Use of HDAC6 protein k52 lactylation modification as a target in preparation of a product for treatment of prostate cancer

Abstract

The application belongs to the technical field of tumor precision medicine, and particularly relates to application of HDAC6 protein K52 lactylation modification as a target in preparation of a product for prostate cancer treatment, experimental results show that the content of lactic acid in PCa cells increases and causes protein pan-lactylation modification; inhibition of pan-lactylation can induce oxidative stress and cause cell apoptosis; compared with the HDAC6 wt group, after transfection of the HDAC6 K52R plasmid, the acetylation level of alpha-Tubulin in PC-3 cells is increased, the FH activity is significantly reduced, and the ROS level is significantly increased; after transfection of the HDAC6 K52R plasmid, the ROS level in PC-3 cells is significantly increased, and the cell apoptosis rate is significantly increased, which provides direct and reliable theoretical and experimental basis for development of a targeted therapeutic drug for HDAC6 K52 lactylation modification, and has important clinical application prospect and market value.

Description

Technical Field

[0001] This invention belongs to the field of precision oncology technology, specifically involving the application of HDAC6 protein K52 lactation modification as a target in the preparation of products for prostate cancer treatment. Background Technology

[0002] Prostate cancer (PCa) is the second most common malignant tumor in men worldwide. Because androgens and androgen receptor (AR) signaling play a crucial role in the development and progression of PCa, androgen deprivation therapy (ADT) has been an important treatment strategy for PCa patients. First-generation AR antagonists, including bicalutamide, flutamide, and nilumid, bind to AR to exert their antagonistic effect, but long-term use easily leads to drug resistance. Second-generation AR antagonists, mainly including enzalutamide, apatamide, and dalotamide, target the ligand-binding domain of AR, thus exhibiting a stronger inhibitory effect on the androgen-AR axis. However, their high cost and serious side effects such as hypertension, seizures, and joint pain exacerbate the physical, psychological, and economic burden on PCa patients. Therefore, exploring pathogenic mechanisms beyond the androgen receptor and targeted drugs will provide new insights for the clinical treatment of PCa.

[0003] ROS primarily originate from mitochondria and NADPH oxidases, including hydrogen peroxide (H2O2), superoxide anion (O2-), and hydroxyl radicals (OH-). ROS plays a dual role in tumor progression. On one hand, low levels of ROS act as signaling molecules, interfering with the normal regulation of DNA repair mechanisms through interactions with macromolecules such as DNA, RNA, and proteins, leading to impaired genome integrity and activation of redox-related signaling pathways, ultimately promoting tumorigenesis and development. On the other hand, when ROS production is imbalanced with the clearance capacity of antioxidant enzymes, excessive ROS can cause oxidative stress in cells, thereby inducing apoptosis, pyroptosis, autophagy, and other forms of programmed cell death.

[0004] Lactic acid, another key product of metabolic reprogramming, is widely involved in the malignant progression of tumors. Studies have reported that lactate maintains intracellular pH and redox homeostasis by regulating the conversion of NADH to NAD+, thereby sustaining tumor cell survival under stress conditions. Furthermore, lactate-mediated protein lactation also participates in maintaining cellular redox homeostasis.

[0005] HDAC6, a member of the class IIb HDAC family, is primarily located in the cytoplasm. Its overexpression in various cancers is associated with tumor initiation, progression, and prognosis. In prostate cancer (PCa), studies have reported that aberrant activation of HDAC6 is a key factor in promoting tumor progression, resisting apoptosis, and leading to CRPC formation, making it a potential therapeutic target for prostate cancer. Research has confirmed that HDAC6 plays an important role in maintaining cellular oxidative stress homeostasis. It can promote the deacetylation and activation of the key mitochondrial cell division factor Drp1, accelerate the clearance of damaged mitochondria, prevent ROS accumulation, and maintain mitochondrial homeostasis. Furthermore, HDAC6 can regulate cellular adaptive responses to hypoxia stress by deacetyling and stabilizing the hypoxia-inducible factor HIF-1α. However, research on HDAC6 K52 lactation modification and PCa has not yet been reported in the literature.

