Application of PARP inhibitor combined with SPOP inhibitor or CK2 inhibitor in preparation of prostatic cancer inhibitor

Abstract

The invention discloses an application of a PARP inhibitor combined with an SPOP inhibitor or a CK2 inhibitor in preparation of a prostate cancer inhibitor, and is characterized in that the PARP inhibitor is Olaparib, the SPOP inhibitor is SPOP-IN-6B, the CK2 inhibitor is Silmitasertib, and the SPOP inhibitor is SPOP-IN-6B, the CK2 inhibitor is SPOP-IN-6B and the CK2 inhibitor is SPOP-IN-6B. The invention further provides application of the PARP inhibitor and SPOP inhibitor combination or PARP inhibitor and CK2 inhibitor combination in preparation of drugs for promoting apoptosis of prostate cancer cells and application of the PARP inhibitor and CK2 inhibitor combination in preparation of drugs for inhibiting growth of prostate cancer cells, and has the advantages that the SPOP inhibitor or CK2 inhibitor is used for assisting in treatment of the PARP inhibitor in prostate cancer; the kit has the characteristics of strong efficacy, high specificity and the like, and is beneficial to early treatment of prostatic cancer.

Description

Technical Field

[0001] The present invention relates to a prostate cancer inhibitor, and in particular to an application of a PARP inhibitor in combination with a SPOP inhibitor or a CK2 inhibitor in the preparation of a prostate cancer inhibitor. Background Art

[0002] DNA damage repair response (DDR) is a complex DNA repair mechanism that cells have evolved to combat single-strand DNA breaks or double-strand DNA breaks in order to maintain the integrity of the genome. In mammalian cells, homologous recombination repair (HR) and non-homologous end joining (NHEJ) are the two main pathways for repairing double-strand DNA breaks. HR analyzes homologous sequences from other parts of the genome and collects information lost at the break site, while NHEJ directly reconnects the broken ends together. Although the NHEJ mechanism is relatively simple, it sometimes causes rearrangements, while HR is believed to be error-free and successfully repairs damaged DNA. However, if there are defects in DDR, such as overactivation of NHEJ or defects in HR, two results may occur: one is cell death; the other is gene mutation, or subsequent malignant transformation into tumor cells. On the other hand, these defects can also serve as a therapeutic opportunity, and more and more targeted inhibitors of the DDR pathway have emerged, including PARP, ATM, ATR, CHK1, WEE1, and DNA-PK inhibitors.

[0003] The speckle type BTB / POZ protein (SPOP) is a substrate-binding adaptor of the CULLIN3 / RING-box1 E3 ubiquitin ligase complex, which can mediate the degradable and non-degradable polyubiquitination of many substrates with different biological functions. It promotes HR in the DDR, inhibits NHEJ, and ultimately correctly repairs damaged DNA.

[0004] Prostate cancer (PCa) is a group of epithelial malignancies that occur in the prostate. It is the second most common cancer in men, second only to lung cancer, and has a significant impact on the rising mortality rate among men worldwide. Currently, treatments for prostate cancer include surgery, radiotherapy, and chemotherapy. The choice of treatment strategy depends on the patient's clinical evaluation and analysis. If left untreated, prostate cancer cells can spread to nearby tissues, lymph nodes, and even bones. In addition, in tumor cells with defects in a certain DDR pathway, DNA repair will be highly dependent on other bypass pathways, and DDR targeted drugs can also be considered for treatment.

[0005] Studies have shown that the poly (ADP-ribose) polymerase inhibitor (abbreviated as PARP inhibitor) Olaparib can be used in the treatment of prostate cancer. These treatments are effective in improving the survival rate of prostate cancer patients with SPOP gene mutations, but in PCa cells with wild-type SPOP, the use of the PARP inhibitor Olaparib cannot completely block the DDR, because the SPOP-mediated DDR bypass pathway can also successfully repair damaged DNA, allowing PCa cells to continue to survive. The survival rate of cancer cells under its treatment is still high, and it cannot significantly improve the survival rate of patients.

