Use of vcPIP1 inhibitors in the manufacture of a product for the treatment of prostate cancer

Abstract

The present application relates to the field of medicine, and more particularly to the application of VCPIP1 inhibitor in the preparation of products for treating prostate cancer, which is of great significance for promoting the precise treatment of prostate cancer. The present application verifies that the specific inhibitor CAS-12290-201 (CAS) of VCPIP1 can effectively inhibit the proliferation, clone formation, migration, invasion and EMT process of prostate cancer cells in vitro, and inhibit the growth of subcutaneous transplanted tumors in vivo. Therefore, the inhibitor strategy targeting VCPIP1 has potential therapeutic value in rapidly progressing mHSPC.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and more particularly to the use of VCPIP1 inhibitors in the preparation of products for the treatment of prostate cancer. Background Technology

[0002] Prostate cancer is the most common malignant tumor of the male genitourinary system, and its incidence is increasing globally. For localized prostate cancer, the prognosis is generally good after radical surgery or radiotherapy, with a 5-year survival rate as high as 97%, demonstrating a significant advantage of early treatment. However, some prostate cancer patients in my country are diagnosed at an advanced stage, and advanced metastatic prostate cancer remains a challenge in clinical treatment.

[0003] Androgen deprivation therapy (ADT) is the foundational treatment for advanced prostate cancer. Metastatic hormone-sensitive prostate cancer (mHSPC) is a type of metastatic prostate cancer that is sensitive to endocrine therapy. In clinical practice, the vast majority of patients with metastatic prostate cancer who have not undergone endocrine therapy meet the diagnostic criteria for mHSPC. In recent years, significant breakthroughs have been made in the diagnosis and treatment of mHSPC. Novel combination therapies based on ADT (novel endocrine therapies or chemotherapy) have significantly improved the overall survival of mHSPC patients and have become the treatment option with the highest level of evidence and recommendation for this patient population.

[0004] However, almost all prostate cancers inevitably develop into castration-resistant prostate cancer (CRPC) after a period of androgen deprivation therapy. CRPC tumor cells exhibit malignant phenotypes such as increased genomic instability and DNA damage repair defects, resulting in a significantly increased degree of malignancy. Even after comprehensive treatment with multiple regimens, the cancer continues to progress rapidly, leading to an extremely poor prognosis. Therefore, there is an urgent need to find new therapeutic targets and strategies. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide the application of VCPIP1 inhibitors in the preparation of products for the treatment of prostate cancer, which is of great significance for promoting precision treatment of prostate cancer.

[0006] This invention is implemented as follows: This invention provides the use of VCPIP1 inhibitors in the preparation of products for the treatment of prostate cancer.

[0007] Furthermore, the product includes any one of the following products: (1) Products used to inhibit the proliferation and / or cloning of prostate cancer cells; (2) Products used to inhibit the migration and invasion of prostate cancer cells; (3) Products used to inhibit prostate cancer metastasis; (4) Products used to reverse the epithelial-mesenchymal transition of prostate cancer cells.

[0008] Furthermore, the prostate cancer includes metastatic hormone-sensitive prostate cancer and metastatic castration-resistant prostate cancer.

[0009] Furthermore, the VCPIP1 inhibitor is CAS-12290-201.

[0010] This invention has the following advantages: It demonstrates that treatment with the VCPIP1-specific inhibitor CAS-12290-201 (CAS) effectively inhibits the proliferation, colony formation, migration, invasion, and EMT process of prostate cancer cells in vitro, and inhibits the growth of subcutaneous xenografts and lung metastases in vivo. Therefore, the VCPIP1-targeting inhibitor strategy has potential therapeutic value in rapidly progressive mHSPC. Attached Figure Description

[0011] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0012] Figure 1 EdU assay was used to detect the effect of CAS treatment on the proliferation of DU145 and LNCaP cells.

[0013] Figure 2 The clonogenic assay was used to detect the effect of CAS treatment on the clonogenic ability of DU145 and LNCaP cells.