[0006] During their research, the inventors discovered that increased lactate levels in PCa cells lead to pan-lactation modification of proteins; inhibiting pan-lactation can induce oxidative stress and induce apoptosis. By transfecting PCa cell lines with the HDAC6 K52R mutant (mimicking the inactivation of lactation modification at this site), and using flow cytometry, Western blotting, and CCK-8 experiments, they confirmed that inhibiting lactation modification at the K52 site significantly induces oxidative stress in PCa cells and promotes apoptosis. In summary, this discovery reveals for the first time the crucial role of HDAC6 K52 lactation modification in driving the malignant proliferation of prostate cancer cells, thus establishing it as a novel, functionally validated therapeutic target. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a reagent for detecting the expression level of HDAC6 K52 lactation modification in the preparation of products for diagnosing diseases.

[0008] To solve the above-mentioned technical problems, the technical solution of this application is as follows:

[0009] The primary objective of this invention is to provide a drug target, wherein the drug target is the lactation modification of lysine residue 52 of the HDAC6 protein.

[0010] A second objective of the present invention is to provide the use of the aforementioned drug target in the preparation of products for the diagnosis and / or treatment of prostate cancer.

[0011] A third objective of this invention is to provide the use of a reagent for detecting the lactation modification level of lysine 52 of the HDAC6 protein in the preparation of a kit for assessing the prognosis or treatment response of patients with prostate cancer, wherein an elevated level of lactation modification indicates a poor prognosis or treatment resistance.

[0012] A fourth objective of this invention is to provide an agent for promoting apoptosis of prostate cancer cells and / or inhibiting the proliferation of prostate cancer cells, said agent comprising an active ingredient capable of inhibiting the lactation modification level of lysine at position 52 of the HDAC6 protein.

[0013] Preferably, the active ingredient is selected from any one or more of the following:

[0014] (1) Inhibitor of lactate dehydrogenase; (2) Mutant targeting lysine 52 of HDAC6 protein, wherein lysine 52 is mutated to an amino acid that cannot be lactated;

[0015] (3) Antibodies or small molecule compounds that specifically block the lactation modification of lysine at position 52 of HDAC6 protein; (4) Enzymes or their activators that promote the delactation modification of lysine at position 52 of HDAC6 protein.

[0016] Preferably, the lactate dehydrogenase inhibitor is selected from sodium oxalate.

[0017] The fifth objective of this invention is to provide a method for screening candidate drugs that promote apoptosis or inhibit proliferation of prostate cancer cells in vitro, comprising the following steps: (1) contacting the candidate drug with prostate cancer cells expressing HDAC6 protein; (2) detecting the lactation modification level of lysine at position 52 of HDAC6 protein in the cells; (3) if the modification level decreases, then the candidate drug is determined to be the target drug.

[0018] The sixth objective of this invention is to provide a medicament for the prevention and / or treatment of prostate cancer, wherein the active ingredient of the medicament is a lactate dehydrogenase inhibitor.

[0019] The seventh objective of this invention is to provide the use of an inhibitor that inhibits lactation modification of lysine at position 52 of the HDAC6 protein in the preparation of medicaments for the prevention and / or treatment of prostate cancer.

[0020] The beneficial effects of this invention are as follows: This invention provides a drug target, namely, the lactation modification of lysine residue 52 of the HDAC6 protein. Experimental results show that the lactate content in PCa cells increases, leading to pan-lactation modification of the protein; inhibiting pan-lactation can induce oxidative stress and apoptosis; compared with the HDAC6 wt group, transfection with the HDAC6 K52R plasmid increased α-Tubulin acetylation level, significantly reduced FH activity, and significantly increased ROS level in PC-3 cells. This invention systematically elucidates for the first time that HDAC6 K52 lactation modification inhibits oxidative stress and apoptosis in PCa cells. It provides direct and reliable theoretical and experimental basis for the development of targeted therapeutic drugs against HDAC6 K52 lactation modification, and has important clinical application prospects and market value. Attached Figure Description

[0021] Figure 1 Lactic acid content and protein pan-lactic acidification level in human prostate stromal cells and prostate cancer cells.

[0022] Note: (A) L-lactic acid content assay kit was used to detect the lactate content in WPMY-1, PC-3, LNCaP, and 22RV1 cells; (B) Western blot assay was used to detect the level of protein pan-lactic acidification in cells.

[0023] Figure 2 Lactate dehydrogenase inhibitors suppress pan-lactation of proteins in PCa cells.

[0024] Note: (A) Western blot detection of pan-lactation level of protein in PC-3 cells; (B) Quantitative results of Western blot.