[0006] At present, studies have confirmed that SPOP is a new drug target for the treatment of renal cancer, and a series of SPOP small molecule inhibitors have been discovered. Among them, SPOP-IN-6B is a type of SPOP inhibitor, but because SPOP only mediates the bypass pathway of DDR, the blocking effect of SPOP inhibitors on DDR is relatively weak, so it is speculated that the survival rate of prostate cancer cells under its treatment is still high.

[0007] Casein kinase 2 (CK2) is a highly conserved serine / threonine kinase with more than 500 known substrates, accounting for approximately 10% of the human phosphoproteome. CK2 is a key controller of cellular processes that can trigger the DDR. Most DDR-related CK2 substrates such as HP1β, XRCC1, and MDC1 promote DNA acquisition or form scaffolds to support participation in chromatin remodeling. Although there are many CK2 inhibitors, only a few can act as effective selective inhibitors. Silmitasertib (CX-4945) is a new CK2 inhibitor approved by the FDA for the treatment of cholangiocarcinoma, but it has not yet been used in prostate cancer. At present, there are no published reports on the use of PARP inhibitors combined with SPOP inhibitors or CK2 inhibitors as therapeutic targets in prostate cancer. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a PARP inhibitor with significantly enhanced effect combined with a SPOP inhibitor or a CK2 inhibitor for use in the preparation of a prostate cancer inhibitor.

[0009] The technical solution adopted by the present invention to solve the above technical problem is: use of a PARP inhibitor in combination with a SPOP inhibitor or a CK2 inhibitor in the preparation of a prostate cancer inhibitor.

[0010] Furthermore, the PARP inhibitor is Olaparib, the SPOP inhibitor is SPOP-IN-6B, and the CK2 inhibitor is Silmitasertib.

[0011] The present invention also provides the use of the combination of the PARP inhibitor and the SPOP inhibitor in the preparation of a drug for promoting apoptosis of prostate cancer cells, wherein the PARP inhibitor is Olaparib, and the SPOP inhibitor is SPOP-IN-6B.

[0012] The present invention also provides the use of the combination of the PARP inhibitor and the SPOP inhibitor in the preparation of a drug for inhibiting the growth of prostate cancer cells, wherein the PARP inhibitor is Olaparib, and the SPOP inhibitor is SPOP-IN-6B.

[0013] The present invention also provides the use of the combination of the PARP inhibitor and the CK2 inhibitor in the preparation of a drug for promoting apoptosis of prostate cancer cells, wherein the PARP inhibitor is Olaparib and the CK2 inhibitor is Silmitasertib.

[0014] The present invention also provides the use of the combination of the PARP inhibitor and the CK2 inhibitor in the preparation of a drug for inhibiting the growth of prostate cancer cells, wherein the PARP inhibitor is Olaparib and the CK2 inhibitor is Silmitasertib.