[0014] Figure 3 The effect of CAS treatment on the migration ability of DU145 and LNCaP cells was detected by scratch assay.

[0015] Figure 4 Transwell assays were used to investigate the effect of CAS treatment on the invasive ability of DU145 and LNCaP cells.

[0016] Figure 5 Western blot analysis was performed to detect the effect of CAS treatment on the expression of EMT-related molecular markers in DU145 (A) and LNCaP (B) cells.

[0017] Figure 6The effect of CAS treatment on the tumorigenicity of DU145 cells in a subcutaneous xenograft model was detected; representative tumor images (A) tumor volume growth curve (B) tumor weight (C) (D) immunohistochemical detection and quantification of EMT-related protein and Ki67 expression levels in xenograft tumors of each group (E).

[0018] Figure 7 The effect of CAS treatment on the in vivo metastasis ability of DU145 cells was detected using a tail vein transfer model. Detailed Implementation

[0019] The technical solution of the present invention will now be clearly and completely described in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified in the embodiments, conditions are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0020] 1. Experimental Materials DU145 prostate cancer cells (CL-0075) and LNCaP prostate cancer cells (CL-0143) were both purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0021] The molecular formula of the small molecule inhibitor CAS-12290-201 is C 14 H 13 ClFN3O2, purchased from MedChemExpress (MCE), Cat. No.: HY-159000; CAS No.: 3025298-68-1.

[0022] 2 Experimental Methods 2.1 Cloning experiment DU145 and LNCaP cells in logarithmic growth phase were taken at a dose of 1×10⁻⁶. 3 Cells were seeded per well in 6-well plates and treated with the specified concentration of CAS-12290-201 (2uM / 4uM). Cell growth was observed under a microscope and the medium was changed every 48 h. After 10 days of culture, cell colony formation was observed under a microscope. The 6-well plates were removed, residual solution was aspirated from the wells, and the cells were washed with PBS for 5 min. Each well was then fixed in 500 mL of 4% paraformaldehyde for 20 min. After fixation, the paraformaldehyde fixative was aspirated, and the cells were washed with PBS for 5 min. The PBS was then aspirated, and 500 mL of 1% crystal violet was added to each well for staining for 30 min. Each well was washed twice with PBS, and the PBS was aspirated to remove the crystal violet from the wells. Cell colony formation in each well was photographed and counted using a CTL ELISA spectrophotometer, and subsequent analysis and mapping were performed using GraphPad Prism.

[0023] 2.2 EdU proliferation detection After treating DU145 and LNCaP cells with the specified concentration of CAS-12290-201 (2uM / 4uM) for 48 hours, cell proliferation capacity was assessed by EdU assay.

[0024] DU145 and LNCaP cells were harvested at a concentration of 1×10⁻⁶. 4 Seed cells / well in 24-well cell culture slides and incubate overnight. Prepare 2X EdU working solution; the recommended final EdU concentration is 10 μM (1X). Dilute EdU (10 mM) 1:500 with cell culture medium to obtain 2X EdU working solution (20 μM). Add an equal volume of the preheated (37ºC) 2X EdU working solution to the 24-well plate to achieve a final EdU concentration of 1X, and incubate for another 2 hours. After EdU labeling, remove the culture medium, add 500 μl of 4% paraformaldehyde, and fix at room temperature for 15 minutes. Remove the fixative, wash each well three times with 500 μl of washing buffer for 3-5 minutes each time. Remove the washing buffer, and incubate each well with 300 μl of permeabilization buffer (PBS containing 0.3% Triton X-100) at room temperature for 10-15 minutes. Remove the permeabilization buffer. Wash cells 1-2 times per well with 500 μL of washing buffer, 3-5 minutes each time. Prepare the Click Additive Solution: Dissolve one tube of Click Additive in 1.3 mL of deionized water, mix until completely dissolved. This is the Click Additive Solution. After preparation, aliquot appropriately and store at -20°C. Prepare the Click reaction solution (for a total volume of 500 μL): 430 μL Click Reaction Buffer + 20 μL CuSO4 + 1 μL Azide 488 + 50 μL Click Additive Solution. Remove the washing buffer from the previous step. Add 0.5 mL of Click reaction solution to each well, gently shake the culture plate to ensure the reaction mixture evenly covers the sample. Incubate at room temperature in the dark for 30 minutes. Aspirate the Click reaction solution and wash 3 times with washing buffer, 3-5 minutes each time. Stain the cell nuclei with DAPI staining solution and incubate at room temperature in the dark for 10 minutes. Aspirate the DAPI staining solution. Wash 3 times with washing buffer, 3-5 minutes each time. Add one drop of anti-fluorescence quencher to the slide, carefully remove the cell smear, place it on the slide with the cell side down (avoid air bubbles), and finally take random field-of-view photographs under an upright fluorescence microscope, taking care to avoid light.

