Application of combination of LIMK1 and CDK5 inhibitors in preparation of drugs for treating and / or preventing esophageal cancer

Abstract

The invention discloses application of combination of LIMK1 and CDK5 inhibitors in preparation of drugs for treating and / or preventing esophageal cancer, and belongs to the technical field of biological medicines. It is found for the first time that the combination of the LIMK1 inhibitor and the CDK5 inhibitor can inhibit invasion and metastasis of esophageal cancer. The LIMK1 inhibitor and the CDK5 inhibitor provided by the invention, as a medicine for treating and / or preventing esophageal cancer, have a remarkable synergistic effect and good safety.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly to the use of a combination of LIMK1 and CDK5 inhibitors in preparing a drug for treating and / or preventing esophageal cancer. Background Art

[0002] Esophageal cancer is one of the most common malignant tumors of the digestive tract. In 2020, among the 36 types of cancer worldwide, esophageal cancer ranked seventh in incidence (604,000 new cases) and sixth in mortality (544,000 deaths). There are two histological subtypes of esophageal cancer: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EA). The vast majority of cases in the Asia-Pacific region, including my country, are ESCC, and its mortality rate remains high due to recurrence and metastasis. The steps of tumor metastasis are complex and difficult to study, especially the regulatory role of phosphorylation modification in esophageal cancer metastasis is still unclear. Therefore, elucidating the key phosphorylation events that regulate tumor metastasis and developing therapeutic strategies will provide new clues for cancer treatment.

[0003] Surgery, radiotherapy, chemotherapy, and molecularly targeted drugs are the main treatments for esophageal cancer. Surgery and radiotherapy are local treatments, while chemotherapy and molecularly targeted drugs are systemic. However, commonly used anticancer drugs often have toxic side effects, and the development of tolerance during chemotherapy can lead to postoperative recurrence and poor prognosis, making treatment more difficult. Kinase-targeted inhibitors can specifically block phosphorylation-regulated signaling pathways that contribute to tumor growth, proliferation, and metastasis, thereby achieving therapeutic goals.

[0004] In recent years, with the continuous advancement of new oncology theories and the emergence of clinical drug challenges such as tumor metastasis, the development of innovative anticancer drugs has remained a hot topic and focus in oncology research. With the continuous advancement of molecular biology mechanisms and the continued promotion and support of the biotechnology industry, the exploration of tumors has continued to deepen. An increasing number of tumor-specific molecular targets have been identified, and the resulting small molecule targeted anti-tumor drugs have also grown rapidly. Combining different small molecule drugs can achieve therapeutic effects at lower concentrations, in a safer and more effective manner. However, the combined treatment of LIMK1 and CDK5 inhibitors has not yet been demonstrated to inhibit tumor cells, particularly tumor invasion and metastasis. Summary of the Invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a combined use of LIMK1 and CDK5 inhibitors in the preparation of drugs for treating and / or preventing esophageal cancer.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The use of LIMK1 and CDK5 inhibitors in combination for the preparation of drugs for the treatment and / or prevention of esophageal cancer, wherein the LIMK1 inhibitors include reagents used to reduce LIMK1 expression / activity through gene editing technologies such as RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, Cre-loxP gene recombination technology, and also include compounds that reduce LIMK1 expression / activity; CDK5 inhibitors include reagents used to reduce CDK5 expression / activity through gene editing technologies such as RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, Cre-loxP gene recombination technology, and also include compounds that reduce CDK5 expression / activity.

[0008] Furthermore, the compound that reduces LIMK1 expression / activity may include BMS-5 and pharmaceutically acceptable salts thereof, and the compound that reduces CDK5 expression / activity may include Dinaciclib and pharmaceutically acceptable salts thereof.

[0009] The structural formula of the BMS-5 is shown in formula (I), and the structural formula of the Dinaciclib is shown in formula (II):

[0010]

[0011] Furthermore, the effective concentration of BMS-5 is preferably 2.5±0.5 μM.

[0012] Furthermore, the effective concentration of Dinaciclib is preferably 2.5±0.5 nM.

[0013] Furthermore, the LIMK1 inhibitor includes siRNA-LIMK1, and its corresponding target sequence is: 5'-GGCAGCTCTTCTGCAAGAA-3'.

