Yellow croaker swim bladder polypeptide and its application

Abstract

The present invention relates to the field of bioactive peptides, and more specifically to yellow croaker maw peptides and their applications in anti-prostate cancer. The amino acid sequence of the polypeptide is SPSP (i.e., Ser-Pro-Ser-Pro) or GPAR (i.e., Gly-Pro-Ala-Arg). The yellow croaker maw peptides SPSP and GPAR prepared by the present invention are highly safe, can induce apoptosis of prostate cancer cell DU-145 cells, and have excellent anti-prostate cancer effects. At a concentration of 3 mg / mL, the inhibition rates of SPSP and GPAR on DU-145 cells are as high as 88.24% and 91.53%, respectively, and have the potential to be developed into anti-prostate cancer bioactive peptides.

Description

Technical field:

[0002] The present invention relates to the field of bioactive peptides, and in particular to yellow croaker maw peptide and its application in treating prostate cancer. Background technology:

[0004] Prostate cancer is one of the most common malignant tumors in men worldwide. In my country, the incidence and mortality rates of prostate cancer are increasing year by year, and the prevention and treatment of prostate cancer is a major challenge currently facing my country's public health. Early prostate cancer is difficult to detect, and most patients are already in the late stages when diagnosed. Currently, the main treatments for patients with advanced prostate cancer are hormone therapy, radiotherapy, chemotherapy, and endocrine therapy. Although these treatments have achieved certain results, they are prone to causing other adverse reactions such as frequent urination, dysuria, rectal bleeding, and decreased libido. For the treatment of prostate cancer, finding a safe and effective treatment method is currently a hot topic in anti-prostate cancer research.

[0005] Bioactive peptides have numerous advantages, including ease of access, biocompatibility, high efficacy, and low toxicity, and demonstrate significant potential in cancer treatment. Studies have shown that the anti-cancer mechanisms of bioactive peptides include inducing cell cycle arrest, inducing apoptosis, inhibiting angiogenesis, inhibiting tumor stem cells, and activating the immune response.

[0006] Yellow croaker is a farmed marine fish species, rich in protein and highly nutritious, making it a popular choice. The fish maw, located dorsally within the body cavity of bony fish, is a vital organ for maintaining balance. During yellow croaker processing, the maw is often disposed of as waste, resulting in a waste of resources. The high-value utilization of fish maw has attracted attention. Fish maw has a long history of use as both food and medicine in my country. The earliest written record of its use dates back to the Northern Wei Dynasty's "Qimin Yaoshu," and its medicinal use dates back to the Tang Dynasty's "Bencao Shiyi." The main component of fish maw is collagen, and fish maw peptides are enzymatic hydrolysis products of collagen, with various benefits including anti-aging, anti-fatigue, and anti-cancer properties.

[0007] Currently, there is little research on the in-depth development and application of yellow croaker maw peptides, and their application in the fight against prostate cancer is even more insufficient. Therefore, the development of anti-prostate cancer yellow croaker maw peptides can greatly enhance the economic value of yellow croaker maw and has broad market prospects. Summary of the invention:

[0009] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides the use of yellow croaker maw polypeptide in the treatment of prostate cancer. The yellow croaker maw polypeptide prepared by the method of the present invention has high safety, can induce apoptosis of prostate cancer cell DU-145, has excellent anti-prostate cancer effect, and has the potential to be developed into an anti-prostate cancer bioactive peptide.

[0010] The technical solution adopted by the present invention to solve the above technical problems is:

[0011] One of the technical solutions provided by the present invention is a yellow croaker swim bladder polypeptide, wherein the amino acid sequence of the polypeptide is SPSP (i.e., Ser-Pro-Ser-Pro) or GPAR (i.e., Gly-Pro-Ala-Arg).

[0012] The second technical solution provided by the present invention is the application of the yellow croaker swim bladder polypeptide SPSP or GPAR, especially in the fight against prostate cancer, and more particularly in the preparation of drugs for treating prostate cancer.