[0025] 2.3 Transwell Experiment DU145 and LNCaP cells were treated with CAS-12290-201 (2 μM / 4 μM) at a specified concentration for 48 hours, and their invasive ability was assessed by Transwell assay. DU145 and LNCaP cells were harvested, replaced with serum-free medium, and starved for 24 h. Starved cells were digested with trypsin containing 0.25% EDTA, and digestion was terminated with complete medium. Cells were collected in 15 mL centrifuge tubes, centrifuged, and the supernatant was discarded; the cell pellet was collected. The chambers were pretreated with 20 μg Matrigel gel on ice. After cell counting, each group was sputtered at 1–10 × 10⁶ cells / mL. 4 Cells were seeded at a density of 200 μL / well into Transwell chambers. 500 μL of complete culture medium containing 30% FBS was added to the bottom of each chamber, and the chambers were incubated for 48 h for subsequent invasiveness testing. After 48 h, the chambers were removed, residual solution was aspirated from the chambers, and the cells were washed with PBS for 5 min. Residual cells in the upper chamber were gently scraped away with a cotton swab, and the chambers were fixed in 500 mL of 4% paraformaldehyde for 20 min. After fixation, the paraformaldehyde fixative was aspirated, and the cells were washed with PBS for 5 min. The PBS was then aspirated, and 500 mL of 0.1% crystal violet was added for staining for 30 min. The chambers were then rinsed thoroughly with PBS. Subsequently, an appropriate amount of PBS was added to each chamber, and the cells were observed under a microscope. At least three different fields of view were randomly selected for photographing, and cell counting was performed using ImageJ software. Subsequent statistical analysis was then conducted.

[0026] 2.4 Scratch Test DU145 and LNCaP cells were treated with a specified concentration of CAS-12290-201 (2uM / 4uM) for 48 hours, and cell migration ability was assessed by scratch assay. DU145 and LNCaP cells were harvested at 3×10⁻⁶... 5 Cells were seeded per well in 6-well plates and cultured at 37°C in a 5% CO2 incubator. When cell confluence reached 90%, cells were evenly and vertically marked with a 1 ml pipette tip along a ruler; the cells were then gently washed three times with PBS to remove detached cells. After 0 h, the culture medium was replaced with serum-free medium, and the cells were cultured at 37°C in a 5% CO2 incubator. Cell migration was recorded in each group after 24 h.

[0027] 2.5 Western blot detection of protein expression 2.5.1 Use of pre-formed adhesive Dissolve one packet of MOPS-SDS Running Buffer in 1 L of deionized water. Remove the pre-cast gel from its packaging and peel off the pink adhesive strip at the bottom of the gel plate. Gently and parallelly push the comb out of the gel plate in the direction of the arrow. Avoid leaving any liquid residue in the wells when pushing the comb out. Remove the green silicone sealing strip from the inner frame of the electrophoresis tank, then reinsert it into the groove of the inner frame with the flat side facing out. Load the gel. Pour enough MOPS-SDS Running Buffer into the inner tank of the electrophoresis tank to cover the sample wells by 5-7 mm. Add the same electrophoresis buffer to the outer tank to ensure proper cooling. For best results, the buffer in the outer tank should be slightly lower than that in the inner tank, but should not overflow the gel plate. Use a syringe or other tool to draw an appropriate amount of 1X electrophoresis buffer and gently rinse the sample wells to remove air bubbles and residual storage buffer.