[0015] Compared with the prior art, the advantages of the present invention are as follows: the present invention discloses the use of a PARP inhibitor combined with a SPOP inhibitor combination or a CK2 inhibitor in the preparation of a prostate cancer inhibitor, wherein the PARP inhibitor is Olaparib, the SPOP inhibitor is SPOP-IN-6B, and the CK2 inhibitor is Silmitasertib. Flow cytometry revealed that the SPOP inhibitor or the CK2 inhibitor successfully enhanced the apoptosis of prostate cancer cells induced by the PARP inhibitor; in vivo xenograft experiments revealed that the use of the PARP inhibitor combined with the SPOP inhibitor or the CK2 inhibitor can effectively inhibit the growth of prostate cancer tumors. The invention will help guide the clinical use of SPOP inhibitors or CK2 inhibitors to assist PARP inhibitors in the treatment of prostate cancer, and has the characteristics of strong efficacy and high specificity, which is beneficial to the early treatment of prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1To verify the effect of SPOP-IN-6B on Olaparib-induced prostate cancer cells by flow cytometry, A shows the apoptosis of PC-3 and DU145 cells in the control group, Olaparib group, SPOP-IN-6B group, and Olaparib and SPOP-IN-6B group, and B shows the statistical analysis of apoptosis (*P<0.1, **P<0.01); Figure 2 To verify the effect of SPOP-IN-6B on Olaparib-induced prostate cancer cells by fluorescence method, A shows the apoptosis of PC-3 and DU145 cells in the control group, Olaparib group, SPOP-IN-6B group, and Olaparib and SPOP-IN-6B group, and B shows the statistical analysis of apoptosis (*P<0.1, **P<0.01); Figure 3 The results of in vivo xenograft experiments and dynamic calculation of tumor volume, where A is the tumor growth of the control group, Olaparib group, SPOP-IN-6B group, and Olaparib and SPOP-IN-6B group, and B is the statistical analysis of the tumor volume experimental results (*P<0.1, **P<0.01); Figure 4 To verify the effect of SPOP-IN-6B on the degree of DNA damage and apoptosis of prostate cancer cells induced by Olaparib by immunofluorescence, A is the degree of DNA damage and apoptosis of the control group, Olaparib group, SPOP-IN-6B group, and Olaparib and SPOP-IN-6B group, and B is the statistical analysis of the degree of DNA damage and apoptosis (*P<0.1, **P<0.01); Figure 5 To verify the effect of Silmitasertib on Olaparib-induced prostate cancer cells by flow cytometry, A is the apoptosis of PC-3 and DU145 cells in the control group, Olaparib group, Silmitasertib group, and Olaparib and Silmitasertib combination group, and B is the statistical analysis of cell apoptosis (*P<0.1, **P<0.01); Figure 6 To verify the effect of Silmitasertib on Olaparib-induced prostate cancer cells by immunofluorescence, A shows the apoptosis of PC-3 and DU145 cells in the control group, Olaparib group, Silmitasertib group, and Olaparib and Silmitasertib combination group, and B shows the statistical analysis of apoptosis (*P<0.1, **P<0.01); Figure 7 The results of in vivo xenograft experiments and dynamic calculation of tumor volume, where A is the tumor growth of the control group, Olaparib group, Silmitasertib group, and Olaparib and Silmitasertib combination group, and B is the statistical analysis of the tumor volume experimental results (*P<0.1, **P<0.01); Figure 8 Immunofluorescence method was used to verify the effect of Silmitasertib on the degree of DNA damage and apoptosis of prostate cancer cells induced by Olaparib, where A is the degree of DNA damage and apoptosis in the control group, Olaparib group, Silmitasertib group, and the combination of Olaparib and Silmitasertib group; B is the statistical analysis of the degree of DNA damage and apoptosis (*P<0.1, **P<0.01). DETAILED DESCRIPTION

[0017] The present invention is further described in detail below with reference to the accompanying drawings.

[0018] 1. Experimental Methods 1. Cell culture: Prostate cancer cell lines (PC-3, Cat No: TCH-C297; DU145) were purchased from Haixing Bioscience Co., Ltd., Suzhou, Jiangsu Province, China. PC-3 cell line was cultured in Ham's F-12K (Pricella, China, Cat No: PM150910) containing 10% FBS (Vazyme, China, Cat No: F101-01). DU145 cell line was cultured in Minimum Essential Medium (MEM, Pricella, China, Cat No: PM150410 F101-01) containing 10% FBS (Vazyme, China, Cat No: F101-01).

[0019] 2. Flow cytometry (1) Collect cells: aspirate the cell culture medium into a centrifuge tube, wash the adherent cells once with PBS buffer, add an appropriate amount of EDTA-free trypsin cell digestion solution to digest the cells, incubate at room temperature until the adherent cells can be blown off by gently blowing, aspirate the trypsin cell digestion solution (avoid excessive digestion with trypsin), gently blow the cells off, transfer them to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, collect the cells, gently resuspend the cells with PBS buffer and count them. Take 5×10 4 -10×10 4Resuspend the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 100µL 1×Annexin V binding buffer to gently resuspend the cells, then add 5µL FITC-Annexin V (fluorescein isothiocyanate labeled annexin-V), mix gently, add 5µL PI staining solution, mix gently. Incubate at room temperature in the dark for 10-15min, and use aluminum foil to avoid light. Resuspend the cells 2-3 times during the incubation process to improve the staining effect.

