Drug, drug composition and combined drug for inhibiting peripheral nerve invasion

Abstract

The present invention provides a drug, a pharmaceutical composition, and a combined drug for inhibiting peripheral nerve invasion, and belongs to the field of chemical medicine technology. The present invention has found that ApoD is related to the development of tumor peripheral nerve invasion, and ApoD inhibitors can weaken the migration and invasion ability of tumor cells, thereby inhibiting the progression of peripheral nerve invasion. At the same time, the present invention has found that WP1066 and WZ811 can inhibit the progression of peripheral nerve invasion by inhibiting AopD expression. In addition, the present invention has found that the combined use of WP1066 and WZ811 can play a synergistic role in inhibiting tumor cell peripheral nerve invasion, and can prevent and / or treat tumor recurrence and metastasis, thereby improving patient prognosis and improving quality of life. The present invention provides a basis for clinical drug treatment of patients with peripheral nerve invasion of salivary gland adenoid cystic carcinoma, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical medicine, and in particular relates to a drug, a drug composition and a combined drug for inhibiting peripheral nerve invasion. Background Art

[0002] Salivary adenoid cystic carcinoma (SACC) is one of the most common malignant tumors of the salivary glands. It has an ideal short-term survival prognosis and can survive with the tumor for a long time. However, due to the high incidence of recurrence and metastasis, the 20-year survival rate is only 20%. Peripheral nerve invasion is the most significant pathological feature of salivary adenoid cystic carcinoma and one of the most important factors affecting the recurrence and metastasis of salivary adenoid cystic carcinoma. It can be seen in 80% of salivary adenoid cystic carcinoma patients. Peripheral nerve invasion is an adverse histological feature in which cancer cells invade the perineural space and spread closely along the nerve bundles. It is associated with poor prognosis of various cancers (such as lung adenocarcinoma, prostate cancer, pancreatic cancer, head and neck squamous cell carcinoma, etc.), and is also considered to be related to pain and local recurrence of the disease. It is an important prognostic factor for malignant tumors and a sign of extensive tumor spread. At present, the main treatment for salivary adenoid cystic carcinoma is surgical treatment. Due to the characteristics of peripheral nerve invasion, it is difficult to achieve a truly negative resection margin in surgical treatment, which leads to tumor recurrence and metastasis. Currently, there is no effective therapeutic drug targeting the peripheral nerve invasion of salivary gland adenoid cystic carcinoma in clinical practice. Therefore, in order to improve the survival rate of patients, it is an urgent problem to prepare a drug that can target the peripheral nerve invasion of salivary gland adenoid cystic carcinoma.

[0003] Apolipoprotein D (ApoD), the gene is located on human chromosome 3 and encodes a component of high-density lipoprotein, with no significant similarity to other apolipoprotein sequences. ApoD is highly conserved among mammalian species and consists of 169 residues, including a 20-amino acid secretory peptide signal and two glycosylation sites. The molecular weight of the mature protein ranges from 20 to 32 kDa. Whether ApoD is associated with peripheral nerve invasion has not been studied. Summary of the invention

[0004] In order to solve the problems existing in the prior art, a drug is provided to block the occurrence of peripheral nerve invasion, improve the prognosis of patients with salivary gland adenoid cystic carcinoma, and improve the quality of life. The present invention provides a drug, a drug composition, and a combined drug for inhibiting peripheral nerve invasion.

[0005] The present invention provides use of an ApoD inhibitor in preparing a medicament for preventing and / or treating peripheral nerve invasion.

[0006] Furthermore, the drug is a drug for preventing and / or treating peripheral nerve invasion caused by tumors;

[0007] Preferably, the drug is a drug for preventing and / or treating tumor recurrence and / or metastasis;

[0008] More preferably, the tumor is salivary gland adenoid cystic carcinoma, head and neck squamous cell carcinoma, prostate cancer, lung cancer, pancreatic cancer, colorectal cancer or gastric cancer;

[0009] Further preferably, the ApoD inhibitor is selected from WP1066 and WZ811.

[0010] The present invention also provides a pharmaceutical composition for preventing and / or treating peripheral nerve invasion, which is composed of WP1066 and WZ811;

[0011] Preferably, the molar ratio of WP1066 to WZ811 is 1:1 to 10;

[0012] More preferably, the molar ratio of WP1066 to WZ811 is 1:1.

[0013] The present invention also provides a method for preparing the aforementioned pharmaceutical composition, which comprises the following steps: taking WP1066 and WZ811 according to a molar ratio, mixing, and obtaining the pharmaceutical composition.

[0014] The present invention also provides use of the aforementioned pharmaceutical composition in preparing a drug for preventing and / or treating peripheral nerve invasion.

[0015] Furthermore, the drug is a drug for preventing and / or treating peripheral nerve invasion caused by tumors;

[0016] Preferably, the drug is a drug for preventing and / or treating tumor recurrence and / or metastasis;

[0017] More preferably, the tumor is salivary gland adenoid cystic carcinoma, head and neck squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer, colorectal cancer or gastric cancer.

[0018] The present invention also provides a pharmaceutical preparation for preventing and / or treating peripheral nerve invasion, which is a preparation prepared by taking the aforementioned pharmaceutical composition as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0019] The present invention also provides a combined drug for preventing and / or treating peripheral nerve invasion, which contains WP1066 and WZ811 of the same or different specifications administered simultaneously or separately, and a pharmaceutically acceptable carrier;

[0020] Preferably, the molar ratio of WP1066 to WZ811 is 1:1 to 10;

[0021] More preferably, the molar ratio of WP1066 to WZ811 is 1:1.

[0022] The present invention also provides the use of WP1066 and WZ811 in combination in the preparation of a drug for preventing and / or treating peripheral nerve invasion;

[0023] Preferably, the molar ratio of WP1066 to WZ811 is 1:1 to 10;

[0024] More preferably, the molar ratio of WP1066 to WZ811 is 1:1.