[0025] Figure 3 Inhibition of protein pan-lactation can induce oxidative stress in PCa cells.

[0026] Note: (A) The content of ROS was detected by flow cytometry using DHE-labeled probes; (B) The results of flow cytometry of ROS in PCa cells were quantified.

[0027] Figure 4 Inhibiting protein pan-lactation can promote PCa cell apoptosis.

[0028] Note: (A) PC-3 cells were double-stained with Annexin V-FITC / PI and the apoptosis rate of the cells was detected by flow cytometry; (B) Quantitative results of PCa cell apoptosis rate by flow cytometry.

[0029] Figure 5 NAC pretreatment can suppress Oxa-induced oxidative stress.

[0030] Note: (A) The content of ROS was detected by flow cytometry using DHE-labeled probes; (B) The results of flow cytometry of ROS in PCa cells were quantified.

[0031] Figure 6 NAC pretreatment can inhibit Oxa-induced apoptosis.

[0032] Note: (A) PC-3 cells were double-stained with Annexin V-FITC / PI and the apoptosis rate of the cells was detected by flow cytometry; (B) Quantitative results of PCa cell apoptosis rate by flow cytometry.

[0033] Figure 7 PCa cells contain high levels of HDAC6 lactation modification.

[0034] Note: (A) IP-WB assay was used to detect the level of endogenous HDAC6 lactation modification in WPMY-1 cells; (B) IP-WB assay was used to detect the level of endogenous HDAC6 lactation modification in PC-3 cells.

[0035] Figure 8 Proteomics and heatmap analysis identified the K32, K51, and K52 sites of HDAC6 as potentially key lactation sites.

[0036] Note: Differential lactation heatmap at specific lysine residues in proteins involved in oxidative stress.

[0037] Figure 9 K52 may be the main lactation site of HDAC6 protein in PC-3 cells.

[0038] Note: (A) PC-3 cells were transfected with the HDAC6 mutant plasmid for 24 h, and the proliferation capacity of PC-3 cells was detected by CCK-8 assay; (B) IP-WB assay was used to detect HDAC6 lactation in PC-3 cells after transfection with the plasmid; (C)

[0039] PC-3 cells were treated with 20 mM Nala for 24 h and transfected with the mutant plasmid Flag-HDAC6 K52R. HDAC6 lactation in PC-3 cells was detected by IP-WB assay. (D) Conservation analysis of HDAC6K52 site and motif enrichment analysis among different species.

[0040] Figure 10 Delactation of HDAC6 K52 reduces its deacetylation function.

[0041] Note: (A) IP-WB assay was used to detect the acetylation level of HDAC6 substrate protein α-tubulin; (B) Fumarase (FH) activity kit was used to detect the activity of FH in PC-3 cells.

[0042] Figure 11 HDAC6 K52 delactation can induce oxidative stress in PC-3 cells.

[0043] Note: (A) The content of ROS was detected by flow cytometry using DHE-labeled probes; (B) The results of flow cytometry of ROS in PCa cells were quantified.

[0044] Figure 12 HDAC6 K52 delactation can promote apoptosis in PC-3 cells.

[0045] Note: (A) PC-3 cells were double-stained with Annexin V-FITC / PI and the apoptosis rate of the cells was detected by flow cytometry; (B) Quantitative results of PCa cell apoptosis rate by flow cytometry. Detailed Implementation

[0046] The following description, in conjunction with the accompanying drawings, illustrates exemplary embodiments of the present invention, including various details to aid understanding. These details should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the invention. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0047] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are all conventional biochemical reagents; and the experimental methods described are all conventional methods.

[0048] In the following examples, all data are presented as mean ± standard deviation of at least three independent experiments and analyzed using GraphPad Prism (v. 9.0, La Jolla, USA) according to specified statistical methods. A t-test was used when the data were normally distributed and homogeneous in variance; a nonparametric rank-sum test was used when the data were not normally distributed. Differences among multiple groups were compared using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001 indicate statistical significance.

[0049] Example 1: Cell Experiment

[0050] 1. Materials and Methods

[0051] The human prostate cancer cell line PC-3 was cultured in vitro using a special culture medium for the human prostate cancer cell line PC-3, and routinely cultured in an incubator containing 5% CO2 at 37°C.