[0014] Furthermore, the CDK5 inhibitor includes siRNA-CDK5, and its corresponding target sequence is: 5'-GGATTCCCGTCCGCTGTTA-3'.

[0015] Furthermore, the effective concentration of the siRNA-LIMK1 or siRNA-CDK5 is preferably 100±20 nM.

[0016] Furthermore, the drug for treating and / or preventing esophageal cancer is a drug that can inhibit the invasion and / or metastasis of esophageal cancer.

[0017] Furthermore, the metastasis includes but is not limited to lung metastasis and lymph node metastasis.

[0018] Furthermore, the esophageal cancer preferably includes but is not limited to esophageal squamous cell carcinoma.

[0019] A drug for treating and / or preventing esophageal cancer, comprising a LIMK1 inhibitor and a CDK5 inhibitor.

[0020] The combination of LIMK1 inhibitors and CDK5 inhibitors has a significant synergistic effect in inhibiting esophageal cancer metastasis, especially esophageal squamous cell carcinoma.

[0021] Furthermore, the drug for treating and / or preventing esophageal cancer is a drug that can inhibit the invasion and / or metastasis of esophageal cancer.

[0022] Furthermore, the metastasis includes but is not limited to lung metastasis and lymph node metastasis.

[0023] Furthermore, the esophageal cancer preferably includes but is not limited to esophageal squamous cell carcinoma.

[0024] Furthermore, the drug for treating and / or preventing esophageal cancer further comprises pharmaceutically acceptable excipients.

[0025] Furthermore, the pharmaceutically acceptable excipient is preferably at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant and a lubricant.

[0026] Furthermore, the administration of the drug for treating and / or preventing esophageal cancer includes but is not limited to oral administration and injection.

[0027] The present invention has the following advantages and effects compared to the prior art:

[0028] The present invention has demonstrated that the combined use of a LIMK1 inhibitor and a CDK5 inhibitor can effectively inhibit the invasion and metastasis of esophageal cancer cells. The present invention's LIMK1 inhibitor and CDK5 inhibitor possess a favorable safety profile as a drug for the treatment and / or prevention of esophageal cancer. Furthermore, the inventors have discovered that the combined use of a LIMK1 inhibitor and a CDK5 inhibitor exhibits significant synergistic effects in inhibiting the invasion and metastasis of esophageal cancer, particularly esophageal squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Figures 1 and 2 are structural formulas of LIMK1 inhibitors and CDK5 inhibitors; A is the structural formula of LIMK1 inhibitor (BMS-5); B is the structural formula of CDK5 inhibitor (Dinaciclib).

[0030] Figure 2Figure 1 is a graph showing the effects of inhibiting LIMK1 and CDK5 alone or in combination on the invasive ability of esophageal squamous cell carcinoma cells; Figure A is a graph showing the effects of LIMK1 inhibitors and CDK5 inhibitors alone or in combination on the invasive ability of esophageal squamous cell carcinoma cells; Figure B is a graph showing the effects of siRNA targeting LIMK1 or CDK5 alone or in combination on the invasive ability of esophageal squamous cell carcinoma cells.

[0031] Figure 3 Figure 2 shows the effects of LIMK1 inhibitors and CDK5 inhibitors used alone or in combination on the metastatic ability of esophageal squamous cell carcinoma cells; Figure A shows the fluorescence detection results of LIMK1 inhibitors and CDK5 inhibitors used alone or in combination on the lung metastasis ability of esophageal squamous cell carcinoma cells; Figure B shows the fluorescence detection results of LIMK1 inhibitors and CDK5 inhibitors used alone or in combination on the lymph node metastasis ability of esophageal squamous cell carcinoma cells.

[0032] Figure 4 The graph shows the results of the morphological changes in the liver, spleen, and kidney of mice using LIMK1 inhibitors and CDK5 inhibitors alone or in combination.