[0020] (2) Flow cytometer detection: After incubation, 400 μL 1×Annexin V binding buffer can be directly added to resuspend the cells and immediately tested on the machine. FITC-Annexin V is excited by a 488 nm laser, and the fluorescence emission spectrum is detected at 530 nm (FITC channel), and the PI channel emission spectrum is about 617 nm. In the scatter plot of the bivariate flow cytometer, the lower left quadrant shows live cells, which is (FITC-Annexin V- / PI-); the lower right quadrant is early apoptotic cells, which is (FITC-Annexin V+ / PI-); the upper right quadrant is necrotic and late apoptotic cells, which is (FITC-Annexin V+ / PI+); the upper left quadrant shows naked nucleus cells, which is (FITC-Annexin V- / PI+).

[0021] 3. Immunofluorescence detection (1) For adherent cells or cell smears: Fix the cells with Bio-Tech Immunostaining Fixative (P0098) or 4% paraformaldehyde for 30 minutes. Wash once with PBS buffer. Add Bio-Tech Immunostaining Strong Permeabilization Solution (P0097) or PBS buffer containing 0.3% Triton X-100, incubate at room temperature for 5 minutes, and proceed to step (3); (2) For paraffin sections: Dewax the tumor tissue in xylene for 5-10 minutes. Replace with fresh xylene, dewax for another 5-10 minutes, then wash with anhydrous ethanol for 5 minutes and 90% ethanol for 2 minutes. Wash with 70% ethanol for 2 minutes and distilled water for 2 minutes, then add 20 μg / mL DNase-free proteinase K (you can use Biotech's ST532 / ST533 proteinase K) and allow to react at 20-37°C for 15-30 minutes. Wash with PBS 3 times and go to step (3) or (4); (3) TUNEL detection: Taking one sample as an example, 5 μL TdT enzyme + 45 μL fluorescent labeling solution + 50 μL TUNEL detection solution is prepared for TUNEL detection solution. Wash twice with PBS buffer. Add 50 μL TUNEL detection solution to the sample and incubate at 37°C in the dark for 60 minutes (green). After washing three times with PBS buffer, add 60 μL DAPI for nuclear staining (blue) for 30 seconds, wash three times with PBS buffer, seal the slide with anti-fluorescence quenching sealing solution and observe under a fluorescence microscope; (4) γH2AX detection: add 60 μL of primary antibody Phospho-Histone H2AX-S139 monoclonal antibody (AP0687, dilution 1:5000) diluted with antibody diluent, and incubate at 4°C overnight. Remove the primary antibody, wash three times with PBS buffer, remove the PBS buffer, add 60 μL of diluted fluorescently labeled Cy3 goat anti-rabbit IgG (HL) (AS007, dilution 1500), incubate at 4°C in the dark for 60 minutes (red), wash three times with PBS buffer, add 60 μL DAPI for nuclear staining (blue) for 30 seconds, wash three times with PBS, seal the slides with anti-fluorescence quenching sealant and observe under a fluorescence microscope.

[0022] 4. In vivo xenograft experiment: 3-5 week old NOD-SCID mice (weight 15-25 g) were used for in vivo xenograft experiment. The treated stable PC-3 cell line was subcutaneously transplanted into the right side of the back of the mice. The tumor volume was measured every 3 days. When the tumor volume reached 50 mm 3 The inhibitors of different experimental groups were gavaged daily for about 14 days. When the tumors grew to an appropriate size, the mice were euthanized, and the tumors were imaged and weighed. There were 5 mice in each group, and all animal experiments were performed in accordance with the guidelines approved by the Ningbo University Animal Care Committee. Tumor volume = (length × width 2 )×1 / 2.

[0023] 5. Result analysis method: This experiment comprehensively checked, proofread and sorted the experimental data and the collected original clinical data to ensure that the data was as complete, accurate and correct as possible. Then, Excel software was used to establish tissue and cell databases respectively, and the original clinical data involved were grouped, summarized and entered into the table. Finally, SPSS 26.0 statistical software was used to sort and analyze the final experimental data. All data statistical tests were bilaterally probabilistic and statistically tested at the test level of α=0.05. If P<0.05, the difference between the two groups was considered statistically significant, otherwise the difference was considered statistically insignificant. The drawing of experimental result graphs such as bar graphs and line graphs was completed jointly using GraphPad Prism 9.0 and SPSS 26.0 software.

[0024] 2. Analysis of experimental results on the effects of the combination of PARP inhibitors and SPOP inhibitors on prostate cancer cells.