[0052] The immortalized human normal prostate matrix cell line WPMY-1 was purchased from Pronoss (Wuhan, China), and the human prostate cancer cell lines (LNCap and 22RV1 cells) were purchased from the Cell Resource Center of Chinese Academy of Sciences (Beijing, China). The cells were cultured in 1640 medium + 10% FBS (fetal bovine serum) + 1% penicillin (100 μg / mL) / streptomycin (100 μg / mL) at 37°C in a 5% CO2 incubator.

[0053] Wild-type and mutant overexpression plasmids and their corresponding negative control vectors were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0054] Cell treatment method: Overexpress HDAC6 in human prostate cancer cell line PC-3 (the treatment method for wild-type and wild-type HDAC6 plasmids is the same; the wild-type HDAC6 overexpression plasmid is used as an example here):

[0055] (1) Inoculate the cells into 6 cm dishes one day in advance;

[0056] (2) When the cell growth density reaches about 60-70% under a microscope, cell transfection can be performed.

[0057] (3) Disinfect the clean bench and laboratory equipment with ultraviolet light for 30 min, then wipe the workbench with 75% alcohol swabs. At the same time, preheat RPMI 1640 culture medium in a water bath at 37℃;

[0058] (4) Based on the concentrations of HDAC6 wt, HDAC6 K52R, and HDAC6 K52T plasmids extracted, calculate the required plasmid volume and the amount of lipo2000 used.

[0059] (5) Preparation of transfection working solution: Prepare 1.5 mL of enzyme-free EP tube (tube A), add 500 μL of RPMI 1640 medium + 7.5 μL of Lipo2000, mix gently, and incubate for 5 min; prepare another 1.5 mL EP tube (tube B), add 500 μL of RPMI 1640 medium + a certain amount of vector / HDAC6 wt / HDAC6 K52R / HDAC6 K52T overexpression plasmid, mix thoroughly, and incubate for 5 min. Then gently mix the liquids in tubes A and B, and incubate at room temperature for 20 min;

[0060] (6) Remove the cells to be transfected, discard the old culture medium, wash with 1×PBS, add 3 mL of RPMI 1640 culture medium without antibiotics, and the transfection mixture from step (5);

[0061] (7) After culturing in the incubator for 4-6 h, replace with RPMI 1640 medium containing serum (10% FBS + 90% RPMI 1640 medium), and continue culturing for 24 h or 48 h before use for other detections.

[0062] 2. Detection Method

[0063] 2.1 Western Blot

[0064] (1) Extraction of cell proteins: Prepare protein lysis buffer: RIPA:PMSF = 100:1, and prepare the required amount according to the ratio. Discard the old culture medium in the small dish, wash 3 times with 1×PBS, and add 600 μL of protein lysis buffer. Incubate at 4℃ on a shaker for 30 min. Collect the cell lysis buffer and transfer it to a 1.5 mL EP tube, centrifuge at 12000 rpm at 4℃ for 15 min. Take the supernatant, add it to a new EP tube, label it, and use the BCA method to detect the protein concentration. Store at -80℃.

[0065] (2) Preparation of protein loading sample: Protein amount: 4×Loading buffer = 150 μL: 50 μL. Use 1×PBS to balance the protein lysis buffer to a final concentration of (1-1.5 mg / mL). After mixing evenly according to the ratio, place the EP tube in a 100℃ metal bath, and remove the EP tube after 10 min to cool it down quickly for later use;

[0066] (3) Assemble the glue-making frame: Assemble and fix the cleaned thin glass plate, thick glass plate, and glue strip to the glue-making frame, and add double-distilled water for a leak test for 10 minutes. After the leak test is completed, discard the double-distilled water and use filter paper to absorb the remaining double-distilled water in the plate;

[0067] (4) Gel preparation: Based on the molecular weight of the proteins, 10% and 12% separating gels were selected (Servicebio® PAGE Color Gel Ultra-Rapid Preparation Kit). The lower and upper gel solutions were prepared according to the preparation system in Table 3-1 below. First, the prepared lower gel solution was added, and the surface of the lower gel was sealed with double-distilled water. After the lower gel solidified, the water was discarded, and the gel was blotted dry with filter paper. Then, the freshly prepared upper gel solution was added, a comb was inserted, and the gel was allowed to solidify (approximately 20 minutes) before use.