[0025] Furthermore, the drug is a drug for preventing and / or treating peripheral nerve invasion caused by tumors;

[0026] Preferably, the drug is a drug for preventing and / or treating tumor recurrence and / or metastasis;

[0027] More preferably, the tumor is salivary gland adenoid cystic carcinoma, head and neck squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer, colorectal cancer or gastric cancer.

[0028] In the present invention, peripheral nerve invasion (PNI) refers to a common pathological feature in which tumor cells invade the perineural space of peripheral nerves and spread closely along the nerve bundles. Tumor cells can indiscriminately invade different layers within the nerve fiber sheath, can exist independently without lymphatic vessels or blood vessels in the anatomical site, and may be the only metastatic pathway for some tumors, and has become an important pathological feature of many malignant tumors, such as salivary gland adenoid cystic carcinoma, head and neck squamous cell carcinoma, prostate cancer, pancreatic cancer, colorectal cancer, gastric cancer, etc. PNI is considered to be related to pain and local recurrence of the disease, is an important prognostic factor for malignant tumors, and is also considered to be a sign of widespread tumor spread.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention has found that ApoD is related to the development of tumor peripheral nerve invasion, and ApoD inhibitors can weaken the migration and invasion ability of tumor cells, thereby inhibiting the progression of peripheral nerve invasion. At the same time, the present invention has found that WP1066 and WZ811 can inhibit the progression of peripheral nerve invasion by inhibiting AopD expression. In addition, the present invention has found that the combined use of WP1066 and WZ811 can play a synergistic role in inhibiting tumor cell peripheral nerve invasion, and can prevent and / or treat tumor recurrence and metastasis, thereby improving patient prognosis and improving quality of life. The present invention provides a basis for clinical drug treatment of patients with peripheral nerve invasion of salivary gland adenoid cystic carcinoma, and has good application prospects.

[0031] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0032] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Figure 2 is the expression level of ApoD in salivary gland adenoid cystic carcinoma: A is the expression level of ApoD in normal human salivary gland acinar cells HSG and salivary gland adenoid cystic carcinoma cell lines SACC-83 and SACC-LM; B is the expression level of ApoD protein in salivary gland adenoid cystic carcinoma tumor tissue and adjacent control tissue, N is normal adjacent control, T is tumor tissue; C is the comparison of ApoD expression levels in salivary gland adenoid cystic carcinoma tumor tissue (SACC) and normal salivary gland tissue (NSG); D is the comparison of ApoD expression levels in salivary gland adenoid cystic carcinoma tissues with positive and negative peripheral nerve invasion, N is nerve, T is tumor cell, and the green arrow indicates that the tumor cell breaks through the peripheral nerve sheath and invades the nerve.

[0034] Figure 2 Figure 3 shows the role of ApoD in the progression of salivary adenoid cystic carcinoma: A shows the results of the efficiency evaluation of silencing ApoD expression in salivary adenoid cystic carcinoma cells by small interfering RNA (siNC) in the control group and ApoD small interfering RNA (siApoD), with GAPDH as the internal reference; B shows that silencing ApoD expression inhibits the migration and invasion of salivary adenoid cystic carcinoma cells; C shows the statistical analysis of the migration and invasion abilities of salivary adenoid cystic carcinoma cells by siApoD and siNC; D shows that silencing ApoD inhibits the expression of migration and invasion-related markers MMP2 and Vimentin proteins.

[0035] Figure 3 Figure 2 shows the role of ApoD in the peripheral nerve invasion of salivary adenoid cystic carcinoma: A shows that silencing ApoD weakens the nerve invasion ability of salivary adenoid cystic carcinoma cells SACC-83; B shows the statistical test of the nerve invasion ability of SACC-83 cells in the experimental and control groups; C shows that silencing ApoD weakens the nerve invasion ability of salivary adenoid cystic carcinoma cells SACC-LM; D shows the statistical test of the nerve invasion ability of SACC-LM cells in the experimental and control groups.

[0036] Figure 4The effect of inhibitors of ApoD expression on the peripheral nerve invasion of salivary gland adenoid cystic carcinoma: A is the effect of WP1066 on ApoD B is the inhibitory effect of WP1066 on ApoD protein expression level; C is that WP1066 weakened the migration and invasion ability of salivary gland adenoid cystic carcinoma cells, while ApoD reversed the inhibition of migration and invasion ability caused by WP1066; D is the statistical test results of migration and invasion ability of the control group (Con), WP1066 treatment group (WP1066) and WP1066 + exogenous ApoD treatment group (WO1066 + ApoD); E is the result that WP1066 treatment reduced the expression of migration and invasion-related markers MMP2 and Vimentin protein, and exogenous ApoD treatment reversed the expression of MMP2 and Vimentin protein; F is the result that WP1066 treatment weakened the neural invasion ability of SACC-83, and ApoD treatment restored the neural invasion ability of SACC-83; G is the result that WP1066 treatment weakened the neural invasion ability of SACC-LM, and ApoD treatment restored the neural invasion ability of SACC-LM.

[0037] Figure 5 The following are the effects of ApoD derived from salivary adenoid cystic carcinoma cells on nerve cells: A shows the effect of CXCR4 on the axonal elongation of nerve cells, and WZ811 is a specific inhibitor of CXCR4; B shows the identification of CXCR4 expression levels in nerve cells; C shows that ApoD accelerates the invasion of SACC-83 cells into nerve fibers, while the CXCR4 inhibitor weakens the promoting effect of ApoD; D shows that ApoD accelerates the invasion of SACC-LM cells into nerve fibers, while the CXCR4 inhibitor weakens the promoting effect of ApoD.