[0013] The yellow croaker swim bladder polypeptide SPSP or GPAR can be synthesized by solid phase synthesis technology, or can be obtained by enzymatic separation of yellow croaker swim bladder. The specific enzymatic separation method is as follows:

[0014] (1) Dried yellow croaker maw was soaked in distilled water (mass-to-volume ratio: 1-5:10-30), the pH was adjusted to 8-9, and 1% (w / v) of the soaking solution volume of complex enzyme (alkaline protease: neutral protease = 3:1) was added for enzymatic hydrolysis at 45-65 °C for 3-6 h. After the enzymatic hydrolysis was completed, the enzyme was inactivated by heating and the supernatant was obtained by centrifugation to obtain the enzymatic hydrolyzate.

[0015] (2) ultrafiltration of the enzymatic hydrolysate obtained in step (1) using an ultrafiltration membrane with a molecular weight of 1 kDa to collect components smaller than 1 kDa;

[0016] (3) Purify the fraction obtained in step (2) using a Sephadex G-25 gel chromatography column, elute with distilled water, detect the absorbance at 220 nm, collect the elution peak fractions, and determine the inhibitory rate of each elution peak fraction on DU-145 cells using the CCK-8 method;

[0017] (4) The fraction with the highest inhibitory rate on DU-145 cells in step (3) was purified using a DEAE-52 anion exchange column, gradient eluted, and the absorbance was detected at 220 nm. The elution peak fractions were collected and the inhibitory rate of each elution peak fraction on DU-145 cells was determined by CCK-8 method;

[0018] (5) Purify the component with the highest inhibitory rate on DU-145 cells in step (4) by reverse-phase high-performance liquid chromatography, collect the peak components in sequence, and determine the inhibitory rate of each peak component on DU-145 cells by CCK-8 method to obtain the component YCSB-1c with the highest inhibitory rate on DU-145 cells;

[0019] The amino acid sequence of the YCSB-1c component was further determined, and two yellow croaker swim bladder polypeptides with DU-145 cell inhibitory activity were obtained, namely SPSP and GPAR.

[0020] Preferably, the distilled water elution rate in step (3) is 0.7 mL / min.

[0021] Preferably, step (4) uses pure water, 0.1 mol / L, and 0.2 mol / L NaCl solution as gradient elution, and the elution rate is 2 mL / min.

[0022] Preferably, the reverse-phase high-performance liquid chromatography conditions in step (5) are as follows: Pursuit XRs C-18 column (250 mm × 21.2 mm, 10 μm), phase A: water containing 0.1% trifluoroacetic acid, phase B: acetonitrile containing 0.1% trifluoroacetic acid; flow rate is 1 mL / min, column temperature is 35 °C, and the wavelength of the UV detector is 280 nm; gradient elution conditions are as follows: phase A: 0~12 min, 95% → 80%; 12~24 min, 80% → 5%.

[0023] The third technical solution provided by the present invention is the YCSB-1c component obtained by reverse-phase high-performance liquid chromatography purification, and its application in anti-prostate cancer, more particularly, in the preparation of drugs for treating prostate cancer.

[0024] Beneficial effects

[0025] The yellow croaker maw polypeptides SPSP and GPAR prepared by the present invention are highly safe, can induce apoptosis of prostate cancer cell DU-145, and have excellent anti-prostate cancer effect. Under the intervention concentration of 3 mg / mL, the inhibition rates of SPSP and GPAR on DU-145 cells are as high as 88.26% and 91.53%, respectively, and have the potential to be developed into anti-prostate cancer bioactive peptides. Description of the drawings:

[0027] Figure 1 This is the elution curve of Sephadex G-25 gel column;

[0028] Figure 2 is the inhibition rate of YCSB on DU-145 cells;

[0029] Figure 3 This is the elution curve of YCSB through a DEAE-52 anion exchange chromatography column with pure water;