[0068] Table 1. Preparation systems for 10% and 12% gels

[0069] (5) Electrophoresis: Assemble the gel plate onto the electrophoresis tank, add the electrophoresis solution, vertically remove the comb, add the experimental protein sample and marker into the sample well, cover the lid, set the voltage to 80 V and the current to 200 mA, and after the markers separate (about 30 min), change the voltage to 120 V and continue electrophoresis until the markers are clearly separated. The loading buffer should be about 1 cm away from the bottom of the glass plate.

[0070] (6) Electroporation: Prepare a matching PVDF membrane according to the size of the cut electrophoresis gel, activate it by soaking it in methanol for about 2 minutes, and then transfer it to the electroporation buffer for later use. Assemble the gel and PVDF membrane with the clips in the order of "black-gel-membrane-white", place them in the electroporation tank, set the voltage to 100 V, 200 mA, and the electroporation time on ice for 90 minutes (the electroporation time is adjusted according to the molecular weight of the target protein);

[0071] (7) Sealing: Prepare the skim milk powder sealing solution: 2.5 g skim milk powder + 45 mL TBST. After electroporation, remove the PVDF membrane, rinse it twice with TBST, and then place it in the milk powder sealing solution for 2 hours.

[0072] (8) Incubation with primary antibodies: The primary antibodies used in this study were Anti-L-Lactyl, β-Tubulin, α-Tubulin, Acetyl-Tubulin, HDAC6, and DYKDDDDK tag. The antibodies were diluted with 5% BSA solution according to the antibody dilution instructions. Then, the primary antibodies were evenly dropped onto the PVDF membrane and incubated overnight at 4°C.

[0073] (9) Washing the membrane: Wash the incubated PVDF membrane with 1×TBST for 15 min / time, repeat 3 times;

[0074] (10) Incubation of secondary antibodies: Goat anti-rabbit and goat anti-mouse antibodies were diluted with 5% BSA solution at a ratio of 1:10000. The PVDF membrane was placed in the corresponding secondary antibody according to the species of the primary antibody and incubated at room temperature for 1.5-2 h.

[0075] (11) After incubation is complete, repeat step (8);

[0076] (12) Preparation of ECL chemiluminescent solution: Prepare according to the instructions with a ratio of luminescent solution A: luminescent solution B = 1: 1. Prepare fresh each time and use in the dark throughout the process.

[0077] (13) Turn on the WB exposure machine and software. After the displayed temperature drops to -25℃, drop the luminescent liquid onto the PVDF film for exposure and save the data.

[0078] 2.2 IP-WB

[0079] (1) Sample preparation: Taking a 6 cm culture dish as an example, collect 600 μL of protein sample according to the protein extraction steps, take 90 μL of sample and mix it with 30 μL of 4×Loading buffer, put it in a 100℃ metal bath and boil for 10 min, cool it down quickly and set it aside, and label it as Input group sample.

[0080] (2) Antibody and sample incubation: Take a 1.5 mL EP tube, add 500 μL of sample and 2 μL of Flag antibody, mix well, and incubate overnight in a 4℃ inverted mixer (normal IgG needs to be set as a negative control for antibody);

[0081] (3) Preparation of Protein A+G magnetic beads: Following the ratio of 20 μL of magnetic bead suspension per 500 μL sample, take the required volume of magnetic bead suspension into a 1.5 mL EP tube, then add 1×TBS to a final volume of 500 μL. Then, gently resuspend the magnetic beads using a pipette. Place the tube on a magnetic rack for 10 s to separate, discard the supernatant, and repeat the washing process twice. After washing, resuspend the magnetic beads with 1×TBS according to the initial volume of magnetic beads.

[0082] (4) After the incubation in step (2) is completed, add 20 μL of magnetic beads to the EP tube, incubate at 4℃ for 2 h by inversion, and after the incubation is completed, place it on a magnetic rack for 10 s to separate and remove the supernatant. Then add 500 μL of 1×TBS, gently blow to resuspend the magnetic beads, perform magnetic separation, and repeat the washing 3 times;

[0083] (5) Elution: Add 100 μL of 1×Loading buffer to the magnetic bead EP tube after washing in step (4), and heat at 95℃ for 5-10 min. Place on a magnetic rack for separation for 10 s, and collect the supernatant for Western blotting.