[0038] Figure 6 The results of the combined use of WP1066 and WZ811 to inhibit peripheral nerve invasion: A shows the effect of the combined use of WP1066 and WZ811 on the peripheral nerve invasion of SACC-83 compared with the control group and the use of WP1066 or WZ811 alone; B shows the statistical test results of the peripheral nerve invasion ability of the control group, WP1066 group, WZ811 group and WP1066+WZ811 group. DETAILED DESCRIPTION

[0039] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0040] In the present invention, WP1066 is a JAK2 / STAT3 signaling inhibitor, and WZ811 is a CXCR4 inhibitor. The structures of WP1066 and WZ811 are shown below:

[0041]

[0042] The materials used in the present invention are as follows:

[0043] (1) Cells: salivary gland adenoid cystic carcinoma cells SACC-83 and SACC-LM, and normal human salivary gland acinar cells HSG. SACC-83 cells were derived from a 26-year-old female patient with salivary gland adenoid cystic carcinoma. They are epithelial cells with polygonal shape and mosaic arrangement. SACC-LM cells were derived from a 26-year-old female patient with salivary gland adenoid cystic carcinoma. They are epithelial cells. HSG cells were derived from the submandibular gland of a 54-year-old male. HSG cells, SACC-83 cells, and SACC-LM cells were purchased from ATCC, USA.

[0044] (2) Tissue specimens: Salivary gland adenoid cystic carcinoma tissues and normal salivary gland tissues were obtained from the West China Hospital of Stomatology, Sichuan University, and approved by the Ethics Committee of the West China School of Stomatology, Sichuan University. Peritumoral tissues were obtained from more than 1 cm around the tumor and were confirmed to be tumor-free by pathology. Salivary gland adenoid cystic carcinoma tissues were collected from the Department of Head and Neck Oncology, West China Hospital of Stomatology, Sichuan University, and the ApoD expression level was identified by immunohistochemical staining. H&E staining was further used to identify whether the tumor had peripheral nerve invasion, and the relationship between the ApoD expression level between specimens positive and negative for peripheral nerve invasion was analyzed.

[0045] (3) C57 mice: C57 mice were purchased from Jicui Yaokang Biotechnology Co., Ltd. and cultured in the SPF Animal Experiment Management Center of West China Hospital, Sichuan University. The animal experiments were approved by the Animal Ethics Management Committee of West China School of Stomatology, Sichuan University.

[0046] The experimental method involved in the present invention is as follows:

[0047] (1) qRT-PCR: The treated cells were washed 2-3 times with PBS, lysed with TRIZOL at room temperature for 10 min, and total RNA was extracted by isopropanol precipitation. The total RNA concentration was then detected. An equal amount of RNA (about 800 ng) was added to each group and reversed into cDNA using a reverse transcription kit. The relative expression of the target gene was detected using a Roche qPCR instrument.

[0048] (2) Protein immunoblotting: The treated cells were washed 2-3 times with PBS, and proteins were extracted with RIPA lysis buffer. The protein concentration was detected by BCA assay and then balanced. Proteins were separated by polyacrylamide gel electrophoresis and transferred to PVDF membrane. After blocking with 5% skim milk, primary and secondary antibodies were incubated. The reactive proteins were detected with ECL substrate and imaged with gel electrophoresis imager. The dilution ratio of primary antibody was 1:1000, and the dilution ratio of secondary antibody was 1:10000.

[0049] (3) H&E staining: paraffin sections were dewaxed and rehydrated in xylene and graded alcohol (100%, 95%, 90%, 80%, 70%, 50%), and washed with tap water three times, 5 min each time; paraffin sections were placed in hematoxylin staining solution for 2-3 min, washed with water, differentiated in 1% hydrochloric acid alcohol for 1-2 s, washed again with water, and blued with 0.6% ammonia solution; eosin staining was performed for 1 min; gradient alcohol dehydration was performed to make it transparent, and after drying, the sections were sealed with neutral gum.

[0050] (4) Immunohistochemical staining: Paraffin sections were dewaxed and rehydrated in xylene and graded alcohol (100%, 95%, 90%, 80%, 70%, 50%), and washed with tap water three times for 5 min each time. They were immersed in 3% hydrogen peroxide in the dark to remove endogenous catalase, and then washed with water three times for 5 min each time after 10 min. Antigen retrieval was performed with sodium citrate antigen retrieval solution, and microwaved on high heat for 5 min. After the retrieval solution boiled, the heat was adjusted to medium-high heat and maintained for 5 min. After the retrieval solution cooled to room temperature naturally, the sections were washed with PBS three times for 5 min each time, blocked with 5% BSA at room temperature for 1 hour, incubated with primary antibody at 4°C overnight, incubated with secondary antibody at room temperature for 40 min, washed with PBS three times for 5 min each time, developed with DAB colorimetric solution, stained with hematoxylin solution, differentiated with 1% hydrochloric acid alcohol for 1-2 s, and blued with 0.6% ammonia water. The sections were dehydrated to make them transparent, and then mounted after drying. They were observed under an upright microscope. The sections were dehydrated with graded alcohol to make them transparent, and then mounted with neutral gum after drying.

[0051] (5) Migration and invasion assay: The migration and invasion ability was evaluated by Transwell assay. The number of cells digested and counted after treatment was about 5×10 4 The cells were resuspended in 200 μl serum-free medium and planted in the Transwell chamber. 600 μl 10% complete medium was added to the lower chamber of the Transwell culture plate. The chamber was cultured in the Transwell culture plate and the air bubbles between the chamber and the culture plate were emptied. Transwell positive cells were detected by crystal violet staining after 24-48 hours. The invasion experiment required adding 1:8 diluted matrix gel to the chamber before adding cells, and then adding 200 μl of 5×10 diluted in serum-free medium after incubation in a 37°C incubator for 2 hours. 4 cell.

[0052] (6) Nerve invasion experiment: Dorsal root ganglia of C57 mice were extracted and placed in matrix gel in a 37°C incubator for 5 min. They were cultured in Neuroalbasal+B27 medium. Single nerve fibers were visible after about 5 days. An equal amount of tumor cells were added for co-culture as appropriate, and the medium was replaced with 10% fetal bovine serum medium. After 5 days of co-culture, the invasion of tumor cells into nerve fibers was observed under an inverted microscope.