[0028] 2.5.2 Sample loading and electrophoresis After thawing, the protein samples were heated at 100℃ for 5 min, cooled, and gently mixed. 10 μg of protein sample was pipetted into each well, and the order of addition was recorded. The power was connected, and electrophoresis was performed at 80V for 20 min, then switched to 120V until the bromophenol blue reached the bottom of the gel, at which point electrophoresis was stopped.

[0029] 2.5.3 Transfer of film Preparation of rapid transfer buffer: 850ml double-distilled water + 100ml anhydrous ethanol + 50ml rapid transfer buffer stock solution. Cut the PVDF membrane to the size of the target gel and pre-soak it in methanol. After electrophoresis, remove the gel plates from the electrophoresis tank. Carefully insert a suitable pry bar into the gap between the gel plates. Slowly pry the gel plates at the top, middle, and bottom until the sides of the gel plates are completely separated. According to the pre-stained protein marker bands, cut the gel of the desired target protein and transfer it to the electrotransfer buffer, keeping it moist. Unfold the transfer clamp and stack the sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge pad in sequence from positive to negative, avoiding air bubbles between the gel and the PVDF membrane, and tighten the transfer clamp. Install the transfer clamp into the electrotransfer tank, ensuring the PVDF membrane is near the anode and the gel is near the cathode. Turn on the power and transfer at a constant current of 400 mA for 30 min (the current and transfer time are determined according to the molecular weight of the target protein).

[0030] 2.5.4 Blocking and Antibody Incubation After the transfer was completed, the PVDF membrane and gel were removed. The PVDF membrane was placed in the rapid blocking buffer and placed on a shaker for 10 min. The PVDF membrane was washed three times with 1×TBST for 10 min each time. The PVDF membrane was then placed in a small tank containing the corresponding primary antibody solution (antibody prepared according to the primary antibody instructions) and incubated overnight at 4°C on a shaker. The PVDF membrane was washed three times with 1×TBST for 10 min each time. The PVDF membrane was then placed in a small tank containing HRP-labeled secondary antibody dilution solution (antibody prepared according to the secondary antibody instructions) and gently shaken at room temperature for 1 hour. The PVDF membrane was washed three times with 1×TBST for 10 min each time, in preparation for subsequent experiments.

[0031] 2.5.5 Chemiluminescence, development, and fixing Mix equal volumes of reagents A and B thoroughly. Place the PVDF membrane, incubated with the antibody, protein side up, on plastic wrap, add an appropriate amount of the mixture, and incubate at room temperature for 1-2 minutes. Drain excess ECL reagent from the PVDF membrane, then place the PVDF membrane on plastic wrap with the protein blot side up in a darkroom and expose it to X-ray film.

[0032] 2.6 Construction of a subcutaneous xenograft model in nude mice DU145 cells in the logarithmic growth phase were digested and counted, and 5×10⁻⁶ cells were prepared. 6 Cells per 200 μl suspension were prepared at a stacking gel: diluent: complete culture medium ratio of 1:1:3, i.e., 40 μl stacking gel + 40 μl diluent + 120 μl complete culture medium, and then resuspended for 5 × 10⁶ cells / 200 μl. 6 5 × 10⁵ cells. Using a 1 ml syringe, administer 5 × 10⁵ cells. 6 200 μl cells / cell suspension were subcutaneously injected into the mid-posterior axillary region of nude mice. All mice were fed for 7 days after inoculation until tumors formed. The mice were then randomly divided into two groups of 5 mice each. Treatment began: Control group: intraperitoneal injection of physiological saline, volume identical to the treatment group, once daily; CAS group: intraperitoneal injection of CAS-12290-201 (40 mg / kg), once daily. Tumor size was measured weekly and tumor growth curves were plotted. Tumor volume was calculated using the following formula: V = π / 6 × major axis × minor axis. 2 Mice were sacrificed 28 days after cell injection, and tumor tissue was removed to measure the average tumor weight.