[0030] Figure 4 This is the elution curve of YCSB after DEAE-52 anion exchange chromatography column with 0.1 mol / mL NaCl;

[0031] Figure 5 This is the elution curve of YCSB after DEAE-52 anion exchange chromatography column with 0.2 mol / mL NaCl;

[0032] Figure 6 is the inhibition rate of YCSB-1, YCSB-2, and YCSB-3 on DU-145 cells;

[0033] Figure 7 is the reversed-phase high performance liquid chromatogram of YCSB-1;

[0034] Figure 8 is the inhibition rate of YCSB-1 (af) on DU-145 cells;

[0035] Figure 9 LC-MS / MS secondary mass spectrum of YCSB-1c, upper figure: SPSP secondary mass spectrum, lower figure: GPAR secondary mass spectrum;

[0036] Figure 10 AO / EB staining images of DU-145 cells after treatment with YCSB-1c, A: blank group, B: 50 μg / mL, C: 250 μg / mL, D: 750 μg / mL;

[0037] Figure 11 Effects of YCSB-1c on apoptosis of DU-145 cells, A: blank group, B: 50 μg / mL, C: 250 μg / mL, D: 750 μg / mL;

[0038] Figure 12 The cell cycle distribution diagram of DU-145 cells after treatment with YCSB-1c, A: blank group, B: 750 μg / mL, C: 250 μg / mL, D: 50 μg / mL;

[0039] Figure 13 Protein expression in DU-145 cells after treatment with YCSB-1c, A: blank group, B: 750 μg / mL, C: 250 μg / mL, D: 50 μg / mL;

[0040] Figure 14 is the inhibition rate of SPSP and GPAR on DU-145 cells. Specific implementation method:

[0042] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0043] Some of the materials and methods involved in the embodiments of the present invention are as follows:

[0044] 1. Materials

[0045] Yellow croaker maw was purchased from the seafood market, washed with clean water, impurities removed, and air-dried naturally.

[0046] 2. Reagents and instruments

[0047] Prostate cancer cell line DU-145 and DU-145 cell-specific culture medium were purchased from Wuhan Punosai Life Science Co., Ltd.; trypsin digestion buffer was purchased from Amresco, USA; dithiothreitol (DTT) and iodoacetamide (IAA) were purchased from Sigma, USA; AO / EB staining kit, Annexin V-FITC kit, PI kit, and BCA protein quantification kit were purchased from Sangon Biotech (Shanghai) Co., Ltd.; RIPA lysis buffer was purchased from Beyotime Biotechnology Co., Ltd.; CCK-8 reagent was purchased from Guangzhou Songshu Biotechnology Co., Ltd.; Bax, Caspase-3, Caspase-9, and β-actin antibodies were purchased from Cell Signaling Technology, USA; neutral protease and alkaline protease were purchased from Guangzhou Botang Trading Co., Ltd.; Sephadex G-25 gel filler and DEAE-52 gel filler were purchased from Beijing Ruida Henghui Technology Development Co., Ltd.

[0048] 1260 high-performance liquid chromatograph and 1290-6470 ultra-high-pressure liquid-mass spectrometry chromatograph, Agilent, USA; CytoFLEX flow cytometer, Beckman Coulter, USA; VarioskanTM LUX multifunctional microplate reader, Thermo Fisher Scientific (China) Co., Ltd.; TCS SP8 laser confocal fluorescence microscope, Leica, Germany; Alpha 2-4 LD plus vacuum freeze dryer, Christ, Germany.

[0049] Example 1 Preparation of Yellow Croaker Maw Polypeptide

[0050] (1) Enzymatic hydrolysis

[0051] Accurately weigh 50 g of dried yellow croaker swim bladder, add 1000 mL of distilled water and soak for 2 hours, adjust the pH to 8.5 with 0.1 mol / L sodium hydroxide solution, add 1% ( w / v ) was immersed in a mixture of enzymes (alkaline protease:neutral protease mass ratio = 3:1) for enzymatic hydrolysis at 55°C for 4 hours. After completion, the enzymes were inactivated at 90°C for 10 minutes. The supernatant was collected for enzymatic hydrolysis, vacuum concentrated, and freeze-dried to obtain freeze-dried fish maw hydrolysate powder, which was stored at -20°C until further use.