[0053] (7) Cell immunohistochemistry and immunofluorescence: Cell slides were prepared in 6-well cell culture plates, fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, 5 min each time; treated with 0.1% Triton X-100 for 10 min; washed three times with PBS, 5 min each time; blocked with 5% BSA at room temperature for 1 hour, rinsed with PBS; incubated with primary antibody at 4°C overnight, incubated with secondary antibody at room temperature for 40 min, washed three times with PBS, 5 min each time; immunohistochemistry: DAB color development under optical microscope, hematoxylin staining for 40 s, hydrochloric acid alcohol differentiation for 1-2 s, ammonia anti-blueing and then washed with water, tissue sections were dried, sealed and dehydrated for transparency; immunofluorescence: DAPI stained cell nuclei for 10 min; after drying in the dark, anti-fluorescence quencher was added to seal the sections, and observed and analyzed under a fluorescence microscope.

[0054] Example 1. Expression level of ApoD in salivary adenoid cystic carcinoma

[0055] 1. Experimental Methods

[0056] qRT-PCR: HSG cells, SACC-83 cells and SACC-LM cells in the logarithmic growth phase were collected, washed with PBS three times, total RNA of cells was extracted, and the total RNA concentration was detected; an equal amount of RNA (about 800 ng) was added to each group, reversed to cDNA using a reverse transcription kit, and the expression of ApoD relative to glyceraldehyde triphosphate dehydrogenase (GAPDH) in the three cell lines of HSG, SACC-83 and SACC-LM was detected by Roche qPCR instrument. The expression of ApoD relative to GAPDH is shown in the figure. Figure 1 As shown in A.

[0057] Salivary gland adenoid cystic carcinoma tissues and normal adjacent tissues were obtained from the Department of Head and Neck Oncology, West China Hospital of Stomatology, Sichuan University. The tissues were ground with liquid nitrogen, and tissue proteins were extracted with RIPA lysis buffer. Tumor tissues were marked as T, and normal adjacent tissues were marked as N. Western blotting was used to detect the expression levels of ApoD in tumor tissues and normal adjacent tissues. The detection and results are shown in Figure 1 As shown in B. Casein kinase 2 (CK2) was used as a positive control for salivary gland adenoid cystic carcinoma tumor tissue, and βactin was used as an internal reference.

[0058] Salivary gland adenoid cystic carcinoma tissue and normal salivary gland tissue were obtained from the Department of Head and Neck Oncology, West China Hospital of Stomatology, Sichuan University. The tissues were fixed with 4% paraformaldehyde, dehydrated and immersed in wax to prepare paraffin sections. Paraffin sections were dewaxed and rehydrated in xylene and gradient alcohol (100%, 95%, 90%, 80%, 70%, 50%), washed with tap water 3 times, 5 min each time; soaked in 3% hydrogen peroxide in the dark to remove endogenous catalase, washed with water 3 times after 10 min, 5 min each time; antigen repair was performed with sodium citrate antigen repair solution, microwave oven high heat for 5 min, and the repair solution was adjusted to medium-high heat and maintained for 5 min after boiling; after the repair solution naturally cooled to room temperature, washed with PBS 3 times, 5 min each time, blocked with 5% BSA at room temperature for 1 hour, incubated with primary antibody at 4°C overnight, incubated with secondary antibody at room temperature for 40 min, washed with PBS 3 times, 5 min each time, developed with DAB colorimetric solution, stained with hematoxylin solution, differentiated with 1% hydrochloric acid alcohol for 1-2s, blued with 0.6% ammonia water, dehydrated to make it transparent, sealed after drying, and observed under an upright microscope; dehydrated with alcohol gradient to make it transparent, sealed with neutral gum after drying, and observed under an upright microscope. Normal salivary gland tissue is marked as NSG, and tumor tissue is marked as SACC. The expression level of ApoD was observed and recorded under an upright fluorescence microscope. The darker the yellow coloration and the wider the coloration range, the higher the ApoD expression. The results are shown in Figure 2. Figure 1 As shown in C.

[0059] H&E staining: Paraffin sections were made from tissues that were positive or negative for peripheral nerve invasion. The paraffin sections were dewaxed and rehydrated in xylene and gradient alcohol (100%, 95%, 90%, 80%, 70%, 50%), and washed with tap water three times, 5 minutes each time. The paraffin sections were placed in hematoxylin staining solution for 2-3 minutes, and then differentiated in 1% hydrochloric acid alcohol for 1-2 seconds after washing. After washing again, they were blued with 0.6% ammonia water. They were stained with eosin for 1 minute. The sections were dehydrated with gradient alcohol until transparent, and sealed with neutral gum after drying. The invasion of the tumor on the peripheral nerves was observed under an upright fluorescence microscope. Tumor cells were marked as T, nerves were marked as N, samples with negative peripheral nerve invasion were SACC PNI-, and samples with positive peripheral nerve invasion were SACC PNI+. The difference in ApoD expression levels between negative and positive tissue specimens was compared. The results are as follows: Figure 1 As shown in D.

[0060] 2. Experimental results

[0061] The experimental results are shown in Figure 1 : RT-PCR experiments showed that compared with normal salivary gland acinar cells, ApoD was upregulated in adenoid cystic carcinoma cells. In the common adenoid cystic carcinoma cell line SACC-83, ApoD was upregulated by about 2-3 times; in the metastatic adenoid cystic carcinoma cell line SACC-LM, ApoD was upregulated by 5-6 times; compared with SACC-83, ApoD expression in SACC-LM was significantly increased ( Figure 1A). Western blot was used to randomly detect 3 cases of salivary gland adenoid cystic carcinoma and adjacent control tissues. The results showed that the ApoD protein level was significantly increased in the cancer tissues ( Figure 1 B). Immunohistochemistry results showed that ApoD expression levels were increased in tumor tissues compared with normal salivary gland tissues ( Figure 1 C). H&E staining was used to identify tissue specimens with positive peripheral nerve invasion and to analyze the difference in ApoD expression levels between specimens with positive and negative peripheral nerve invasion. The results showed that ApoD protein expression was also elevated in specimens with positive peripheral nerve invasion ( Figure 1 D). The above results indicate that ApoD is significantly upregulated in salivary gland adenoid cystic carcinoma and is involved in the peripheral nerve invasion and progression of salivary gland adenoid cystic carcinoma.