[0084] 2.3 Determination of lactic acid content

[0085] Prostate cancer cells (PC-3, LNCaP, 22RV1 cells) and prostate stromal cells (WPMY-1 cells) were collected, approximately 2.5 × 10⁻⁶ cells. 6 Cells were collected, added to extraction buffer, and dissolved by sonication. The cells were centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The absorbance of the supernatant and standard solution was measured at 570 nm using a lactate assay kit (BC2235, Solarbio) according to the manufacturer's instructions. Lactate concentration was calculated using a standard curve.

[0086] 2.4 CCK-8

[0087] (1) Seed the cells to be tested in 96-well plates at a seeding density of 8000 cells per well, with 4 replicates per group. Add 100 μL of PBS around the perimeter of the plate to reduce water evaporation;

[0088] (2) Drug treatment was carried out according to experimental needs: After cells adhered for 12 h, different concentrations of GA (0.2-1 mM), 20 mM Nala, and 40 mM Oxa were added. The treatment times were 24 h and 48 h, respectively.

[0089] (3) Add 10 μL of CCK-8 solution (G4103, Servicebio) to every 100 μL of cell culture medium (RMPI 1640 medium) and incubate in a cell culture incubator for 2 h.

[0090] (4) After incubation, cell OD was measured at 450 nm using an ELISA reader;

[0091] (5) Calculate the OD value and cell viability of the control group and use GraphPad Prism software to draw graphs.

[0092] 2.5 ROS content detection

[0093] (1) Preparation of DHE working solution: Taking a 6-well plate as an example, the working solution is prepared according to the reactive oxygen species detection kit (C1300-2, Beijing Pulilai Gene Co., Ltd.), with the ratio of DHE probe stock solution: PBS = 2 μL: 5 mL.

[0094] (2) Add DHE probe: Remove the cell culture medium in the 6-well plate, wash 3 times with PBS, add the diluted DHE probe, 1 mL per well, and set up a blank control.

[0095] (3) After incubating the cells at 37°C for 30 min, discard the upper culture medium and wash twice with PBS to remove probes that have not entered the cells.

[0096] (4) Collect cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend in 500 μL PBS in each well;

[0097] (5) The flow cytometer was used to detect the fluorescence intensity of the probe at an excitation wavelength of 535 nm and an emission wavelength of 610 nm.

[0098] 2.6 Apoptosis Detection

[0099] (1) Treat cells with 0.4 mM / 0.6 mM GA, 20 mM Nala, 40 mM Oxa, and NAC respectively. Alternatively, transfect cells one day in advance as needed;

[0100] (2) According to the Annexin V-FITC / PI apoptosis kit (AP101, Lianke Biotechnology) instructions, dilute 5× Binding Buffer with double-distilled water to prepare 1× working solution for later use;

[0101] (3) Instrument parameter adjustment: Collect cells from 3 wells of a 6-well plate, wash twice with pre-cooled PBS by centrifugation, discard the supernatant, add 500 μL of Apoptosis Positive Control Solution to resuspend the cells, and incubate on ice for 30 min. After incubation, discard the supernatant and add 500 μL of 1×Binding Buffer to each tube. One tube is a blank control tube, and two tubes are single-stain tubes. Then add 5 μL of Annexin V-FITC or 10 μL of PI to the single-stain tubes respectively, incubate in the dark for 5 min, and then detect them together with the experimental tubes in step (4);

[0102] (4) Discard the culture medium in the 6-well plate, wash the cells with pre-cooled PBS by centrifugation, and collect the cells in the wells into 1.5 mL EP tubes (double staining tubes for experimental groups). Take 500 μL of 1×Binding Buffer to resuspend the cells.

[0103] (5) In the experimental double staining tubes, add 5 μL Annexin V-FITC and 10 μL PI to each tube for double staining, and incubate at room temperature in the dark for 5 min.

[0104] (6) Detection on flow cytometer: On the flow cytometer, the voltage and compensation of the fluorescence channel were adjusted using blank tubes and single-stain tubes. Annexin V-FITC was detected through the FITC detection channel and PI was detected through the PI detection channel for apoptosis analysis. The results were then processed using Flowjo software.

[0105] 2.7 Fumarase Activity Detection

[0106] (1) Collect cells from 6 mm culture dishes and divide them into two tubes (tube A / tube B). The cells in tube A are quantified by BCA assay (PC0020, Solarbio).