[0033] Figure 5 This figure shows the changes in blood routine test results after esophageal cancer cells are treated with LIMK1 inhibitors and CDK5 inhibitors alone and / or in combination. DETAILED DESCRIPTION

[0034] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0035] Experimental Materials:

[0036] The LIMK1 inhibitor used in the following examples is BMS-5 (CAS No.: 1338247-35-0), and the CDK5 inhibitor used in the following examples is Dinaciclib (CAS No.: 779353-01-4), both of which were synthesized by Shanghai Taosu Biochemical Technology Co., Ltd., with the structural formula shown below. Figure 1 As shown, dissolved in 20% cyclodextrin.

[0037] Human esophageal squamous cell carcinoma cells KYSE150 and KYSE410 were purchased from German Microorganisms and Cell Culture Co., Ltd.; nude mice (BALB / C-nu / nu) were purchased from Yusibeifu (Beijing) Biotechnology Co., Ltd.; NOD-SCID mice were purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.; RPMI 1640 medium was purchased from Gibco, USA; DMSO was purchased from Sigma, USA.

[0038] Human esophageal squamous cell carcinoma luciferase-labeled cells KYSE150-LUC have been published in the literature "Wen Wen Xu et al. Direct Targeting of CREB1 with Imperatorin Inhibits TGFβ2-ERK Signaling to Suppress Esophageal Cancer Metastasis, Advanced Science, 2020. DOI: 10.1002 / advs.202000925".

[0039] Human esophageal squamous cell carcinoma luciferase-labeled cells KYSE410-LUC and KYSE410-I3 have been disclosed in the literature “Identification of miR-515-3p and its targets, vimentin and MMP3, as a key regulatory mechanism in esophageal cancer metastasis: functional and clinical significance”.

[0040] KYSE150-LUC-LM5 cells have been disclosed in the document "Application of CN114259496A--G699-0288 in the preparation of drugs for treating and / or preventing esophageal cancer".

[0041] siRNA transient transfection method:

[0042] siLIMK1 (5'-GGCAGCTCTTCTGCAAGAA-3') and siCDK5 (5'-GGATTCCCGTCCGCTGTTA-3') were synthesized by Shanghai Quanyang Biotechnology Co., Ltd. Well-growing luciferase-labeled human esophageal squamous cell carcinoma (KYSE150-LUC) and KYSE410-LUC cells were digested and plated in 6-well plates. When the cells reached 50%-60% confluence, they were transiently transfected with LIMK1 / CDK5 expression plasmids or siRNAs (siLIMK1 and siCDK5). 48 hours later, the cells were used for the corresponding experiments.

[0043] Example 1: Functional validation of the combined effect of LIMK1 inhibitor and CDK5 inhibitor on the treatment of esophageal cancer metastasis

[0044] (1) Invasion experiment

[0045] Esophageal squamous cell carcinoma cells were treated with given concentrations of LIMK1 inhibitor and CDK5 inhibitor alone and / or in combination, and then the invasion ability of esophageal squamous cell carcinoma cells was detected to study the effect of combined treatment with LIMK1 inhibitor and CDK5 inhibitor on the invasion ability of esophageal squamous cell carcinoma cells; esophageal squamous cell carcinoma cells were treated with 100nM siLIMK1 and siCDK5 alone and / or in combination, and then the invasion ability of esophageal squamous cell carcinoma cells was detected to study the effect of combined treatment with siLIMK1 inhibitor and siCDK5 inhibitor on the invasion ability of esophageal squamous cell carcinoma cells:

[0046] 100 μL of 5% Matrigel was placed in an invasion chamber and placed in a 37°C incubator until ready for use. Highly invasive human esophageal squamous cell carcinoma cells, KYSE150-LUC-LM5 and KYSE410-I3, were digested with 0.25% trypsin to reduce cells to single cell suspension. After counting, 500,000 KYSE150-LUC-LM5 cells and 200,000 KYSE410-LUC-I3 cells were added to the upper chamber, and 500 μL of complete culture medium was added to the lower chamber, along with a LIMK1 inhibitor (2.5 μM) and / or a CDK5 inhibitor (2.5 nM). The cells were incubated at 37°C in 5% CO₂. After 36 hours, the chambers were removed and washed twice with PBS buffer. 500 μL of methanol was added to the lower chamber and fixed for 10 minutes. The methanol was aspirated, and the cells were stained with 0.2% crystal violet for 10 minutes. The crystal violet was aspirated, washed with water to remove any residual violet, and then air-dried and photographed.