[0062] Example 2: Role of ApoD in the progression of salivary adenoid cystic carcinoma

[0063] 1. Experimental Methods

[0064] ApoD gene expression silencing: SACC-83 cells (83) and SACC-LM cells (LM) were cultured in cell culture plates. When the cells grew to the logarithmic growth phase, they were transfected with the control group small interfering RNA (siNC) and ApoD small interfering RNA (siApoD), respectively. Two days after transfection, the cells were collected to extract cellular RNA and protein, and the ApoD silencing efficiency was detected by qRT-PCR and protein immunoblotting, respectively. The relative RNA expression level of the control group siNC was standardized to 1, and the relative expression level of ApoD in the ApoD gene silencing group siApoD was calculated. GAPDH was used as an internal control for protein immunoblotting. The results are shown in Figure 2 As shown in A.

[0065] Migration and invasion assay: SACC-83 cells (83) and SACC-LM cells (LM) were cultured in cell culture plates. When the cells grew to the logarithmic growth phase, they were transfected with the control group small interfering RNA (siNC) and ApoD small interfering RNA (siApoD), respectively. Two days after transfection, the cells were collected and counted. The migration and invasion ability of tumor cells was evaluated by Transwell assay. (1) Migration assay: 200 μl serum-free medium was added to the upper chamber, and the cells were wetted in the Transwell chamber at 37°C. The medium was discarded after incubation in the incubator for 10 min. 50,000 siNC group cells or siApoD group cells diluted in 200 μl serum-free medium were added and cultured in the upper chamber. 600 μl complete medium containing 10% serum was added to the lower chamber. The upper chamber was placed in the lower chamber and cultured in a cell culture incubator. After 1-2 days, crystal violet staining was used to identify the cells that passed through the chamber. The cells were observed and counted under an inverted microscope. The difference in the number of positive cells between the control group (siNC) and the experimental group (siApoD) was statistically analyzed. (2) Invasion assay: After wetting the chamber, add 1:8 diluted matrix gel, incubate in a cell culture incubator at 37°C for 2 h, then add 50,000 siNC group cells or siApoD group cells diluted in 200 μl serum-free culture medium and culture them in the upper chamber. Add 600 μl complete culture medium containing 10% serum to the lower chamber, place the upper chamber in the lower chamber, and culture in a cell culture incubator. After 1-2 days, crystal violet staining was used to identify cells that passed through the chamber, and the cells were observed and counted under an inverted microscope. The difference in the number of positive cells between the control group (siNC) and the experimental group (siApoD) was calculated. The results of migration and invasion assays are shown in Figure 2. Figure 2 B and 2C.

[0066] Detection of migration and invasion related markers: The control group small interfering RNA (siNC) and ApoD small interfering RNA (siApoD) were transfected respectively. Two days after transfection, the cells were collected to extract total cell protein, and the changes in the expression levels of migration and invasion related markers MMP2 and Vimentin between the control group (siNC) and the experimental group (siApoD) were detected. The test results are shown in Figure 2 As shown in D.

[0067] 2. Experimental results

[0068] Lipofectamine transfection with siRNA targeting ApoD successfully silenced salivary gland adenoid cystic carcinoma cell line SACC83 ( Figure 2 83) and SACC-LM ( Figure 2 ApoD mRNA and protein expression in LM ( Figure 2 A). Migration and invasion experiments found that after silencing ApoD expression, the migration and invasion abilities of salivary gland adenoid cystic carcinoma cells were significantly reduced ( Figure 2B and 2C). Western blot results showed that silencing ApoD led to a decrease in the levels of MMP2 and Vimentin proteins in tumor cells ( Figure 2 D), thereby inhibiting tumor cell migration and invasion. The above results indicate that silencing ApoD expression can inhibit the progression of salivary gland adenoid cystic carcinoma.

[0069] Example 3: Role of ApoD in the neural invasion of salivary adenoid cystic carcinoma

[0070] 1. Experimental Methods

[0071] In vitro ganglion culture: 4-week-old C57 mice were purchased from Jicui Yaokang Biotechnology. After being killed, they were soaked in 75% alcohol for 15 minutes for disinfection. After cutting the back skin, the entire spine was removed. The spine was cut along the central axis and the spinal cord was opened. The white ganglia located in the intervertebral foramen were visible, which were dorsal root ganglia. The ganglia were removed and washed 2-3 times with sterile PBS solution and placed in a 6-well cell culture plate containing 10μl of matrix gel. After incubation in a 37℃ incubator for 5 minutes, Neronbasal medium containing B27 was added. After culturing for 3-5 days, a single nerve filament emitted from the ganglion was visible, which can be regarded as a single nerve fiber to simulate the in vitro peripheral nerve invasion model.

[0072] SACC-83 cells and SACC-LM cells were cultured in cell culture plates. When the cells grew to the logarithmic growth phase, they were transfected with control group small interfering RNA (siNC) and ApoD small interfering RNA (siApoD), respectively. The cells were collected 2 days after transfection. Freshly extracted dorsal root ganglia were cultured in 6-well plates. After culturing for 5 days according to the above method to obtain an in vitro peripheral nerve invasion model, 10,000 control group cells (siNC) and experimental group cells (siApoD) were added to each well, and the culture medium was replaced with DMEM complete culture medium containing 10% fetal bovine serum. After continuing to culture for 5 days, the nerve invasion was observed under an inverted fluorescence microscope (the results are shown in Figure 3 A and 3C), and the differences in tumor invasion of nerves between the control group and the experimental group were statistically analyzed (the results are shown in Figure 3 B and 3D).