[0025] 1. Flow cytometry detection: In order to examine the effect of combined induction of SPOP inhibitors and PARP inhibitors on prostate cancer cells, we used flow cytometry experiments.

[0026] Negative control group: prostate cancer cells PC-3 and DU145 treated with DMSO for 24 h; SPOP inhibitor group: prostate cancer cells PC-3 and DU145 treated with 2 μM SPOP-IN-6B for 24 h; PARP inhibitor group: prostate cancer cells PC-3 and DU145 treated with 10 μM Olaparib for 24 h; SPOP inhibitor and PARP inhibitor combination group: prostate cancer cells PC-3 and DU145 were treated with 2μM SPOP-IN-6B and 10μM Olaparib for 24h.

[0027] The results are as follows Figure 1 As shown in A, compared with the control group, the number of early apoptotic cells in the lower right quadrant and necrotic and late apoptotic cells in the upper right quadrant in the PARP inhibitor group increased, while there was no significant change in the SPOP inhibitor group; compared with the SPOP inhibitor group and the PARP inhibitor group, the number of early apoptotic cells and necrotic and late apoptotic cells in the combined SPOP inhibitor and PARP inhibitor group increased significantly.

[0028] The results are as follows Figure 1 As shown in B, the combination of SPOP inhibitor and PARP inhibitor has a synergistic effect, and the effect of promoting apoptosis of prostate cancer cells is significantly greater than the sum of the two used alone.

[0029] In summary, compared with the SPOP inhibitor group and the PARP inhibitor group alone, the number of apoptotic prostate cancer cells increased significantly when the SPOP inhibitor and PARP inhibitor were used in combination, indicating that the SPOP inhibitor can significantly enhance the apoptosis of prostate cancer cells induced by the PARP inhibitor.

[0030] 2. Immunofluorescence: To further verify the effect of the combined use of SPOP inhibitors and PARP inhibitors on prostate cancer cell apoptosis, immunofluorescence experiments were performed on the prostate cancer cells PC-3 and DU145 after treatment in the following groups, respectively. TUNEL represents apoptosis.

[0031] Negative control group: prostate cancer cells PC-3 and DU145 treated with DMSO for 24 h; SPOP inhibitor group: prostate cancer cells PC-3 and DU145 treated with 2 μM SPOP-IN-6B for 24 h; PARP inhibitor group: prostate cancer cells PC-3 and DU145 treated with 10 μM Olaparib for 24 h; SPOP inhibitor and PARP inhibitor combination group: prostate cancer cells PC-3 and DU145 were treated with 2μM SPOP-IN-6B and 10μM Olaparib for 24h.

[0032] The results are as follows Figure 2 As shown in A, compared with the control group, the number of apoptotic cells in the PARP inhibitor group increased, while the difference in the number of apoptotic cells in the SPOP inhibitor group was not significant; compared with the SPOP inhibitor group and the PARP inhibitor group, the number of apoptotic cells in the combination of the SPOP inhibitor and the PARP inhibitor group increased significantly.

[0033] The effects of SPOP inhibitors and PARP inhibitors on prostate cancer cell apoptosis were further studied. Figure 2 As shown in B, the combination of SPOP inhibitor and PARP inhibitor has a synergistic effect, and the effect of promoting apoptosis of prostate cancer cells is greater than the sum of the two alone.

[0034] In summary, through the comparison of the negative control group, PARP inhibitor group and SPOP inhibitor group, it was found that the number of apoptotic cells in the combination group of SPOP inhibitor and PARP inhibitor was significantly increased.

[0035] 3. In vivo xenograft experiment: To further verify the effect of the combined use of SPOP inhibitors and PARP inhibitors on prostate cancer tumor growth, an in vivo xenograft experiment was conducted and the tumor volume was dynamically calculated. Five replicate groups were set up and the tumors were arranged.

[0036] Negative control group: intragastrically administered with DMSO (dimethyl sulfoxide) every day; SPOP inhibitor group: SPOP inhibitor (SPOP-IN-6b at a dose of 40 mg / kg) was intragastrically administered daily; PARP inhibitor group: PARP inhibitor (Olaparib at a dose of 50 mg / kg) was intragastrically administered daily; SPOP inhibitor and PARP inhibitor combination group: SPOP inhibitor and PARP inhibitor combination (SPOP-IN-6b at a dose of 40 mg / kg and Olaparib at a dose of 50 mg / kg) was administered orally every day.