[0047] 100 μL 5% Matrigel was spread on invasion chambers and placed in a 37°C incubator until ready for use. Highly invasive human esophageal squamous cell carcinoma cells KYSE150-LUC-LM5-CON / KYSE150-LUC-LM5-siLIMK1 / KYSE150-LUC-LM5-siCDK5 / KYSE150-LUC-LM5-siLIMK1+siCDK5 and KYSE410-I3 / KYSE410-I3-siLIMK1 / KYSE410-I3-siCDK5 / KYSE410-I3-siLIMK1+siCDK5 were digested with 0.25% trypsin to produce suspended single cells. After counting, 500,000 KYSE150-LUC-LM5 cells and 200,000 KYSE410-LUC-I3 cells were added to the upper layer of each chamber, and 500 μL of complete culture medium was added to the lower layer. The cells were cultured at 37°C in 5% CO2. After 36 hours, the chamber was taken out and washed twice with PBS buffer. 500 μL of methanol was added to the lower chamber and fixed for 10 min. After the methanol was removed, 0.2% crystal violet was added for staining for 10 min. After the crystal violet was removed, the residual crystal violet was washed away with clean water. After drying, the sample was photographed.

[0048] Depend on Figure 2 The results of the invasion experiment showed that the invasion ability of esophageal squamous cell carcinoma cells was significantly reduced when the LIMK1 inhibitor and CDK5 inhibitor were combined and treated, compared with the LIMK1 inhibitor and CDK5 inhibitor treatment groups alone or the control group. At the same time, the invasion ability of esophageal squamous cell carcinoma cells was significantly reduced when the siLIMK1 and siCDK5 inhibitors were combined and treated, compared with the siLIMK1 and siCDK5 treatment groups alone or the control group.

[0049] (2) In vivo experiments

[0050] Twenty-four 6-week-old male mice (NOD-SCID) and male nude mice (BALB / C-nu / nu), including 6 mice in the control group and 18 mice in the experimental group, were selected to construct tumor lung metastasis models and foot pad lymph node metastasis models.

[0051] 1) Human esophageal squamous cell carcinoma luciferase-labeled cells KYSE150-LUC-LM5 were digested into single cells using 0.25% trypsin. 5×10 6 Cancer cells were resuspended in 100 μL PBS buffer.

[0052] 2) Before the experiment, nude mice were anesthetized and the degree of anesthesia was assessed by painless and painful stimulation to ensure that the nude mice were in an anesthetized state;

[0053] 3) Cells were resuspended using a 25G microsyringe and injected into the tail vein of mice to establish a lung metastasis model. A total of 24 mice were included. Cells were resuspended using a 25G microsyringe and injected into the paw pad of mice to establish a paw pad lymph node metastasis model. A total of 24 mice were included.

[0054] 4) Treatment with LIMK1 inhibitors and CDK5 inhibitors, alone and / or in combination: One week after tumor cell injection, mice were randomly divided into groups and treated with 10 mg / kg LIMK1 inhibitor and 15 mg / kg CDK5 inhibitor by intraperitoneal injection twice a week. Two weeks after administration, D-Luciferin (purchased from GOLDBIO) was injected intraperitoneally, and in vivo fluorescence photography was performed to observe cancer cell metastasis.

[0055] 5) Mouse tissue and organ morphology and blood biochemistry routine tests

[0056] The liver, spleen, lung, and kidney of the mice were sent to Wuhan Google Biotechnology Co., Ltd. for tissue and organ morphology testing. Blood was drawn from the mice and centrifuged to obtain plasma. The obtained plasma samples were then sent to Wuhan Google Biotechnology Co., Ltd. for blood biochemical analysis (detection of white blood cell (WBC), lymphocyte, monocyte, granulocyte, red blood cell (WBC), red blood cell (HGB), hematocrit (HCT), platelet (PLT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels). The plasma obtained after centrifugation of blood from the mice in the 0 mg / kg group was used as a control.