[0073] 2. Experimental results

[0074] Primary dorsal root ganglia of C57 mice were extracted and a 3D co-culture model was constructed. After simulating nerve fiber formation in vitro, tumor cells were added to observe the neural invasion ability of tumor cells. The experimental results showed that in the SACC-83 cell line, silencing ApoD expression inhibited tumor cell-nerve cell signal crosstalk, resulting in a decrease in bead-like structures, indicating that silencing ApoD led to a weakening of the neural invasion ability of tumor cells ( Figure 3A and 3B). In the salivary gland adenoid cystic carcinoma cell line SACC-LM, silencing ApoD expression also reduced the neural invasion ability of tumor cells ( Figure 3 C and 3D). The above results indicate that silencing ApoD expression can inhibit the peripheral nerve invasion and progression of salivary gland adenoid cystic carcinoma.

[0075] Example 4: Screening for inhibitors that inhibit ApoD expression

[0076] 1. Experimental Methods

[0077] SACC-83 cells (83) and SACC-LM cells (LM) were cultured in 6-well plates. DMSO (control group Con) and WP1066 (experimental group WP1066) were added at a concentration of 1 μM during the logarithmic growth phase. After 1 day of treatment, the cells were collected to extract RNA, and the changes in the expression levels of STAT3 and ApoD between the control group (Con) and the experimental group (WP1066) were detected. The test results are shown in Figure 4 As shown in A. WP1066 is a specific inhibitor targeting JAK2 / STAT3.

[0078] SACC-83 cells (83) and SACC-LM cells (LM) were cultured in 6-well plates. DMSO (control group Con), WP1066 (experimental group WP1066), and WP1066+exogenous recombinant ApoD (experimental group WP1066+ApoD) were added during the logarithmic growth phase, with WP1066 concentration of 1 μM and ApoD concentration of 200 ng / ml. After 1 day of treatment, the cells were collected to extract proteins, and the changes in STAT3 and ApoD protein expression levels in the DMSO group (control group Con), WP1066 group (experimental group WP1066), and WP1066+exogenous recombinant ApoD group (experimental group WP1066+ApoD) were detected. The test results are shown in Figure 2 Figure 4 As shown in B.

[0079] Migration and invasion assay: SACC-83 cells (83) and SACC-LM cells (LM) were cultured in 6-well plates. DMSO (control group Con), WP1066 (experimental group WP1066), and WP1066 + exogenous recombinant ApoD (experimental group WP1066 + ApoD) were added during the logarithmic growth phase. The concentration of WP1066 was 1 μM and the concentration of ApoD was 200 ng / ml. The cells were collected after 1 day of treatment. The migration and invasion ability of tumor cells was evaluated by Transwell assay. (1) Migration assay: 200 μl serum-free culture medium was added to the upper chamber, and the cells were wetted in the Transwell chamber at 37°C. The culture medium was discarded after incubation in the incubator for 10 min. 50,000 cells diluted in 200 μl serum-free culture medium were added to the DMSO group (Con), WP1066 treatment group (WP1066) and WP1066 + exogenous recombinant ApoD treatment (WP1066 + ApoD) respectively and cultured in the upper chamber. 600 μl of complete culture medium containing 10% serum was added to the lower chamber. The upper chamber was placed in the lower chamber and cultured in a cell culture incubator. After 1-2 days, crystal violet staining was used to identify the cells that passed through the chamber, and the cells were observed and counted under an inverted microscope. The difference in the number of positive cells among the DMSO group (Con), WP1066 treatment group (WP1066) and WP1066 + exogenous recombinant ApoD treatment group (WP1066 + ApoD) was counted. (2) Invasion assay: After wetting the chamber, add 1:8 diluted matrix gel, incubate in a cell culture incubator at 37°C for 2 h, add 50,000 DMSO group (Con) cells and WP1066 group (WP1066) cells or WP1066 + exogenous recombinant ApoD (WP1066 + ApoD) cells diluted in 200 μl serum-free culture medium, and culture them in the upper chamber. Add 600 μl complete culture medium containing 10% serum to the lower chamber, place the upper chamber in the lower chamber, and culture in a cell culture incubator. After 1-2 days, crystal violet staining is used to identify cells that have passed through the chamber, and the cells are observed and counted under an inverted microscope. The difference in the number of positive cells among the DMSO group (Con), WP1066 group (WP1066) and WP1066 + exogenous recombinant ApoD (WP1066 + ApoD) cell groups is counted. The results of migration and invasion assays are shown in Figure 2. Figure 4 C and 4D.

[0080] Detection of migration and invasion related markers: DMSO (control group Con), WP1066 (experimental group WP1066), WP1066 + exogenous recombinant ApoD (experimental group WP1066 + ApoD) were used to extract total cell protein, and the changes in the expression levels of migration and invasion related markers MMP2 and Vimentin were detected between the DMSO group (Con), WP1066 treatment group (WP1066), and WP1066 + exogenous recombinant ApoD treatment group (WP1066 + ApoD). The test results are shown in Figure 2. Figure 4 As shown in E.

[0081] SACC-83 cells (83) and SACC-LM cells (LM) were cultured in cell culture plates. When the cells grew to the logarithmic growth phase, DMSO (control group Con), WP1066 (experimental group WP1066), and WP1066 + exogenous recombinant ApoD (experimental group WP1066 + ApoD) were added respectively. The concentration of WP1066 was 1 μM and the concentration of ApoD was 200 ng / ml. The cells were collected after 1 day of treatment. Freshly extracted dorsal root ganglia were cultured in 6-well plates. After culturing for 5 days according to the method described in Example 3 to obtain an in vitro peripheral nerve invasion model, 10,000 cells of the DMSO treatment group (control group Con), the WP1066 treatment group (experimental group WP1066), and the WP1066+exogenous recombinant ApoD treatment group (experimental group WP1066+ApoD) were added to each well, respectively. The concentration of WP1066 was 1 μM, and the concentration of ApoD was 200 ng / ml. After 1 day, the cells were collected and the culture medium was replaced with DMEM complete culture medium containing 10% fetal bovine serum. After further culturing for 5 days, the nerve invasion was observed under an inverted fluorescence microscope, and the difference in nerve invasion between the control group and the experimental group was statistically analyzed (the results are shown in Figure 2). Figure 4 F and 4G).