[0037] The results are as follows Figure 3As shown in A, compared with the control group, the tumor volume growth rate of mice in the PARP inhibitor group was slowed down, and there was no significant difference in the tumor volume growth rate of mice in the SPOP inhibitor group; compared with the SPOP inhibitor group and the PARP inhibitor group, the tumor volume growth rate of mice in the combination of SPOP inhibitor and PARP inhibitor group was significantly slowed down.

[0038] The effects of SPOP inhibitors and PARP inhibitors on tumor volume were further studied. Figure 3 As shown in B, the combination of SPOP inhibitor and PARP inhibitor has a synergistic effect, and the inhibitory effect on the growth rate of mouse tumor volume is significantly greater than the sum of the two used alone.

[0039] In summary, through the comparison of the negative control group, PARP inhibitor group and SPOP inhibitor group, it was found that the growth rate of tumor volume in the combination group of SPOP inhibitor and PARP inhibitor was significantly slowed down.

[0040] 4. Immunofluorescence: To further verify the effect of the combined use of SPOP inhibitors and PARP inhibitors on the degree of DNA damage and apoptosis of mouse prostate cancer tumor cells, paraffin sections of mouse prostate cancer tumor tissues were taken from the following groups 29 days after oral gavage for immunofluorescence experiments. γH2AX represents the degree of DNA damage and TUNEL represents apoptosis.

[0041] Negative control group: intragastrically administered with DMSO (dimethyl sulfoxide) every day; SPOP inhibitor group: SPOP inhibitor (SPOP-IN-6b at a dose of 40 mg / kg) was intragastrically administered daily; PARP inhibitor group: PARP inhibitor (Olaparib at a dose of 50 mg / kg) was intragastrically administered daily; SPOP inhibitor and PARP inhibitor combination group: SPOP inhibitor and PARP inhibitor combination (SPOP-IN-6b at a dose of 40 mg / kg and Olaparib at a dose of 50 mg / kg) was administered orally every day.

[0042] The results are as follows Figure 4 As shown in A, compared with the control group, the degree of DNA damage and cell apoptosis in the PARP inhibitor group increased, while there was no significant difference in the degree of DNA damage and cell apoptosis in the SPOP inhibitor group; compared with the SPOP inhibitor group and the PARP inhibitor group, the degree of DNA damage and cell apoptosis in the combined SPOP inhibitor and PARP inhibitor group increased significantly.

[0043] The effects of SPOP inhibitors and PARP inhibitors on DNA damage and cell apoptosis were further studied. Figure 4As shown in B, the combination of SPOP inhibitor and PARP inhibitor has a synergistic effect, and the promoting effect on the degree of DNA damage and cell apoptosis is greater than the sum of the two used alone.

[0044] In summary, through the comparison of the negative control group, PARP inhibitor group and SPOP inhibitor group, it was found that the degree of DNA damage and cell apoptosis in the combination group of SPOP inhibitor and PARP inhibitor were significantly increased.

[0045] This invention detects the effects of SPOP inhibitors and PARP inhibitors used alone or in combination on prostate cancer cell apoptosis and tumor growth, and combines statistical principles and modern biological techniques to provide a detection method with high sensitivity, strong specificity, short cycle and stable results, providing a scientific basis for the treatment of prostate cancer patients and the possibility of molecular targeted therapy.

[0046] 3. Analysis of experimental results on the effects of the combination of PARP inhibitors and CK2 inhibitors on prostate cancer cells.

[0047] 1. Flow cytometry detection: In order to examine the effect of combined induction of CK2 inhibitor and PARP inhibitor on prostate cancer cells, we used flow cytometry experiments.

[0048] Negative control group: prostate cancer cells PC-3 and DU145 treated with DMSO for 24 h; CK2 inhibitor group: prostate cancer cells PC-3 and DU145 treated with 1 nM Silmitasertib for 24 h; PARP inhibitor group: prostate cancer cells PC-3 and DU145 treated with 10 μM Olaparib for 24 h; CK2 inhibitor and PARP inhibitor combination group: Prostate cancer cells PC-3 and DU145 were treated with 1 nM Silmitasertib and 10 μM Olaparib for 24 h.

