Drugs that reduce the activity of bile duct cancer cells; Drugs that induce ferroptosis in cholangiocarcinoma cells; Drugs that increase sensitivity to chemotherapy.

9. The use according to claim 3, characterized in that: The cholangiocarcinoma cell is at least one of human cholangiocarcinoma cell QBC-939, human hepatocholangiocarcinoma cell RBE, human cholangiocarcinoma cell QBC-939 with chemotherapy drug resistance, and human hepatocholangiocarcinoma cell RBE with chemotherapy drug resistance.

10. The use according to claim 9, characterized in that: The chemotherapy drug is pemmetinib.

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