[0107] (2) The B tube cells were prepared into mitochondria according to the instructions of the fumarate activity assay kit (G0869W, Grace Biotech) (the entire mitochondrial extraction process was carried out at 4°C). The cells were then sonicated in an ice bath to break them into liquid for later use.

[0108] (3) Set the microplate reader to a wavelength of 340 nm to measure the absorbance;

[0109] (4) Place the liquid in tube B to be tested into a 96-well plate (set 4 replicates), add the working solution of the reagent kit, and immediately measure the absorbance value A1. After incubating at 37°C for 30 min, measure the absorbance value A2 again.

[0110] (5) Finally, the fumarate activity was calculated based on the protein concentration of the sample.

[0111] 3. Results

[0112] Human prostate stromal cells WPMY-1 and prostate cancer cells PC-3, LNCaP, and 22RV1 were cultured in vitro, and the lactate content and pan-lactation modification level of each cell line were detected. The results showed that the lactate content and pan-lactation level of the three PCa cell lines were significantly higher than those of WPMY-1 cells. Figure 1 A and Figure 1 B) The above differences were statistically significant (P<0.05).

[0113] PC-3 cells were treated with 20 mM sodium lactate (Nala) and 40 mM sodium oxalate (Oxa), respectively. Oxa, as a lactate dehydrogenase inhibitor (LDHi), selectively inhibited LDHA and thus lactate production. Western blot results showed that, compared with the control group, Nala treatment significantly promoted pan-lactic acidification in PC-3 cells, while Oxa treatment significantly inhibited pan-lactic acidification. Flow cytometry results showed that, after Nala treatment, there was no significant difference in intracellular ROS levels and apoptosis rate between PC-3 cells and the control group, while Oxa treatment significantly increased intracellular ROS levels and apoptosis rate in PC-3 cells. Figure 2-4 The differences between A and B were statistically significant (P < 0.05). To verify whether Oxa promotes apoptosis in prostate cancer cells through oxidative stress, PC-3 cells were pretreated with 5 mM N-acetylcysteine ​​(NAC) 2 h before Oxa treatment. Compared with the Oxa-treated group, the ROS level and apoptosis rate in PC-3 cells were significantly reduced after NAC pretreatment. Figure 5 A, B and Figure 6 The differences in A and B were all statistically significant (P<0.05). These results indicate that increased lactate levels in PCa cells lead to pan-lactic acidification of proteins; inhibition of pan-lactic acidification can induce oxidative stress and apoptosis.

[0114] PC-3 and WPMY-1 cells were cultured in vitro and subjected to IP and WB experiments. Results showed that lactation bands were observed at the HDAC6 site in PC-3 cells. Figure 7 A), while no lactation bands were detected at the same location in WPMY-1 cells ( Figure 7 B) suggests the presence of high levels of HDAC6 lactation modification in PCa cells. Heatmap analysis showed that Oxa treatment significantly downregulated lactation levels at HDAC6 sites K32, K51, and K52. Figure 8 To identify the lysine lactation site of HDAC6, wild-type and mutant plasmids of HDAC6 with Flag markers were constructed. The mutant plasmids included K32R, K51R, K52R, and K52T (lysine K mutated to arginine R, which can be used to simulate delactation; lysine K mutated to threonine T, which can be used to simulate lactation). These plasmids were transfected into PC-3 cells. CCK-8 results showed that, compared with the blank control group, transfection with the HDAC6 wt plasmid significantly promoted the proliferation of PC-3 cells, while the proliferation of cells in the HDAC6 K52R group was significantly inhibited. Figure 9 A). IP-WB results showed that compared with the HDAC6 wt transfection group, HDAC6 lactation in PC-3 cells was downregulated after transfection with the HDAC6 K52R plasmid, while transfection with K32R and K51R mutant plasmids had no significant effect on HDAC6 lactation levels. Figure 9 B). Compared with the group without Nala treatment, Nala treatment of PC-3 cells transfected with the HDAC6 wt plasmid increased HDAC6 lactation levels, while transfection with the K52R plasmid decreased HDAC6 lactation levels. These results indicate that K52 may be the main lactation site of HDAC6 protein in PC-3 cells. Figure 9 C). Interspecific locus conservation analysis using MEGA software showed that the HDAC6 K52 locus is highly conserved across different species. Figure 9 D).