[0052] (2) Ultrafiltration

[0053] The above-mentioned lyophilized powder was reconstituted with distilled water and then ultrafiltered using an ultrafiltration membrane with a molecular weight of 1 kDa to collect the components smaller than 1 kDa and freeze-dried for later use.

[0054] (3) The present invention uses CCK-8 method to detect the proliferation inhibition rate of samples on DU-145 cells

[0055] DU-145 cells were cultured in a DU-145 cell-specific medium at 37°C and 5% CO2. Cell monolayers were grown to about 80% of the bottom of the flask for use in experiments. 100 μL of DU-145 cells were plated at 2×10 5 The cells were inoculated at a concentration of 10 μL / mL in a 96-well plate. After 24 hours of incubation, 10 μL of the sample to be tested was added. After 24 hours of incubation, 10 μL of CCK-8 solution was added and the reaction was continued for 2 hours. The absorbance was measured at 450 nm using a microplate reader. The inhibition rate was calculated as follows:

[0056]

[0057] Where: A0 is the absorbance value of the above system without adding DU-145 cells and containing only culture medium + CCK-8 solution; A1 is the absorbance value of the above system without adding the test sample and containing only cell culture medium + CCK-8 solution; A2 is the absorbance value of the above system with cell culture medium + sample + CCK-8 solution added simultaneously.

[0058] (4) Purification by Sephadex G-25 gel chromatography column

[0059] Dissolve the lyophilized powder of the ultrafiltration fraction (less than 1 kDa) in distilled water to a concentration of 10 mg / mL. Filter through a 0.45 μm microporous membrane and purify by passing through a Sephadex G-25 gel chromatography column. Elute with distilled water using an automated collector at a flow rate of 0.7 mL / min for 8 minutes per tube. Measure absorbance at 220 nm and plot the elution curve, with the number of eluted tubes as the horizontal axis and the absorbance as the vertical axis.

[0060] Elution curve Figure 1 As shown in Figure 3, a fraction was collected and named YCSB, which was concentrated under reduced pressure at 55 °C and freeze-dried for 72 h. The inhibitory rate of YCSB (1, 10, 100 and 1000 μg / mL) on DU-145 cells was determined according to the CCK-8 method described in (3). The results are shown in Figure 3. Figure 2As shown, the inhibition rate increased with increasing YCSB concentration, showing a concentration-dependent pattern. At a YCSB concentration of 1000 μg / mL, the inhibition rate was 28.4%, demonstrating a moderate inhibitory effect on DU-145 cells, suggesting the presence of anti-prostate cancer peptides in YCSB.

[0061] (5) DEAE-52 anion exchange column purification

[0062] YCSB was purified by DEAE-52 anion exchange column, using pure water, 0.1 mol / L, and 0.2 mol / L NaCl solutions as eluents for gradient elution at a flow rate of 2 mL / min. The collection volume of each tube was 5 mL. The absorbance value was measured at 220 nm, and the elution curve was drawn with the number of elution tubes as the horizontal axis and the absorbance value as the vertical axis. Figure 3 As shown, there is only one peak in the pure water elution curve, which was collected and named YCSB-1; Figure 4 As shown in Figure 2, there are two peaks, one high and one low, in the 0.1 mol / L NaCl elution curve. The peak fraction was collected and named YCSB-2. Figure 5 As shown in Figure 2, two peaks appeared in the 0.2 mol / L NaCl elution curve. The peak fraction was collected and named YCSB-3. The collected fractions were concentrated under reduced pressure at 55 °C and freeze-dried in a vacuum for 72 h. After reconstitution with distilled water, the inhibitory rates of the three fractions (1000 μg / mL) on DU-145 cells were determined according to the CCK-8 method described in (3). Figure 6 As shown, YCSB-1 had the highest inhibition rate of 48.3%, so YCSB-1 was further purified.