[0057] The results are as follows Figure 3 、 Figure 4 、 Figure 5 As shown, from Figure 3 It can be seen that compared with the LIMK1 inhibitor and CDK5 inhibitor single treatment group and the control group, the metastatic ability of esophageal squamous cell carcinoma cells was significantly reduced when LIMK1 inhibitor and CDK5 inhibitor were combined for treatment. Figure 4 It can be seen that after drug treatment, the liver, spleen and kidney of the mice were taken out for immunohistochemistry. The results showed that the combination of LIMK1 inhibitor and CDK5 inhibitor had no effect on the liver, spleen and kidney function of mice. Figure 5 In the study, routine blood tests of mice using LIMK1 inhibitors and CDK5 inhibitors alone or in combination showed no significant differences in various indicators, indicating that LIMK1 inhibitors and CDK5 inhibitors have no obvious toxic side effects on nude mice and have good safety as potential anti-tumor drugs.

[0058] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of a combination of a LIMK1 and a CDK5 inhibitor in the preparation of a drug for treating and / or preventing esophageal cancer, characterized in that: LIMK1 inhibitors include reagents used to reduce LIMK1 expression / activity through RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, and Cre-loxP gene recombination technology, and also include compounds that reduce LIMK1 expression / activity; CDK5 inhibitors include reagents used to reduce CDK5 expression / activity through RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, and Cre-loxP gene recombination technology, and also include compounds that reduce CDK5 expression / activity.

2. Use of the LIMK1 and CDK5 inhibitors in combination for preparing a drug for treating and / or preventing esophageal cancer according to claim 1, characterized in that: The compounds that reduce LIMK1 expression / activity include BMS-5 and pharmaceutically acceptable salts thereof; The compound that reduces CDK5 expression / activity includes Dinaciclib and pharmaceutically acceptable salts thereof; The structural formula of the BMS-5 is shown in formula (I), and the structural formula of the Dinaciclib is shown in formula (II):

3. Use of the LIMK1 and CDK5 inhibitors in combination for preparing a drug for treating and / or preventing esophageal cancer according to claim 2, characterized in that: The effective concentration of BMS-5 is 2.5±0.5μM; The effective concentration of Dinaciclib is 2.5±0.5 nM.

4. Use of the LIMK1 and CDK5 inhibitors in combination for preparing a drug for treating and / or preventing esophageal cancer according to claim 1, characterized in that: The LIMK1 inhibitor includes siRNA-LIMK1, and its corresponding target sequence is: 5'-GGCAGCTCTTCTGCAAGAA-3'; The CDK5 inhibitor includes siRNA-CDK5, and its corresponding target sequence is: 5'-GGATTCCCGTCCGCTGTTA-3'.

5. Use of the combination of LIMK1 and CDK5 inhibitors in the preparation of a drug for treating and / or preventing esophageal cancer according to claim 4, characterized in that: The effective concentration of the siRNA-LIMK1 or siRNA-CDK5 is 100±20 nM.

6. Use of the LIMK1 and CDK5 inhibitors according to any one of claims 1 to 5 in the preparation of a drug for treating and / or preventing esophageal cancer, characterized in that: The drug for treating and / or preventing esophageal cancer is a drug that can inhibit the invasion and / or metastasis of esophageal cancer.

7. Use of the combination of LIMK1 and CDK5 inhibitors in the preparation of a drug for treating and / or preventing esophageal cancer according to claim 6, characterized in that: The metastasis includes lung metastasis and lymph node metastasis.

8. Use of a combination of a LIMK1 and CDK5 inhibitor according to any one of claims 1 to 5 in the preparation of a drug for treating and / or preventing esophageal cancer, characterized in that: The esophageal cancer includes esophageal squamous cell carcinoma.

9. Use of a combination of a LIMK1 and CDK5 inhibitor according to any one of claims 1 to 5 in the preparation of a drug for treating and / or preventing esophageal cancer, characterized in that: The drug for treating and / or preventing esophageal cancer further comprises a pharmaceutically acceptable excipient; The pharmaceutically acceptable excipient is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant, and a lubricant; The administration methods of the drug for treating and / or preventing esophageal cancer include oral administration and injection.

10. A drug for treating and / or preventing esophageal cancer, characterized in that: The invention comprises the LIMK1 inhibitor and CDK5 inhibitor according to any one of claims 1 to 9.

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