[0115] To investigate the effect of HDAC6 K52 lactation modification on HDAC6 protein function, PC-3 cells were transfected with HDAC6 wt, HDAC6 K52R, and HDAC6 K52T plasmids, respectively. Compared with the HDAC6 wt group, transfection with HDAC6 K52R plasmid increased the level of α-Tubulin acetylation in PC-3 cells. Figure 10 A), FH activity was significantly reduced ( Figure 10 B), ROS levels increased significantly ( Figure 11A); Flow cytometry results showed that transfection with HDAC6 K52R plasmid significantly increased ROS levels and apoptosis rate in PC-3 cells. These differences were statistically significant (P<0.05). Transfection with HDAC6 K52T plasmid did not significantly change ROS levels or apoptosis rate in PC-3 cells. Figure 11 (A, B, and 12A, B). These results indicate that lactate and pan-lactic acidification levels are higher in prostate cancer cells than in normal prostate stromal cells, and HDAC6 lactation modification is highly expressed in PCa cells. Combined with proteomics analysis, it was found that lactation modification at the HDAC6 K52 site is a key lactation site in PC-3 cells. Further research revealed that delactation at the HDAC6 K52 site reduces deacetylation function, inducing oxidative stress and apoptosis. This could potentially be a target for developing related products for the diagnosis and / or treatment of prostate cancer.

[0116] In summary, this invention provides a drug target: lactation modification of lysine residue 52 in the HDAC6 protein. Experimental results show that increased lactate content in PCa cells leads to pan-lactation modification of the protein; inhibition of pan-lactation induces oxidative stress and apoptosis; compared with the HDAC6 wt group, transfection with the HDAC6 K52R plasmid increased α-Tubulin acetylation, significantly decreased FH activity, and significantly increased ROS levels in PC-3 cells; transfection with the HDAC6 K52R plasmid also significantly increased ROS levels and apoptosis rate in PC-3 cells. This invention systematically elucidates for the first time how HDAC6K52 lactation modification inhibits oxidative stress and apoptosis in PCa cells. It provides direct and reliable theoretical and experimental evidence for the development of targeted therapeutics against HDAC6 K52 lactation modification, and has significant clinical application prospects and market value.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A drug target, characterized in that, The drug target is the lactation modification of lysine residue 52 in the HDAC6 protein.

2. The use of the drug target as described in claim 1 in the preparation of products for the diagnosis and / or treatment of prostate cancer.

3. The use of a reagent for detecting the lactation modification level of lysine 52 of the HDAC6 protein in the preparation of a kit for assessing the prognosis or treatment response of patients with prostate cancer, wherein an elevated level of lactation modification indicates a poor prognosis or treatment resistance.

4. An agent for promoting apoptosis of prostate cancer cells and / or inhibiting the proliferation of prostate cancer cells, characterized in that, The formulation contains an active ingredient capable of inhibiting the lactation modification level of lysine at position 52 of the HDAC6 protein.

5. The formulation as described in claim 4, characterized in that, The active ingredient is selected from any one or more of the following: (1) Inhibitor of lactate dehydrogenase; (2) A mutant targeting lysine 52 of the HDAC6 protein, wherein lysine 52 is mutated to an amino acid that cannot be lactated; (3) Antibodies or small molecule compounds that specifically block the lactation modification of lysine at position 52 of the HDAC6 protein; (4) Enzymes or their activators that promote the delactation of lysine at position 52 of HDAC6 protein.

6. The formulation as described in claim 5, characterized in that, The lactate dehydrogenase inhibitor mentioned is selected from sodium oxalate.

7. A method for in vitro screening of candidate drugs that promote apoptosis or inhibit the proliferation of prostate cancer cells, characterized in that, Includes the following steps: (1) Contact the candidate drug with prostate cancer cells expressing HDAC6 protein; (2) Detect the lactation modification level of lysine at position 52 of the HDAC6 protein in the cells; (3) If the level of modification is reduced, the candidate drug is determined to be the target drug.

8. A drug for the prevention and / or treatment of prostate cancer, characterized in that, The active ingredient in the drug is a lactate dehydrogenase inhibitor.

9. Application of inhibitors that inhibit lactation modification of lysine at position 52 of HDAC6 protein in the preparation of drugs for the prevention and / or treatment of prostate cancer.

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