[0063] (6) Reverse-phase high performance liquid chromatography purification

[0064] The YCSB-1 fraction was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using a Pursuit XRs C-18 column (250 mm × 21.2 mm, 10 μm). Phase A consisted of water containing 0.1% trifluoroacetic acid, and phase B consisted of acetonitrile containing 0.1% trifluoroacetic acid. The flow rate was 1 mL / min, the column temperature was 35°C, and UV detection was performed at 280 nm. The gradient elution conditions were as follows: Phase A: 95% to 80% (0–12 min); 80% to 5% (12–24 min). The chromatogram is shown in Figure 7, revealing six peaks, designated YCSD-1a, YCSD-1b, YCSD-1c, YCSD-1d, YCSD-1e, and YCSD-1f. Fractions were collected and concentrated under reduced pressure at 55°C and freeze-dried in vacuo for 72 h. After reconstitution with distilled water, the inhibition rate of the six components (1000 μg / mL) on DU-145 cells was determined according to the CCK-8 method described in (3). Figure 8As shown, YCSB-1c exhibited the highest inhibition rate of 45.8%. Therefore, the amino acid sequences of the YCSB-1c components were determined.

[0065] (7) Amino acid sequence determination

[0066] The YCSB-1c fraction was added to a DTT solution to a final concentration of 10 mmol / L and reduced in a 56°C water bath for 1 h. IAA solution was then added to a final concentration of 50 mmol / L. After reaction in the dark for 40 min, the fraction was desalted and the solvent evaporated to dryness in a vacuum centrifuge at 45°C. The fraction was resuspended in 10 mL of 0.1% formic acid before LC-MS / MS analysis. Chromatographic conditions were as follows: a column (150 μm × 15 cm) packed with ReproSil-Pur C18-AQ resin (1.9 μm, 100 Å), an injection volume of 5 μL, phase A: water containing 0.1% formic acid, phase B: acetonitrile containing 0.1% formic acid, and a flow rate of 0.6 μL / min. Gradient elution conditions were as follows: Phase A: 0–2 min, 96% → 92%; 2–45 min, 92% → 72%; 45–55 min, 72% → 60%; 55–66 min, 60% → 5%. Mass spectrometry conditions: Samples were analyzed using a Q Exactive™ UHMR hybrid quadrupole-Orbitrap™ mass spectrometer at 2.2 kV, 270°C, a CID detector, and a precursor ion scan range of m / z 100~1500.

[0067] As shown in Table 1 , two tetrapeptides were detected, named SPSP and GPAR.

[0068] Figure 9 The following are the MS spectra of two tetrapeptides. The amino acid sequence of SPSP is Serine-Proline-Serine-Proline (Ser-Pro-Ser-Pro), and the amino acid sequence of GPAR is Glycine-Proline-Alanine-Arginine (Gly-Pro-Ala-Arg).

[0069] Table 1 Amino acid sequence identification of YCSB-1c

[0070]

[0071] (8) Determination of apoptosis of DU-145 cells by YCSB-1c samples

[0072] ① AO / EB method

[0073] DU-145 cells in the logarithmic growth phase were collected and 2×10 5Cells were seeded at a concentration of 100 μg / mL in a 6-well plate, with 2 mL per well. After 24 hours of culture, the culture medium was replaced with culture medium containing 50, 250, and 750 μg / mL YCSB-1c. A blank control was treated with the original culture medium and cultured for another 24 hours. The 6-well plate was removed, the supernatant aspirated, and the cells were washed twice with PBS, the wash solution discarded. 10 μL of an AO / EB mixture (AO:EB = 1:1) was added, and the cells were incubated at room temperature in the dark for 5 minutes. The cells were observed and photographed under a laser confocal fluorescence microscope.