[0082] 2. Experimental results

[0083] qRT-PCR showed that WP1066 successfully inhibited JAK2 / STST3 signaling, leading to a significant decrease in ApoD mRNA and protein levels in salivary gland adenoid cystic carcinoma cells ( Figure 4 A and 4B). Migration and invasion experiments found that WP1066 treatment led to a significant decrease in the migration and invasion abilities of tumor cells, while ApoD stimulation was able to restore the migration and invasion abilities of tumor cells ( Figure 4 C and 4D). Similar to silencing ApoD, WP1066 treatment inhibited the expression levels of MMP2 and Vimentin proteins in tumor cells ( Figure 4 E), resulting in decreased migration and invasion abilities. Similarly, the neural invasion ability of tumor cells treated with WP1066 was also significantly weakened ( Figure 4 F and 4G). The above results indicate that WP1066 can target ApoD and inhibit the expression of ApoD mRNA and protein, thereby weakening the migration and invasion ability of tumor cells and hindering the progression of peripheral nerve invasion.

[0084] Example 5. Effects of ApoD receptor inhibitors

[0085] 1. Experimental Methods

[0086] Conditioned medium (CM) collection: SACC-83 cells (83) and SACC-LM cells (LM) were cultured in 10 cm cell culture dishes. The culture medium was replaced with serum-free medium during the logarithmic growth phase. After 24 h of continuous culture, the cell culture supernatant was collected and centrifuged at 2000 g, 4°C for 20 min to obtain the supernatant to make 83 conditioned medium (83-CM) and LM conditioned medium (LM-CM).

[0087] Cell immunofluorescence: DRG cells were donated by the National Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University. DRG cells were cultured in DMEM medium containing 10% serum. DRG cells in the logarithmic growth phase were digested and resuspended in DMEM medium containing 10% serum and counted. 10,000 DRG cells were cultured in 12-well plates. After 12 hours of culture, the cells were treated with DMSO (control group Con), CXCR4 inhibitor WZ811 treatment group (WZ811), 83 conditioned medium treatment group (83-CM), 83 conditioned medium + CXCR4 inhibitor WZ811 treatment group (83-CM+WZ811), LM conditioned medium treatment group (LM-CM), LM conditioned medium + CXCR4 inhibitor WZ811 treatment group (LM-CM+WZ811), exogenous ApoD treatment group (ApoD) and exogenous ApoD+WZ811 treatment group. The concentration of ApoD was 200 ng / ml and the concentration of WZ811 treatment was 1 μM. After 1 day of treatment, cells were collected for Tuj1 immunofluorescence staining (Tuj1 is a marker of neuronal axons). The treated cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 15 minutes, washed 3 times with PBS, 5 minutes each time; treated with 0.1% Triton X-100 for 10 minutes; washed 3 times with PBS, 5 minutes each time; blocked with 5% BSA at room temperature for 1 hour, rinsed with PBS; incubated with primary antibody at 4°C overnight, incubated with secondary antibody at room temperature for 40 minutes, washed 3 times with PBS, 5 minutes each time; dried in the dark, and sealed with anti-fluorescence quencher, and observed and analyzed with a fluorescence microscope. The results are shown in the figure. Figure 5 As shown in A.

[0088] Cell immunohistochemistry: Digest DRG cells in the logarithmic growth phase, add freshly digested DRG cells to a 6-well cell culture plate after adding a sterile coverslip, and culture for 12 hours until the cells adhere to the wall before collecting the cells for immunohistochemistry. Wash the cells 3 times with PBS, fix with 4% paraformaldehyde for 15 minutes, wash with PBS 3 times, 5 minutes each time; treat with 0.1% Triton X-100 for 10 minutes; wash with PBS 3 times, 5 minutes each time; block with 5% BSA at room temperature for 1 hour, rinse with PBS; incubate with primary antibody at 4°C overnight, incubate with secondary antibody at room temperature for 40 minutes, wash with PBS 3 times, 5 minutes each time; develop color with DAB under an optical microscope, stain with hematoxylin for 40 seconds, differentiate with hydrochloric acid and alcohol for 1-2 seconds, reverse blue with ammonia water and wash with water, and dry and seal the tissue sections before dehydration and transparency. Observe and analyze under an inverted fluorescence microscope, the results are as follows Figure 5 As shown in B.

[0089] In vitro peripheral nerve invasion model: 4-week-old C57 mice were purchased from Jicui Yaokang Biological. After being killed, they were soaked in 75% alcohol for 15 minutes for disinfection. After cutting the back skin, the entire spine was removed. The spine was cut along the central axis and the spinal cord was opened. The white ganglia located in the intervertebral foramen were visible, which were dorsal root ganglia. The ganglia were removed and washed 2-3 times with sterile PBS solution and placed in a 6-well cell culture plate containing 10μl of matrix gel. After incubation in a 37°C incubator for 5 minutes, Neronbasal medium containing B27 was added. After culturing for 5 days, 10,000 SACC-83 cells (83) or SACC-LM cells (LM) in the logarithmic growth period were added, and the culture medium was replaced with DMEM complete culture medium containing 10% serum. The control group (Con) was added with sterile PBS solution, the experimental group was added with 200 ng / ml ApoD exogenous recombinant protein (ApoD), and the experimental group was added with 200 ng / ml ApoD exogenous recombinant protein + 1 μM WZ811 (ApoD+WZ811). After 3 days of continuous culture, the peripheral nerve invasion was observed, and the differences in nerve invasion between the groups were statistically analyzed. The results are as follows Figure 5 C and 5D.