[0049] The results are as follows Figure 5 As shown in A, compared with the control group, the numbers of early apoptotic cells in the lower right quadrant and necrotic and late apoptotic cells in the upper right quadrant in the CK2 inhibitor group and the PARP inhibitor group alone increased; compared with the CK2 inhibitor group and the PARP inhibitor group alone, the numbers of early apoptotic cells and necrotic and late apoptotic cells in the upper right quadrant in the CK2 inhibitor and PARP inhibitor combination group increased significantly.

[0050] The results are as follows Figure 5 As shown in B, the results were statistically analyzed and it was found that the combination of CK2 inhibitor and PARP inhibitor had a synergistic effect, and the effect of promoting apoptosis of prostate cancer cells was significantly greater than that of using either alone.

[0051] In summary, compared with the CK2 inhibitor group and the PARP inhibitor group used alone, the number of apoptotic prostate cancer cells increased significantly when CK2 inhibitor and PARP inhibitor were used in combination, indicating that PARP inhibitor can significantly enhance the apoptosis of prostate cancer cells induced by CK2 inhibitor.

[0052] 2. Immunofluorescence experiment: To further verify the effect of the combined use of CK2 inhibitors and PARP inhibitors on prostate cancer cell apoptosis, TUNEL cell apoptosis detection was performed on the prostate cancer cells PC-3 and DU145 after treatment in the following groups, respectively. TUNEL represents apoptosis and green fluorescence represents cell apoptosis.

[0053] Negative control group: prostate cancer cells PC-3 and DU145 treated with DMSO for 24 h; CK2 inhibitor group: prostate cancer cells PC-3 and DU145 treated with 1 nM Silmitasertib for 24 h; PARP inhibitor group: prostate cancer cells PC-3 and DU145 treated with 10 μM Olaparib for 24 h; CK2 inhibitor and PARP inhibitor combination group: Prostate cancer cells PC-3 and DU145 were treated with 1 nM Silmitasertib and 10 μM Olaparib for 24 h.

[0054] The results are as follows Figure 6 As shown in A, compared with the control group, the number of apoptotic cells increased when PARP inhibitors and CK2 inhibitors were used alone; compared with the CK2 inhibitor group and PARP inhibitor group, the number of apoptotic cells in the CK2 inhibitor and PARP inhibitor combination group increased significantly.

[0055] The results are as follows Figure 6 As shown in B, the results were statistically analyzed and it was found that the combination of CK2 inhibitor and PARP inhibitor had a synergistic effect, and the effect of promoting apoptosis of prostate cancer cells was greater than that of either alone.

[0056] In summary, through the comparison of the negative control group, PARP inhibitor group and CK2 inhibitor group, it was found that the number of apoptotic cells in the combination group of CK2 inhibitor and PARP inhibitor was significantly increased.

[0057] 3. In vivo xenograft experiment: To further verify the effect of the combined use of CK2 inhibitors and PARP inhibitors on prostate cancer tumor growth, an in vivo xenograft experiment was conducted and the tumor volume was dynamically calculated. Five replicate groups were set up and the tumors were arranged.

[0058] Negative control group: intragastrically administered with DMSO (dimethyl sulfoxide) every day; CK2 inhibitor group: CK2 inhibitor (Silmitasertib at a dose of 40 mg / kg) was intragastrically administered daily; PARP inhibitor group: PARP inhibitor (Olaparib at a dose of 50 mg / kg) was intragastrically administered daily; CK2 inhibitor and PARP inhibitor combination group: CK2 inhibitor and PARP inhibitor combination (Silmitasertib at a dose of 40 mg / kg and Olaparib at a dose of 50 mg / kg) were gavaged daily.

[0059] The results are as follows Figure 7 As shown in A, compared with the control group, the tumor volume growth rate of mice in the PARP inhibitor and CK2 inhibitor groups alone was slowed down; compared with the CK2 inhibitor and PARP inhibitor groups alone, the tumor volume growth rate of mice in the CK2 inhibitor and PARP inhibitor combination group was significantly slowed down.