[0074] The results are as follows Figure 10 As shown, acridine orange (AO) can penetrate cells with intact cell membranes, staining the nuclei with a uniform green fluorescence. In contrast, ethidium bromide (EB) can only penetrate cells with damaged cell membranes, intercalating into nuclear DNA and causing it to emit orange-red fluorescence. Cells in the blank group exhibited uniform green fluorescence, indicating the presence of a large number of viable cells. As the concentration of YCSB-1c increased, the number of viable cells decreased and the number of apoptotic cells increased, indicating that YCSB-1c can induce apoptosis in DU-145 cells in a concentration-dependent manner.

[0075] ② Flow cytometry to determine cell apoptosis

[0076] DU-145 cells in the logarithmic growth phase were collected and 2×10 5 Cells were seeded in 6-well plates at a concentration of 50, 250, and 750 μg / mL of YCSB-1c. Culture medium containing 50, 250, and 750 μg / mL YCSB-1c was added, respectively. A blank control was treated with the original culture medium and cultured for 48 hours. The pellet was collected by centrifugation at 2500 rpm at 4°C for 5 minutes. Pre-chilled PBS at 4°C was added and the pellet was collected by centrifugation at 2500 rpm for 5 minutes. This was repeated twice. The cells were resuspended in 500 μL of Annexin V Binding Buffer, followed by 5 μL of Annexin V-FITC solution. After reacting in the dark for 5 minutes, 5 mL of PI solution was added and the cells were allowed to stand in the dark for 5 minutes. The cells were filtered through a 300-mesh sieve, and apoptosis was analyzed by flow cytometry.

[0077] The results are as follows Figure 11As shown, the four quadrants in the figure represent different cell states: upper left, necrotic cells; lower left, normal cells; upper right, late apoptotic cells; lower right, early apoptotic cells. Compared to the blank control, the YCSB-1c group showed a significant increase in apoptotic cells. Early apoptotic cells showed a modest increase in the 50 μg / mL and 250 μg / mL YCSB-1c groups, but increased significantly in the 750 μg / mL YCSB-1c group, reaching 10.92%. The proportions of late apoptotic cells in the 50, 250, and 750 μg / mL YCSB-1c groups were 7.97%, 14.44%, and 16.25%, respectively, representing increases of 342.78%, 702.22%, and 802.78%, respectively, compared to the blank control (1.80%). YCSB-1c induces DU-145 cells into apoptosis, and the effect is positively correlated with concentration, consistent with the results of AO / EB staining.

[0078] ③ Flow cytometry to measure cell cycle

[0079] DU-145 cells in the logarithmic growth phase were collected and 2×10 6 Cells were seeded into 6-well plates at a concentration of 100 μg / mL and culture medium containing 50, 250, and 750 μg / mL YCSB-1c, respectively. A blank control was treated with the original culture medium and cultured for 48 hours. The cells were harvested by centrifugation at 2500 rpm at 4°C for 5 minutes, washed twice with 4°C pre-chilled PBS, and then 1 mL of -20°C 70% ethanol was added, pipetted evenly, and allowed to stand overnight. The cells were centrifuged at 2500 rpm at 4°C for 5 minutes, the supernatant discarded, and the cells were washed with 4°C pre-chilled PBS. 0.5 mL of PI staining solution was added to each tube of cells and incubated at 37°C in the dark for 30 minutes. Cell cycle analysis was performed by flow cytometry.

[0080] The results are as follows Figure 12 As shown in Table 2, the cell cycle can be divided into five phases: G0 phase (cell quiescence), G1 phase (pre-DNA synthesis phase), S phase (DNA synthesis phase), G2 phase (late DNA synthesis phase), and M phase (cell division phase). As shown in Table 3, compared with the blank group, the number of G0 / G1 phase cells in the YCSB-1c group increased significantly ( p <0.05, and showed concentration-dependent behavior; the number of cells in the S and G2 / M phases decreased significantly ( p <0.05). This indicates that YCSB-1c arrests the cell cycle of DU-145 cells at the G0 / G1 phase, prevents cells from entering the S phase to synthesize DNA, and thus induces apoptosis of DU-145 cells.