[0033] 2.7 Construction of a nude mouse tail vein transfer model DU145 cells in logarithmic growth phase were transfected with the Luciferase vector for later imaging. A 1ml disposable sterile syringe was used to slowly inject a 2×10⁶ cell / 100 μl cell suspension into the tail vein of mice. All mice were fed for 7 days after inoculation and then randomly divided into two groups of 5 mice each. Treatment began as follows: control group: intraperitoneal injection of physiological saline, volume identical to the treatment group, once daily; CAS group: intraperitoneal injection of CAS-12290-201 (40 mg / kg), once daily. After 6 weeks, mice were intraperitoneally injected with D-Luciferin (15 mg / ml) at a dose of 10 μl / g. 15-20 minutes later, mice were anesthetized with isoflurane, and then fluorescent imaging was performed using a bio-imaging system to record tumor metastasis.

[0034] 2.8 Statistical Analysis Data processing was performed using SPSS 26.0 software (IBM Corp, Armonk, NY, USA), and graph plotting was performed using GraphPad Prism 8.0 software. Normally distributed measurement data were expressed as mean ± standard deviation. The t-test was used for comparisons between two groups, the chi-square test was used for contingency table analysis, and one-way ANOVA was used for comparisons of three or more groups. P <0.05 indicates a statistically significant difference. Significant differences between groups are represented by: * P <0.05,** P <0.01, *** P <0.001, **** P <0.0001; ns indicates no significant difference.

[0035] 3. Experimental Results and Analysis 3.1 The VCPIP1 inhibitor CAS-12290-201 (CAS) inhibits the proliferation and clonogenic ability of prostate cancer cells. DU145 and LNCaP cells were treated with the VCPIP1-specific inhibitor CAS-12290-201 (CAS). EdU assay results showed that CAS treatment significantly reduced the proliferation of DU145 and LNCaP prostate cancer cells compared to the control group. Figure 1 Colony formation experiments further confirmed that CAS treatment significantly reduced the number of colonies formed in DU145 and LNCaP cells. Figure 2 These results indicate that inhibiting VCPIP1 can effectively reduce the proliferation and clonogenic ability of prostate cancer cells.

[0036] 3.2 VCPIP1 inhibitor CAS inhibits the migration and invasion of prostate cancer cells. The effects of CAS on the migration and invasion abilities of prostate cancer cells were evaluated using scratch healing assays and Transwell invasion assays. The scratch healing assay results showed that CAS treatment significantly delayed the scratch repair rate of DU145 and LNCaP cells. Figure 3 Transwell assays further confirmed that CAS treatment significantly reduced the ability of DU145 and LNCaP cells to penetrate the basement membrane. Figure 4 The above findings suggest that targeted inhibition of VCPIP1 can effectively weaken the migration and invasion characteristics of prostate cancer cells.

[0037] 3.3 VCPIP1 inhibitor CAS reverses epithelial-mesenchymal transition in prostate cancer cells This study aimed to elucidate the regulatory mechanism of CAS treatment on phenotypic transformation of prostate cancer cells by detecting molecular markers related to epithelial-mesenchymal transition (EMT) using Western blotting. Experimental data showed that, compared with the control group, CAS treatment significantly increased E-cadherin levels while decreasing the expression levels of N-cadherin and Vimentin. Figure 5 These molecular-level changes suggest that inhibiting VCPIP1 can effectively reverse the EMT process.

[0038] 3.4 VCPIP1 inhibitor CAS inhibits prostate cancer tumorigenesis in vivo To further confirm the effect of targeting VCPIP1 on prostate cancer progression, we constructed a subcutaneous xenograft model in nude mice using the DU145 cell line and treated it with CAS (carcinoma occult blood). The results showed that the tumor growth rate, volume, and weight in the CAS-treated group were significantly lower than those in the solvent control group. Figure 6 ).