[0090] 2. Experimental results

[0091] Immunofluorescence revealed that salivary gland adenoid cystic carcinoma cell culture supernatant and exogenous ApoD stimulation promoted the elongation of nerve cell axons, but this promotion effect depended on the activation of CXCR4 receptor ( Figure 5 A). Furthermore, immunohistochemistry identified that CXCR4 was widely expressed in the cytoplasm and axons of neurons ( Figure 5 B). The results of the neuroinvasion experiment showed that exogenous ApoD stimulation enhanced the neuroinvasion ability of tumor cells SACC-83, but the CXCR4 inhibitor WZ811 could weaken the promoting effect of exogenous ApoD ( Figure 5C), WZ811 can also inhibit the promotion of SACC-LM neuroinvasion by ApoD ( Figure 5 D) The above results indicate that WZ811 blocks the receptor CXCR4 of tumor cell-derived ApoD on nerve cells, thereby blocking the peripheral nerve invasion and progression.

[0092] Example 6: Inhibitory effect of combined use of WP1066 and WZ811 on peripheral nerve invasion of salivary adenoid cystic carcinoma

[0093] 1. Experimental Methods

[0094] SACC-83 cells were cultured in a cell culture plate. When the cells grew to the logarithmic growth phase, DMSO (control group Con) and WP1066 (experimental group WP1066) were added, respectively, with a WP1066 concentration of 1 μM, and the cells were collected after 1 day of treatment. 4-week-old C57 mice were purchased from Jicui Yaokang Biological. After being killed, they were soaked in 75% alcohol for 15 minutes for disinfection and sterilization. After cutting the back skin, the entire spine was removed. The spine was cut along the central axis and the spinal cord was opened. The white ganglia located in the intervertebral foramen were visible, which were the dorsal root ganglia. The ganglion was removed and washed 2-3 times with sterile PBS solution, then placed in a 6-well cell culture plate containing 10 μl of matrix gel. After incubation at 37°C for 5 minutes, Neronbasal medium containing B27 was added. After 5 days of culture, 10,000 control group cells (Con) or WP1066-treated group cells (WP1066) were added, and WZ811 was added to the 3D culture model. The concentration of WZ811 was 1 μM. The control group plus WZ811 was marked as WZ811, and the WP1066-treated group plus WZ811 was marked as WP1066+WZ811. After 3 days of culture, the peripheral nerve invasion was observed, and the differences in nerve invasion between the groups were statistically analyzed. The results are shown in Figure 2. Figure 6 A and 6B.

[0095] 2. Experimental results

[0096] To investigate the inhibitory effect of WP1066 and WZ811 combined on the invasion of peripheral nerves of salivary gland adenoid cystic carcinoma. Nerve-tumor 3D co-culture showed that compared with the control group, WP1066 treatment inhibited the invasion of tumor cells into peripheral nerves, and WZ811 treatment also weakened the invasion of tumor cells into peripheral nerves. In addition, the combined use of WP1066 and WZ811 had a better inhibitory effect on the invasion of tumor cells into peripheral nerves than WP1066 or WZ811 alone, playing a synergistic role ( Figure 6 A and 6B).

[0097] In summary, the present invention has found that ApoD is related to the development of tumor peripheral nerve invasion, and ApoD inhibitors can weaken the migration and invasion ability of tumor cells, thereby inhibiting the progression of peripheral nerve invasion. At the same time, the present invention has found that WP1066 and WZ811 can inhibit the progression of peripheral nerve invasion by inhibiting AopD expression. In addition, the present invention has found that the combined use of WP1066 and WZ811 can play a synergistic role in inhibiting tumor cell peripheral nerve invasion, and can prevent and / or treat tumor recurrence and metastasis, thereby improving patient prognosis and improving quality of life. The present invention provides a basis for clinical drug treatment of patients with peripheral nerve invasion of salivary gland adenoid cystic carcinoma, and has good application prospects.

Claims

1. A pharmaceutical composition for treating peripheral nerve invasion caused by salivary gland adenoid cystic carcinoma, characterized in that: The pharmaceutical composition consists of WP1066 and WZ811.

2. The pharmaceutical composition according to claim 1, characterized in that: The molar ratio of WP1066 to WZ811 is 1:1-10.

3. The pharmaceutical composition according to claim 2, characterized in that: The molar ratio of WP1066 to WZ811 is 1:

1.

4. The method for preparing the pharmaceutical composition according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: taking WP1066 and WZ811 according to a molar ratio, mixing, and obtaining.

5. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a medicament for treating peripheral nerve invasion caused by salivary gland adenoid cystic carcinoma.

6. A pharmaceutical preparation for treating peripheral nerve invasion caused by salivary gland adenoid cystic carcinoma, characterized in that: The pharmaceutical preparation is a preparation prepared by taking the pharmaceutical composition according to any one of claims 1 to 3 as an active ingredient and adding pharmaceutically acceptable excipients.

7. A combined drug for treating peripheral nerve invasion caused by salivary gland adenoid cystic carcinoma, characterized in that: The combination drug contains WP1066 and WZ811 of the same or different strengths, which are administered simultaneously or separately, and a pharmaceutically acceptable carrier.

8. The combined drug according to claim 7, characterized in that: The molar ratio of WP1066 to WZ811 is 1:1-10.

9. The combined drug according to claim 8, characterized in that: The molar ratio of WP1066 to WZ811 is 1:

1.

10. Use of WP1066 and WZ811 in combination for the preparation of a drug for treating peripheral nerve invasion caused by salivary gland adenoid cystic carcinoma.

11. The use according to claim 10, characterized in that: The molar ratio of WP1066 to WZ811 is 1:1-10.

12. The use according to claim 11, characterized in that: The molar ratio of WP1066 to WZ811 is 1:1.

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