[0081] Table 2 Cell cycle distribution of DU-145 cells after treatment with YCSB-1c

[0082]

[0083] Note: Data in the same column with different superscript letters indicate significant differences ( p <0.05).

[0084] ④ Expression of apoptosis proteins

[0085] DU-145 cells in the logarithmic growth phase were collected and 2×10 6 Cells were seeded in 6-well plates at 400 μg / mL concentrations and culture medium containing 50, 250, and 750 μg / mL YCSB-1c was added. A blank control was treated with the original culture medium and cultured for 48 h. Cells were harvested by centrifugation at 2500 rpm at 4°C for 5 min, and 1 mL of PBS was added. The cells were centrifuged at 2500 rpm at 4°C for 5 min, and the supernatant was discarded. Cells were washed twice with pre-chilled PBS and centrifuged at 2500 rpm at 4°C for 5 min. The supernatant was discarded and the pellet was placed on ice. 200 μL of RIPA lysis buffer was added, mixed repeatedly by pipetting, vortexed for 45 s, and placed on ice for 1 h. The cells were centrifuged at 2500 rpm at 4°C for 30 min, and the supernatant was transferred to a clean, pre-chilled EP tube. Protein expression was quantified using a BCA protein quantification kit, and the expression of Bax, Caspase-3, and Caspase-9 proteins was determined by Western blot.

[0086] The results are as follows Figure 13 As shown in Table 3, Bax is a pro-apoptotic gene that can activate the mitochondrial apoptosis pathway, while Caspase-3 and Caspase-9 are two key proteases that execute apoptosis. The three apoptotic protein bands are clear without obvious tailing ( Figure 13 Compared with the blank group, the expression of Bax, Caspase-3, and Caspase-9 proteins increased in YCSB-1c groups at different concentrations in a concentration-dependent manner (Table 3). This suggests that YCSB-1c can upregulate the expression of Bax, Caspase-3, and Caspase-9 proteins, thereby promoting apoptosis in DU-145 cells.

[0087] Table 3 Protein expression in DU-145 cells after YCSB-1c treatment

[0088]

[0089] Note: Data in the same column with different superscript letters indicate significant differences ( p <0.05).

[0090] Example 2 Inhibitory effect of synthetic peptides on DU-145 cells

[0091] The two identified peptides, SPSP (Ser-Pro-Ser-Pro) and GPAR (Gly-Pro-Ala-Arg), were synthesized using solid-phase synthesis. The inhibitory rates of the two synthetic peptides on DU-145 cells were determined using the CCK-8 assay described in step (3) of Example 1. High (3 mg / mL), medium (2 mg / mL), and low (1 mg / mL) dose groups were set.

[0092] The results are as follows Figure 14 As shown in the figure, both synthetic peptides showed excellent inhibitory ability against DU-145 cells in a dose-dependent manner. At a high dose (3 mg / mL), the inhibition rates of SPSP and GPAR on DU-145 cells reached 88.26% and 91.53%, respectively.

[0093] In summary, the yellow croaker maw peptides (SPSP and GPAR) prepared in this invention can induce apoptosis in DU-145 cells, arrest the cell cycle in the G0 / G1 phase, and upregulate the expression of the apoptotic proteins Bax, Caspase-3, and Caspase-9 in DU-145 cells. Yellow croaker maw peptides have excellent anti-prostate cancer effects and have the potential to be used in the development of anti-prostate cancer drugs.

[0094] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0095] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A use of a yellow croaker maw polypeptide in the preparation of a drug for treating prostate cancer, characterized in that: The amino acid sequence of the polypeptide is SPSP, namely Ser-Pro-Ser-Pro, or the amino acid sequence of the polypeptide is GPAR, namely Gly-Pro-Ala-Arg.

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