[0039] Immunohistochemical analysis showed that after CAS treatment, the intermediate phenotypic markers N-cadherin and Vimentin in xenograft tumors were simultaneously downregulated, while the expression of the epithelial marker E-cadherin was significantly increased, and the proportion of Ki67-positive cells was significantly reduced. This evidence suggests that targeting VCPIP1 can inhibit the in vivo malignant progression of prostate cancer by regulating the EMT process and cell proliferation activity.

[0040] 3.5 VCPIP1 inhibitor CAS inhibits prostate cancer metastasis in vivo To further verify the effect of VCPIP1 targeting on prostate cancer metastasis in vivo, we constructed the DU145 / luciferase cell line and established a nude mouse metastasis model via tail vein injection, followed by CAS treatment. Six weeks after injection, we observed the metastasis of prostate cancer cells in vivo using a small animal in vivo imaging system. The results showed that the bioluminescent signal intensity of metastatic lesions in the CAS treatment group was significantly lower than that in the control group, with a statistically significant difference, indicating that the CAS treatment group had significantly fewer metastatic lesions. This confirms that targeting VCPIP1 can significantly inhibit the in vivo metastasis of prostate cancer cells. Figure 7 ).

[0041] 4. Discussion This invention utilizes the VCPIP1-specific small molecule inhibitor CAS-12290-201 (CAS) to functionally intervene in prostate cancer cells. We found that CAS treatment can significantly inhibit the proliferation, colony formation, migration, and invasion of prostate cancer cells in vitro and reverse the epithelial-mesenchymal transition process; simultaneously, it effectively inhibits tumor growth in an in vivo subcutaneous xenograft model.

[0042] In the previous mechanism analysis of this invention, we clearly established that VCPIP1 regulates the K48 ubiquitination modification of lysine 711 of UBE3A through deubiquitination, inhibiting the degradation of UBE3A via the proteasome pathway, thereby upregulating UBE3A protein levels, promoting p53 ubiquitination and degradation, and ultimately driving the malignant phenotype of prostate cancer. Based on this, the inhibition of VCPIP1 enzyme activity after CAS treatment should block the above-mentioned regulatory axis. The decreased cell proliferation, migration, and invasion abilities and the reversal of EMT markers observed in this invention after CAS treatment are highly consistent with the phenotype after VCPIP1 knockdown, suggesting that CAS may reduce the protein stability of UBE3A and restore p53 expression by inhibiting the deubiquitination function of VCPIP1, thereby antagonizing the malignant progression of prostate cancer.

[0043] In this invention, CAS significantly inhibited the growth of DU145 subcutaneous xenografts in vivo, indicating its in vivo efficacy, and no significant toxicity was observed. This provides preliminary preclinical evidence for the subsequent development of small molecule drugs targeting VCPIP1 for the treatment of rapidly progressive mHSPC. Currently, there are few reports on the application of VCPIP1 inhibitors in solid tumors. We are the first to apply CAS to a prostate cancer model and verify its anti-tumor activity, expanding the feasibility of VCPIP1 as a therapeutic target for prostate cancer.

[0044] In summary, this invention verifies the functional dependence of VCPIP1 as a driver of malignant progression in prostate cancer from the perspective of pharmacological intervention, and suggests that the VCPIP1-specific inhibitor CAS has the potential to become a precision treatment for rapidly progressive mHSPC.

Claims

1. Application of VCPIP1 inhibitors in the preparation of products for the treatment of prostate cancer.

2. The application according to claim 1, characterized in that: The product includes any one of the following products: (1) Products used to inhibit the proliferation and / or cloning of prostate cancer cells; (2) Products used to inhibit the migration and invasion of prostate cancer cells; (3) Products used to inhibit prostate cancer metastasis; (4) Products used to reverse the epithelial-mesenchymal transition of prostate cancer cells.

3. The application according to claim 1, characterized in that: The prostate cancers mentioned include metastatic hormone-sensitive prostate cancer and metastatic castration-resistant prostate cancer.

4. The application according to claim 1, characterized in that: The VCPIP1 inhibitor is CAS-12290-201.

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