[0060] The results are as follows Figure 7 As shown in B, the results were statistically analyzed and it was found that the combination of CK2 inhibitor and PARP inhibitor had a synergistic effect, and the inhibitory effect on the growth rate of mouse tumor volume was significantly greater than that of either alone.

[0061] In summary, through the comparison of the negative control group, PARP inhibitor group and CK2 inhibitor group, it was found that the growth rate of tumor volume in the combination group of CK2 inhibitor and PARP inhibitor was significantly slowed down.

[0062] 4. Immunofluorescence detection: To further verify the effect of the combined use of CK2 inhibitors and PARP inhibitors on the degree of DNA damage and apoptosis of mouse prostate cancer tumor cells, paraffin sections of mouse prostate cancer tumor tissues from the following groups 29 days after oral gavage were taken for immunofluorescence experiments. γH2AX represents the degree of DNA damage and TUNEL represents apoptosis.

[0063] Negative control group: intragastrically administered with DMSO (dimethyl sulfoxide) every day; CK2 inhibitor group: CK2 inhibitor (Silmitasertib at a dose of 40 mg / kg) was intragastrically administered daily; PARP inhibitor group: PARP inhibitor (Olaparib at a dose of 50 mg / kg) was intragastrically administered daily; CK2 inhibitor and PARP inhibitor combination group: CK2 inhibitor and PARP inhibitor combination (Silmitasertib at a dose of 40 mg / kg and Olaparib at a dose of 50 mg / kg) were gavaged daily.

[0064] like Figure 8 As shown in A, compared with the control group, the degree of DNA damage and cell apoptosis in the PARP inhibitor group and the CK2 inhibition group increased; compared with the CK2 inhibitor group and the PARP inhibitor group, the degree of DNA damage and cell apoptosis in the CK2 inhibitor and PARP inhibitor combination group increased significantly.

[0065] like Figure 8 As shown in B, the results were statistically analyzed and it was found that the combination of CK2 inhibitor and PARP inhibitor had a synergistic effect, and the promoting effect on the degree of DNA damage and cell apoptosis was greater than that of either alone.

[0066] In summary, through the comparison of the negative control group, the PARP inhibitor group alone and the CK2 inhibitor group, it was found that the degree of DNA damage and cell apoptosis in the CK2 inhibitor and PARP inhibitor combination group were significantly increased.

[0067] This invention detects the effects of CK2 inhibitors and PARP inhibitors used alone or in combination on prostate cancer cell apoptosis and tumor growth, and combines statistical principles and modern biological techniques to provide a detection method with high sensitivity, strong specificity, short cycle and stable results, providing a scientific basis for the treatment of prostate cancer patients and the possibility of molecular targeted therapy.

[0068] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. Use of PARP inhibitors combined with SPOP inhibitors or CK2 inhibitors in the preparation of prostate cancer inhibitors.

2. Use of the PARP inhibitor combined with a SPOP inhibitor or a CK2 inhibitor according to claim 1 in the preparation of a prostate cancer inhibitor, characterized in that: The PARP inhibitor is Olaparib, the SPOP inhibitor is SPOP-IN-6B, and the CK2 inhibitor is Silmitasertib.

3. Use of a combination of a PARP inhibitor and a SPOP inhibitor in the preparation of a drug for promoting apoptosis of prostate cancer cells, characterized in that: The PARP inhibitor is Olaparib, and the SPOP inhibitor is SPOP-IN-6B.

4. Use of a combination of a PARP inhibitor and a SPOP inhibitor in the preparation of a drug for inhibiting the growth of prostate cancer cells, characterized in that: The PARP inhibitor is Olaparib, and the SPOP inhibitor is SPOP-IN-6B.

5. Use of a combination of a PARP inhibitor and a CK2 inhibitor in the preparation of a drug for promoting apoptosis of prostate cancer cells, characterized in that: The PARP inhibitor is Olaparib, and the CK2 inhibitor is Silmitasertib.

6. Use of a combination of a PARP inhibitor and a CK2 inhibitor in the preparation of a drug for inhibiting the growth of prostate cancer cells, characterized in that: The PARP inhibitor is Olaparib, and the CK2 inhibitor is Silmitasertib.

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